PROCESSES IN BIOLOGICAL VISION: including,

ELECTROCHEMISTRY OF THE NEURON

This material is excerpted from the full β-version of the text. The final printed version will be more concise due to further editing and economical constraints. A Table of Contents and an index are located at the end of this paper. James T. Fulton Vision Concepts [email protected]

April 30, 2017 Copyright 2004 James T. Fulton The Retina 3- 1 3 Description of the Retina1

3.1 Introduction

A major finding of this work is that the retinula of the Insecta and some Mollusca eyes do not employ opsin as a substrate for the appropriate chromophores, which remain the rhodopsin()’s of the retinine family (section 3.6). The retinines are deposited directly on the villi emanating from the dendrites of the sensory receptor neurons. This finding has wide ramifications relating to the conventional concepts of the sensory receptors of Chordata. The opsin of these sensory receptors is used to control the orientation of the retinine liquid crystalline chromophores. It does not participate in the transduction process. Innumerable representations of the retina appear in the literature, at all different scales and using every imaginable artistic technique. Most of them do not show an absolute scale or even the coordinates of the retinal area being discussed. They virtually never show the direction and optical quality of the incident radiation or the location of the focal plane of the optical system. When these parameters are used as a foundation of discussion, many sketches, photographs and electron micrographs are found inadequate. Some become interpretable from a different perspective. The purpose of this Chapter is to provide a more comprehensive view of the retina, including a more coordinated description of the different zones of the retina, and a broader understanding of the top level architecture of the visual signaling system pertaining to the retina. Recently, good progress has been made in determining the distribution of different chromophorically sensitive photoreceptors in the retina. However, the results are still largely statistical.

Initially, this chapter will examine all photosensitive surfaces that contribute to the visual capabilities of an animal. In this context, the group of retinula found in the eyes of Arthropoda will be considered a retina. This provides a useful element in the development of an overall framework for discussing the retina.

Traditionally, the eyes of animals have been separated into two categories, vertebrate and invertebrate. Complete books have been written based on this distinction2. Bernhard has provided a comprehensive study of the compound eye3. Laughlin has provided an extensive comparison between these two categories4. However, this use of a dichotomy is constraining. As discussed briefly in Chapter 1, there are clearly three categories of eyes that are well aligned with the conventional phylogenic tree. These are the compound eye, and its prototypical simple eye, of Arthropoda, the complex eye with a direct retina of Mollusca, and the complex eye with a reversed retina of Chordata. Evolution has led to incongruities within this classification but they are minor–and illustrative. This work will employ the above trichotomy instead of the conventional dichotomy.

The above trichotomy provides a much better framework for interpreting both the detailed form of the types of photoreceptor cells found in eyes and the degree of structure in the retinas of these phyla.

The above trichotomy of eye types should not be confused with the spectral capability of eyes. The fundamental architecture of biological vision is tetrachromatic. Provision is made in the photochemistry of vision to form photoreceptors of four spectral types and many superfamilies and families of animals are tetrachromatic. It is only within phyla that one finds trichromats and they are of two distinct types. Arthropoda are generally short wavelength trichromats, employing photoreceptors sensitive to the ultraviolet, short and medium wavelength spectral ranges. Chordata are generally tetrachromatic, except in the physically larger animals and sometimes as a result of evolution to satisfy an ecological niche. Tetrachromats have photoreceptors sensitive to the ultraviolet, short, medium and long wavelength regions of the visible spectrum. Many birds5, fish and rodents are known to be tetrachromatic. Tetrachromaticity is common among fish6. However, many species exhibit tetrachromatic vision as

1Released: April 30, 2017 2Autrum, H. ed. (1979) Comparative Physiology and Evolution of Vision in Invertebrates: A. Invertebrate Photoreceptors. NY: Springer-Verlag 3Bernhard, C. (1965) The Functional Organization of the Compound Eye. NY: Pergamon Press 4Laughlin, S. (1981) Common principles for vertebrate and invertebrate visual systems. In Handbook of Sensory Physiology, Vol. VII/6B, “Comparative physiology and evolution of vision in invertebrates,” Autrum, H. ed. NY: Springer-Verlag pg. 263 5Altshuler, D. et. al. Xxx Evol. Ecol. Res. vol. 3, p 767 6Neumeyer, C. & Arnold, K. (1989) Tetrachromatic colour vision in goldfish and turtle. Xxx In 2 Processes in Biological Vision

juveniles but frequently change to long wavelength trichromats with age (or more fundamentally, as the lens group grows and thickens). The larger members of Chordata, particularly the larger terrestrial members such as Human, have lost their ultraviolet sensitivity due to the thickness of the lens group serving the eye. It is not clear that the retinas of all large Chordata have completely lost their ultraviolet capability through evolution. Humans exhibit at least some ultraviolet capability when the lens group is removed. The large chordates are long wavelength trichromats, having photoreceptors sensitive to the short, medium and long wavelength portions of the visual spectrum. The data available for Mollusca is inadequate to determine their general capability. 3.1.1 Background 3.1.1.1 Order and tetra-chromaticity in the photoreceptor arrays of the retinas

By examining the trichotomy of animal retinas, there is a clear trend with regard to the statistical order of the arrays of photoreceptors. There also appears to be an order with respect to these arrays in many animals as a function of their maturity. Franceschini has provided a striking example of the orderliness of the retinal array in Arthropoda7. This orderliness even extends to the lamina behind the retina. The orderliness is clearly traceable to the prototypical simple eye. The orderliness is reminiscent of that of a crystalline structure.

Bowmaker & Kunz have described a similar level of orderliness in the immature brown trout8. They noted that the orderliness appeared to fall with age and surmised that the ultraviolet photoreceptors tended to disappear from the retina. Their images were primarily of small areas of the retina. Their spectrograms of the four chromophores of vision, although somewhat limited at the extreme wavelengths and plotted on a normalized linear ordinate, are in close agreement with those predicted by this work.

Kouyama & Marshak studied the statistics of two mosaics of retinal neurons in the primate retina9. The areas were in the mid-periphery, typically six millimeters from the macaque fovea, and consisted of about 150 photoreceptors and about twice as many signal processing neurons described as bipolar cells. Unfortunately, they did not employ optical stimuli to excite the arrays. They employed staining of what were believed to be photoreceptors and bipolar cells associated with the short wavelength spectral channels of vision. Therefore, the arrays were not directly correlated with the spectral performance of the visual system. Chan, et. al. provide both morphology and statistics on the distribution of horizontal cells of the 1st lateral matrix in new world monkeys, with some comparisons with the macaque monkey10. They provide a picture of one horizontal cell, at 6.9 millimeters from the fovea, with a neuritic arborization of less than 50 microns in diameter but an axon that is 958 microns long. This length approaches the maximum reported for neurons processing signals in the electrotonic (analog) domain.

Chan, et. al. also reference the paper by Dacey defining two distinct types of horizontal cells that appear to correspond to the P-channel and Q-channel horizontal cells of this work11. The H2 horizontal cells respond to short wavelength stimulation. Both the H1 and H2 type cells respond to mid-wavelength stimulation. The H1 type cells respond to both mid and long wavelength stimulation but do not respond to short wavelength stimulation. The H1 cells generate Q-channel chrominance signals and the H2 cells generate P-channel chrominance signals according to the nomenclature of this work. Chan, et. al. confirm the polymorphism of color vision in the marmoset based primarily on their ability to get it to make color matches following training. They claim trichromatism in females of that family. However, they claim the other animals are dichromats because they did not respond.

7Franceschini, N. (1985) XXX 8Bowmaker, J. & Kunz, Y. (1987) Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the broun trout (Salmo trutta): age-dependent changes. Vision Res. vol. 27, no. 12, pp. 2101-2108 9Kouyama, N. & Marshak, D. (1997) The topographical relationship between two neuronal mosaics in the short wavelength-sensitive system of the primate retina. Visual Neuroscience, vol. 14, pp. 159-167 10Chan, T. Goodchild, A. & Martin, P. (1997) The morphology and distribution of horizontal cells in the retina of the new world monkey, the marmoset Callithrix jacchus. Visual Neuroscience, vol. 14, pp. 125-140 11Dacey, D. Lee, B. Stafford, D. Pokorny, J. & Smith, V. (1996) Horizontal cells of the primate retina: cone specificity without spectral opponency. Science, vol. 271, pp. 656-659 The Retina 3- 3

Kaneko provides a more concise description of several types of horizontal cells based on their spectral sensitivity12. He also notes the high degree of spatial summation associated with these cells and their genealogy traceable back to the S-potentials of Svaetichin in 1953. He describes so-called L-type horizontal cells which hyperpolarize in response to any spectrum of light and would correspond to the bipolar cells forming the R-channel (luminance) signals of this work. He also describes a biphasic C-type cell that is hyperpolarized by short wavelength light and depolarized by long wavelengths. Unfortunately, the paper was in the form of a review and he did not quantify “long” in this context. The C-type cell could correspond to the horizontal cells forming the P-channel chrominance signal of this work. He also described a waveform previously described by Svaetichin and also by Tomita. This “triphasic” C-type cell was hyperpolarized by spectral lights at both ends of the spectrum and depolarized by the intermediate spectral region. He also discussed the fact that the “horizontal cells” occurred in multiple layers of the retina. The “external horizontal” cells connected to photoreceptors and corresponded to the 1st lateral matrix of this work. The intermediate horizontal cells are probably bipolar cells. The “internal horizontal” cells appear to correspond to the 2nd lateral matrix. This organization may account for the triphasic character of the later cells. Cells in this layer are predominantly concerned with the spatial summation of signals from earlier cells. Subtraction of P-channel and Q-channel signals could generate a triphasic output although the ultimate purpose was to provide spatial integration of information. Kaneko also reported intermediate horizontal cells that were axon-less (amercine). Kaneko hints at the time-dispersal processing of information, at least among the amercine cells of the 2nd lateral matrix. He also discusses the “atypical” spike signals found by some investigators when probing the 2nd lateral matrix. Finally, he notes that as of 1979 the nature of any chemical neurotransmitter associated with the photorecpetors had not been identified. He posits that it is not glutamate because of the abundance of this material throughout the neural system. He notes that other investigators have been suggesting GABA because of the reports that horizontal cells take up this chemical from a bathing solution and can also synthesize GABA.

The Chan, et. al. paper also provides considerable statistical information on both the size of horizontal cells and their arborization. It also addresses the ubiquitous question of why some horizontal cells have “axons” and others do not. Clearly there are a variety of types of horizontal cells. Some appear to have short axons, some have long axons and some have an axon sharing an outer coating with neuritic structures over a portion of their length.

The introductory material in the above paper by Kouyama & Marshak repeats much questionable conventional wisdom. It highlights another significant relationship between the retinas of animals, are all photoreceptor arrays formed from and considered part of the brain or are they part of the peripheral nervous system. Most texts consider the chordate retina to be formed of a multilayer extension of the neural tissue of the central nervous system. However, the photoreceptors of Arthropoda are frequently described as more peripheral in nature with their axons extending to contact the structurally remote lamina. Mollusca is also frequently shown as employing photoreceptors with minimal local neural support prior to long axons extending to a separate structure that is generally considered part of the brain. On the other hand, the chordate retina is usually shown as a multilayer laminate with considerable similarity to the laminate found in the chordate brain. Whereas, Houyama & Marshak identify this laminate as consisting of three layers, it can be considered as five layers by separating the inner nuclear layer into a 1st lateral layer, a bipolar layer and a 2nd lateral layer (see Section 2.6). This brings the number of layers more in line with the conventional view of the LGN and cortical structures. Note the 2nd lateral layer is far less well developed in primates than it is in felines and other hunting members of Chordata.

It is important to note the tetrachromatic capability, and polarization detection capability designed into the basic retina of vision. This capability is frequently limited through evolution based on the local environment. In the paper of Bowmaker & Kunz, the fish, Salmo trutta, a member of Chordata, has little need for an ultraviolet capability as it matures and moves into deeper water that ultraviolet light does not penetrate. Furthermore, as it matures, the thickness of its lens and corneal cover become too thick to transmit ultraviolet light effectively. It is possible that the ultraviolet photoreceptors would atrophy. However, recent data from humans shows that the ultraviolet sensitive photoreceptors remain active in the retina through old age. It is only the lens that restricts ultraviolet vision in humans (See Section 17.2.4). When the retinas of the chordates are examined, the orderliness of the photoreceptor arrays is seen to be much less than in Arthropoda. There appears to be a definite fractal appearance to the arrays when an area of several hundred cells on a side is examined. Frequently there appears to be a hexagonal grouping on a local basis within the overall array. There are also additional local arrangements associated with the development of the fovea and foveola in some animals. These arrangements, which frequently are not circularly symmetrical, also appear to be evolutionary responses to environmental conditions.

12Kaneko, A. (1979) Physiology of the retina. Ann. Rev. Neurosci., vol. 2, pp. 169-191 4 Processes in Biological Vision

As a result of these modifications, the orderliness of photoreceptor arrays in chordate retinas cannot be easily described. Attempts at description of these retinas quickly enter the field of complex statistical notation. Examples of the trend in these statistics are found in Kageyama & Wong-Riley13. That article also hints at the near complete breakdown in the correlation between the statistics of the photoreceptor arrays and subsequent neural arrays of the signal processing stage. The extremely high degree of spatial signal summation performed by the bipolar cells and lateral cells of the 1st lateral matrix destroys any one-to-one correlations based on physical geometry in the signal path of chordates except for the cells of the foveola. The size of these spatial receptive fields at the ganglion level has been studied by Benardete & Kaplan, again falling back on statistics to describe their findings14. The Benardete & Kaplan papers also introduce another interesting topic. When performing center-surround experiments, they used two different stimulus configurations. In one case, the center and surround shared a common border. In another, there was a (uncontrolled?) gap between the two stimuli. They do not discuss the uncontrolled variable related to tremor that may have led them to these configurations. They did however, paralyze the optical system of the animals under test. Whether they paralyzed the tremor as well as the larger saccades, which have different origins within the brain, was not discussed. Although they used a color CRT monitor, they did not describe any measures to control or quantify the spectral content of their stimuli. Their papers used the M & P pathway designations. There was no discussion of the P-channel and Q-channels related to the chrominance signals defined in this work. 3.1.1.2 Comparison of retinas of different phyla

Most of the literature does not associate the photoreceptors of vision directly with brain tissue. In Arthropoda, the axons of the photoreceptors are shown traveling to a distinctly separate structure, the lamina for additional processing15. In the case of Mollusca, there is less data available. However, it appears that the photoreceptors are supported locally by at most one or two layers of neurons before the signals travel to the structures associated with the animals brain. Only further study will determine whether these retinas can be considered to be made by material intimately associated with the brain of the animal. If they are, the neural pathways from the retinas would be properly described as association fibers within the brain. Otherwise, they would be considered peripheral neural paths. The case is more clear for Chordata. The retinas are extensions of the surface of the brain and both the optic nerve and optic radiation are clearly classed as association fibers. There is a significant difference in the topology of the retinas and the rest of the brain. In most of the brain, the association fibers originate and terminate in layer 4. Placing these terminals in a middle level of the brain tissue provides maximum oppportunity for interconnection with both decoding and encoding circuits. In the case of the retinas, there is no decoding requirement. The ganglion cells connecting to the association fibers are found in the final layer of the retina.

Laughlin has provided a broad though dated comparison of retinas, and other elements of vision, between vertebrates and invertebrates in an index to a volume devoted to invertebrate vision16. Use of this dichotomy does not present the data for Arthropoda and Mollusca in the proper context and leads to concepts and interpretations not adopted in this work, such as multiple spatial images being passed along the signaling system. However, the coverage is extensive and much of the data is useful. On page 5 of the same work, Autrum discusses conforming the nomenclature used in the study of the retinas and brains of arthropods and molluscs to eliminate confusion.

Sections 3.5 & 3.6 have been added to this chapter for further discussion of the ommatidia of members of Mollusca and Insecta respectively. These sections focus on the morphology and function of these ommatidia. Little data has been uncovered relating to the metabolism and potential regeneration of these ommatidia and their component photoreceptor neurons to date (2016). Section 3.6 has become quite large because of the surprising diversity of the elements of the visual modalities of the millions of Orders, SubOrders and Families of Insecta. Some of this diversity has called for a taxonomy of Insecta based specifically on features within the visual modalities of various species (Section xxx).

13Kageyama, G. & Wong-Riley, M. (1984) The histological localization of the cytochrome oxidase in the retina and lateral geniculate nucleus of the ferret, cat, and monkey. J. Neuroscience. vol. 4, no. 10, pp. 2445-2459 14Benardete, E. & Kaplan, E. (1997) The receptive field of the primate P retinal ganglion cell. Visual Neuroscience, vol. 14, pp. 169-185 & 187-205 15Gouras, P. (1991) The perception of color. Boca Raton, FL: CRC Press 16Laughlin, S. (1981) Common principles for vertebrate and invertebrate visual systems. In Handbook of sensory physiology, Vol VII/6B “Comparative physiology and evolution of vision in invertebrates,” Autrum, H. ed. NY: Springer-Verlag, pp. 263-267 The Retina 3- 5 3.1.2 A framework for discussion of the chordate retina

The remainder of this Chapter will focus on the chordate retina. In 1979, Kaneko made the bold statement that “The main neuronal circuitry of the retina has now been made clear, but most of our understanding still remains superficial17.” He did this after providing tentative answers to only eight of the twenty questions Stell had placed on the table in 197218. To broaden this understanding and combine it with the morphology of the retina requires a more sophisticated framework. It is suggested that this can be done by examining the retina from three major perspectives, a morphological plan view, a morphological profile view, and a signaling architecture view.

3.1.2.1 The plan view perspective of the retina

The retina is far from a symmetrical structure. When discussing the plan view of a retina, it is important to consider a global view of the whole retina as well as local views that are referenced with respect to the coordinates of the global view. It is not adequate to specify that a recording is made at a distance from the fovea. The angle must also be specified. It is also of interest to make recordings of the various layers of the retina after staining. It is critical that such recordings specify the same dimensions as mentioned above. Considerable effort should also be made to specify the absolute or relative location of the imagery in terms of the cross-section of the retina from Bruch’s membrane or some other surface near the retinal pigmented epithelium (RPE) that is relatively stable. Recording as a function of the depth from the vitreous humor makes the results highly location dependent.

There are two primary problems with describing the plan view of a retina. First, it is a spherical surface that is difficult to represent using a two dimensional figure. It is also frequently more useful to represent the plan view with respect to object space, as it is normally done in optometry. If it is presented with respect to object space, the correct index of refraction associated with the normal object space environment should be used. Observing this precaution insures the data represents the functional characteristics of the retina. Notice that the angles in object space are significantly different from those of the terrestrial chordate retina in image space due to the difference in index of refraction between the vitreous humor and air. Failing to maintain the appropriate indexes significantly distorts the angles relative to the retina with reference to the pupil as measured in object space. Calculating distances on the retina using incorrect angles is counterproductive. 3.1.2.1.1 A panoramic view of the photoreceptors of the retina

In many experiments attempting to record the plan view of a retina, there is an interesting option. A panoramic image of the retina covering 180 degrees or more in object space can be recorded over a swath of only ten degrees or less. The resulting image will show the effective sizes of the Outer Segments of the photoreceptors throughout the field of the retina. They will appear quite small near the foveola and quite large near the periphery even though all photoreceptors in a given retina are nearly the same size (typically within a factor of two). The larger apparent difference is due to the variable focal length of the physiological optical system of the animal.

3.1.2.1.2 Global recording of other layers of the retina

Techniques are available for recording layers of the retina other than the photoreceptors. Such recordings are usually performed in-vitro after dissection of the ocular, and frequently after slicing the retina to make it lay flat. These processes severely distort the actual geometry of the retina relative to its performance in object space. The resulting recorded data should be carefully related to the equivalent object space data. Alternately, the retina should be stained, the vitreous humor replaced and the physiological lens system replaced so that a panoramic recording can be made as outlined above. When discussing the layers of the retina, it is suggested that the layer notation discussed in Chapter 2 be used to help codify the database, recognizing that the 2nd lateral layer is poorly developed in human retinas and probably in all primates optimized for an arboreal environment.

17Kaneko, A. (1979) Op. Cit. pg. 185 18Stell, W, (1972) The morphological organization of the vertebrate retina. In Handbook of Sensory Physiology. Vol. VII/2. Ed. Fuortes, M. NY: Springer-Verlag. Pp. 111-213 6 Processes in Biological Vision

3.1.2.1.3 Local recordings of the plan view of the retina

The literature is replete with local recordings of the retina. Some are made through the physiological optical system and some are made after dissection. For the human, a terrestrial chordate, the angles discussed with respect to these recordings are seldom specific and are frequently misstated or calculated erroneously. If the recording is made through the physiological optics, the angles refer to object space and not image space. As discussed above, the surface dimensions on the retina must be calculated with respect to the image space angles. 3.1.2.2 The profile view perspective of the retina

It is seldom useful to attempt to record a global view of the retina in profile because it is so thin relative to its overall dimensions. However, it is very useful to make local recordings. These can show the orientation of the Outer Segments of the photoreceptors relative to the pupil of the eye as well as define the density of neurons in the different layers of the retina laminate as a function of location relative to the optical axis, the fovea or other landmarks. It is very important to indicate specifically what landmark was used for reference and where the image was taken relative to that landmark in the spherical coordinates of the retina (with specific reference to object or image space). Linear dimensions relative to the retina are usually inadequate in defining a location on the retina for purposes of understanding function. As mentioned above, it is suggested that the layers of the laminate be defined in terms of those in Chapter 2 to support and maintain a consistent database. It is also suggested that measurements be made relative to Bruch’s membrane or some other feature near the RPE that is stable relative to the layers.

Measurements that can be precisely located by others can support detailed investigations into the signal architecture of the brain. This architecture is poorly understood at this time beyond the simplest level. This work has found that the system makes use of certain spatial relationships in the signal processing of the retina to avoid the need for the brain to employ trigonometric calculations. It is fairly obvious that if more detailed information concerning the retina was known as a function of location, the precise mapping of this type of spatial layout could be recognized. There are other mappings that are currently believed likely but cannot be recognized with the limited precision of the data available on a global context. It is this level of detail that is needed to help understand the extensive arborization associated with the neurons of different layers of the retina. In many cases, this knowledge may not lead to defining sublayers of the retina. It may however lead to the description of multiple maps that represent different groups of neurons within a single layer. 3.1.2.3 The signaling architecture of the retina

The literature presents a pretty clear picture of the gross functional responsibilities of each major layer of the chordate retina. There are some differences in emphasis between the various major families (humans have a poorly developed 2nd lateral matrix layer). It is the opportunity to extend the functional subdivision of these responsibilities which calls for the type of spatial accuracy in data recording discussed in the above sections. As these studies proceed, it becomes ever more important to observe and update the best available signaling architecture diagrams and detailed signaling schematics of the retina. It also becomes extremely important to recognize that the signal processing architecture employs both temporal, chromatic and spatial processing–frequently within the same layer of the retina. The so-called tri-phasic waveforms of Svaetichin and of Tomita may be examples of this. These have been recorded based on the spectral characteristics of large area stimuli. It has not been shown that these waveforms are considered spectrally important by the brain. They might actually be of significant spatial importance instead. In the absence of any broad band (black and white) photoreceptors in the animal retina, the signal processing must take advantage of the original spectral signals from the photoreceptors. If these signals are differenced, either in the 1st or 2nd lateral matrices, and then further processed without concern for their spectral properties, spatial pre-emphasis of important elements of the scene in object space can be obtained. This is very likely to be the situation found in big and small felines. They are known to generate or extract signals in the cortex that show both angular preference and selectivity to the pitch of gratings at a particular orientation19. Laboratory activity in the next few years should show great progress in understanding the still mind numbing complexity in the architecture of the retina of Chordata. However, a greater degree of precision in locating the relevant groups of neurons within a given layer of the retina will be required.

3.1.2.3.1 Expansion of spatial processing within the Top Level Schematic

19Hubel, D. (1988) Eye, brain, and vision. NY: Scientific American Library The Retina 3- 7

Although Hubel, et. al20. and others have made strides in recording neural activity in the cortex of felines reflecting a response to spatial orientation and complexity within a scene, it is unlikely, based on the Top Level Architecture of the visual system that the processing associated with these responses originates in the cortex or LGN. It most likely originates in what has been labeled the 2nd lateral matrix of this work. This matrix probably generates a series of signals, of both monophase and biphase character, that can be transmitted in compact form over the optic nerve before being processed further by the engines of the old brain and the Cortex. As discussed in the sections on the Visual Block Diagram, this spatial encoding has not been explicitly presented in this work because of the limited data available concerning its specific architecture and signaling paths. These paths, carrying primarily correlated spatial information, have been assigned the Z-channel designation (Z1, Z2, Z3, etc.) at this time pending further specificity. Eventually, it is hoped that these channels can be correlated with the investigations of the above authors. The signals of the Z-channels are assumed to originate in the 2nd lateral matrix. However, the processing matrices of the retina have been shown as three distinct entities primarily for pedagogical reasons. As hinted at above, there is no guarantee that these matrices are discreet with respect to the layers of the retina. They may involve multiple groups of neurons within one or more layers. It is not even clear yet whether this type of processing is concentrated in the various regions associated with the foveola, the periphery or both.

3.1.2.3.2 Subdivision of retinal layers or interdigitation

Up through the 1970's, the chordate retina was generally considered to consist of three neural layers. The interneural layer, INL, was frequently described as the bipolar layer. With a total thickness of only about 50-60 microns, it was very difficult to subdivide this layer based on electrophysiological probing. More recently, finer probes and more precise positioning mechanisms have become available. Accompanied by a variety of staining techniques, these improvements have led to recognition of additional regions within the INL. Currently, this layer is seen to consist of three sublayers of neural cells,

+ the 1st lateral matrix layer consisting of what are generally labeled as horizontal cells and appear to be primarily concerned with generating chrominance signals,

+ the bipolar layer consisting primarily of bipolar cells concerned with the generation of luminance signals, and

+ the 2nd lateral matrix layer consisting of a variety of cells grouped around the designation Amacrine cells. This layer is poorly developed in the higher primates but appears to be primarily concerned with the generation of appearance signals.

Beyond the above coarse subdivision of the signal processing layers of the retina, each of these sublayers exhibits a variety of functional cell types. The literature has suffered from a lack of a model in this area with many authors providing glimpses of the signals found in one sublayer based on the probing of a small number of samples. Some of the papers providing the best signal characteristics have assumed the simplest possible architecture21. Kaneko & Tachibana open their paper with the presumptive statement that “bipolar cells provide the only pathway from photoreceptor cells to ganglion cells.” Their analyses do not provide for or address any other type of cell. However, much of their data can be interpreted as involving the horizontal cells of another paper of theirs22 and those of many other authors23. They do stress that the first paper deals specifically with “bipolar cells” which have highly complicated receptive fields. This condition typifies the problem of sorting out the wide variety of functional cell types within these regions.

As an example of the difficulty of drawing conclusions concerning the architecture of the individual layers (or sublayers) of the signal processing region of the retina, Kaneko & Tachibana24 studied the receptive fields of 85 “bipolar cells.” Parentheses appear around this term because their article did not recognize the existence of any other type of signal processing cells nor did they precisely define what they meant by this designation. They did not define the location of their probe with respect to the retina and any fovea if present. They found that only one

20Hubel, D. (1988) Op. Cit. 21Kaneko,, A. & Tachibana, M. (1983a) Double color-opponent receptive fields of carp bipolar cells. Vision Res. vol. 23, pp. 381-388 22Kaneko,, A. & Tachibana, M. (1983b) Double color-opponent receptive fields of carp bipolar cells. Vision Res. vol. 23, pp. 371-380 23Dacey, D. Lee, B. Stafford, D. Pokorny, J. & Smith, V. (1996) Horizontal cells of the primate retina: cone specificity without spectral opponency. Science, vol. 271, pp. 656-659 24Kaneko, A. & Tachibana, M. (1983a) Op. Cit. 8 Processes in Biological Vision

quarter of these cells exhibited “double opponent receptive fields. Of these 15 represented “on-center cells” and three represented “off-center cells.” Kaneko & Tahibana presented interesting data, in the same paper, describing the performance of the off-center double opponent cells as a function of stimulus spot size. This data clearly showed that the cells were involved in a spatial integration processing role. No data was offered as to whether the cells studied provided signals, either simultaneously or exclusively, to the chrominance or the appearance channels of the visual system. See Section 3.4.2.2 for details.

3.1.2.3.3 Initial tabulation of signal processing roles within the retina

Although the majority of the interconnections between the photoreceptors of the signal detection stage and the initial neurons of the signal processing stage are pretty well understood, it will be a long time before the interconnections between the initial signal processing neurons and the ganglion cells is understood to any level of detail. This situation is exacerbated by several conditions. First, we do not have a clear conceptual understanding of the roles of time, space and wavelength in the algorithms used in signal processing within the retina. Second, we do not know if the same algorithms are used in the foveola, the fovea and the periphery of the retina. It appears that the algorithms vary significantly. Third, the input structures of the ganglion cells appear to form part of the signal processing stage of vision. These structures clearly perform a significant signal integration role prior to encoding.

Whereas it is easy to record signals as a function of spectral wavelength, it is much more difficult to record signals with fine precision as a function of spatial location. Most spatial location data involve the integration of data from retinal electrophysical probing in response to finite size (gross) spectral stimuli. On the other hand most attempts to obtain finer data have employed gratings or sharp edges in psychophysical, not electrophysical, experiments. There has been very little activity attempting to correlate the results of the probing of the retina with the form of the signals actually transmitted to and perceived by the cortex. A probe signal that exhibits spectral characteristics but is processed by the signal projection stage as appearance information is not perceived in the same context as the laboratory investigator might presume.

Because of the above problems, the literature contains a great deal of data employing a great variety of notation, much of it based on ordinal numbers, type 1, type 2, H1, H2, etc. or on other convenient designations. Correlating these designations over a period of 50 years, based on the limited specificity in the published articles, becomes quite difficult. However, it is possible to discern classes of functional signal processing within the retina based on the literature. These will be grouped in TABLE 3.1.2-1 for purposes of discussion. The Retina 3- 9

TABLE 3.1.2-1 FUNCTIONAL CLASSES OF SIGNAL PROCESSING IN THE RETINA OF CHORDATA OTHER THAN IN THE FOVEOLA AND WITHOUT CONSIDERATION OF TIME-DISPERSAL

Predominant layer Major Predominant % of Function (Designation in vision) role cell type layer

SIGNAL DETECTION I. Molecular Spectral sig. gen. Photoreceptor 100 Transduce light into electricity and (Photoreceptor) normalize the signal amplitudes SIGNAL PROCESSING II. External granular Chrom. sig. gen. Horizontal High Compose the N, O, P & Q signals as (1st Lateral matrix) appropriate (typically associated with “H1” and “H2” cells)

III. Pyramidal Lumin. sig. gen. Bipolar ? Compose the monophase R signal (Bipolar) ? Relay biphase signals from the 1st lateral matrix to the ganglion cells*

IV. Internal granular Appear. sig. gen. Horizontal. ? Compose the currently poorly defined nd (2 Lateral matrix) (Amercine) Zn signals describing specific spatial properties (static and dynamic) of the scene

25** Perform spatial signal integration for as yet poorly understood purposes

? Compose the “threat location” signals for transmission to the Precision Optical System of the mid-brain***

SIGNAL PROJECTION V. Ganglionic Encoding of Ganglion Threshold and encode monophase (Ganglion) analog signals signals from all sources

Encode biphase signals from all sources.

* In general, literary references to biphase or inverting bipolar cells are considered lateral cells in this table. There is an open question concerning whether some or all axons from the 1st lateral layer connect directly to ganglion cells. An alternate situation would require some bipolar cells to be biased for the transmission of biphase signals from the 1st lateral layer to the ganglion cells. ** The percentage is taken from Kaneko & Tachibana (1983b) *** Threat location signals describe the angular distance of a threat from the line of fixation in terms of the axes of the ocular muscle pairs. 10 Processes in Biological Vision

3.1.3 Additional concerns in experiment design

All of the experimental effort to date associated with the organization of the retina has been exploratory. Lacking a detailed model with which to work, most of it has involved less than rigorous experimental design. The result has been a large amount of data collected in the presence of uncontrolled variables. Under these conditions, it is understandable that most of the published data is statistical in character. As Flitcroft has noted, “Several studies of the colour coding in cells of the primary visual cortex in primates have described cells that have double opponent receptive fields (with references)25.” This condition is not limited to the primates and does not originate in the cortex. The earliest cells of the signal processing stage exhibit both a chromatic opponent and a spatial opponent characteristic that are not normally separated into independent variables within the experimental design. Beyond this dual character is the additional factor of timing, both with regard to the time dispersal of signals due to their point of origination in the retina and to the impact of tremor on the conversion of spatial information into temporal signals. In many animals, there is also the parameter of polarization of the incident radiation to contend with. To understand the operation of the signaling system beginning at the retina, it is mandatory that these variables be recognized as independent and treated accordingly during experiments. Flitcroft’s analyses all assume a linear visual system. This assumption essentially limits his analyses to small signal conditions. 3.1.3.1 Lack of a detailed model

DeVries & Baylor typified the experimental design problem when they opened their paper26. They began “Rod signals in the mammalian retina are thought to. . . .” and then continued with: “A possible alternative pathway involves. . . .” [underlines added] Their position is not unique. Without a comprehensive view of a large volume of the literature, it is difficult to define the signaling architecture of the retina. After such a study, it is clear that very meticulous experimental design and parameter control is required. The model of this work can aid in this respect. DeVries & Baylor conclude with “evidence that an alternative pathway transmits signals to ganglion cells in parallel with the classical RDB (rod-depolarizing bipolar) pathway.” That finding is compatible with the model of this work. However, it appears that a detailed understanding of the circuitry of the retina may still be beyond the state-of-the are. This is primarily because of our lack of a complete understanding of the fundamental signaling architecture of the visual system 3.1.3.2 Lack of consistent control of the motion of the eyes

The experimental literature provides a variety of experimental conditions relative to control of the eyes under test. In general, the procedures do not address the subject of tremor directly. In some experiments, the eye motions are halted by drugs. However, there was no discussion of what motions were halted and what motions, if any, remain. Needless to say, tremor plays a large part in understanding the operation of the retina with respect to edges between various center and surround stimuli. 3.1.3.3 Lack of precise control of stimuli

A large amount of the experimental work has involved the use of wide spectral band stimuli that have not been correlated with the absorption characteristics of the chromophores of vision. Under these conditions, the signals recorded from the neural layers of the retina are hopelessly compromised before data reduction is begun. It has become possible to obtain more discreet data in recent years with the use of tricolor cathode ray displays. However, many investigators have defined colors for their experiments that do not limit the color of the display to that of only one phosphor at a time. Most experiments do not describe the edges of the stimulus fields. Without knowing the sharpness of the edges involved, it is difficult to completely interpret the electrophysiological signals generated.

25Flitcroft, D. (1985) The interactions between chromatic aberration, defocus and stimulus chromaticity. Vision Res. vol. 29, no. 3, pp. 349-360 26DeVries, S. & Baylor, D. (1995) An alternative pathway for signal flow from rod photoreceptors to ganglion cells in mammalian retina. Proc. Natl. Acad. Sci. USA, vol. 92, pp. 10658-10662 The Retina 3- 11 3.1.3.4 Importance of controlling the stimuli orientation

It has only recently been found that the direction of the light falling on photoreceptor cells is a critically important parameter in signal generation. These cells exhibit two separate absorption spectra. The isotropic spectrum is due to the absorption of the chromophores by an intrinsic non-functional mechanism. It always exhibits a peak absorption near 500nm regardless of the type of chromophore involved. A an-isotropic spectrum is realized when light is applied to the cells along the long axis of the Outer Segments. This is the functional spectrum that differs in peak absorption depending on the chromophore present. This is the explanation why observations made through the pupil of the eye give different characteristics for the individual photoreceptors than do experiments involving transverse illumination of individual cells. The absorption characteristics of the photoreceptor cells are also affected by the F/# of the illumination applied to them. The spectral differentiation of cells in the retinal mosaic becomes poorer if the pupil is dilated and is almost entirely lost if the ocular is dissected to allow flood illumination to be applied to the retina. 3.1.4 Matters specific to the organizational structure of the chordate retina 3.1.4.1 Matters of scale

The language used in the literature to describe the retina is quite broad ranging. This reflects the broad range of backgrounds of the investigators. When one attempts to provide a review of the overall field, the problem of terminology is a difficult one. The comprehensive review by Wassle & Boycott27, although already dated, is both an excellent source and an excellent example of this problem. It summarizes the three sets of names used to define “two” classes of ganglion cells of the macaque retina (pg. 449). It includes over 400 references. However, many of the figures are caricatures. To provide a comprehensive discussion of the retina requires both a comprehensive and consistent vocabulary of independent discrete terms.

A first order example of the problem involves the terms, anatomy, morphology, histology and cytology. These terms relate to the structure and shape of bodily parts. However, they apply at different levels of scale. The problem is complicated by the fact that animals vary so widely in scale. In this work, anatomy will relate to both morphology and physiology at the visual system level. Histology will apply to the morphology and physiology at the light microscope level. Cytology will encompass morphology and physiology at the electron microscope level. Cytology generally relies on the electron microscope for both adequate definition and magnification. Topography will apply to the external features of an element at any of the above levels.

Many investigators have tried to select especially large samples of a given cell type from among a group of animals of a given scale. This procedure makes many laboratory procedures easier. However, one should keep in mind that a “larger” than average sample is necessarily an atypical sample. The investigator should be aware of the consequences of this fact and make note of it in his dissertation.

Except for the two-dimensional extent of the complete retina, it is best described in terms of histology. To study the individual cells of the retina, the coarser surface features, i.e., topography, can be described at the histological level. The finer surface features, and questions about the actual formation, structure and function of a cell require the electron microscope and thus fall within the purview of cytology. 3.1.4.2 Fovea versus other terms

It serves no purpose to have different names for the area of most acute angular vision in the phylum Chordata. This has arisen where investigators have chosen arbitrarily to adopt a new element name rather than consult the literature. The most general definition of the word fovea does not specify its detailed shape, only that it involves a small depression. Prince has used the word fovea to describe a variety of features related to high acuity vision, using additional adjectives where needed. This is most appropriate. Reference to a “central area” in cats should be replaced by fovea or fovea centralis. References to a “visual streak” in rabbits should similarly be replaced by fovea or elongated fovea. These terms would then be consistent with double fovea and other specific descriptors. The fovea is seen to be a feature: + with a shape that is highly variable and species specific, possibly even family specific. + with a location that can be defined in terms of the optical axes of the eye. This location is also species specific.

27Wassle, H. & Boycott, B. (1991) Functional Architecture of the Mammalian Retina. Physiol. Rev. vol. 71 no. 2 pp. 447-480 12 Processes in Biological Vision

With the term fovea applied to all members of the chordate phylum, the terms parafovea, perifovea, foveola, etc. take on their normal meanings independent of species or family. In this work, the inner zone of the fovea, known as the foveola, takes on a more important role than previously. The photoreceptors of this zone enjoy a unique signal path to the cortex. 3.1.4.3 Amercine cells

The designation of a lateral cell in the neural layer of the retina by the Greek descriptor, amercine (without an axon), based on its apparent topography was unfortunate. It left the audience with the impression that the cell had no functional axon. The fact that the axon and at least part of the dendritic structures shared a common external cell wall was not recognized for a very long time. Recently, synapses believed to be associated with an axon of an amercine cell have been discussed. This work will present the cytology and the functional characteristics of the amercine cell that should clarify this descriptor generated difficulty. There has been a tendency to describe a layer of the retina in terms of the label amercine. This is very unfortunate. It is important to note that many types of neurons and many types of “amercine” cells have now been identified in this putative layer. Many have fully arborized neuritic structures and identifiable axons. 3.1.4.4 Matters of architecture

The retina exhibits two separate and distinct architectural profiles that can be related to its plan and profile views. Each of these should be addressed individually in order to understand how the resultant neural signals are created for transmission to the brain. As noted in Chapter 2, one of the features of this architecture is the introduction of time- dispersal into the signaling algorithm. The second is the introduction of tremor to allow the fundamental change detection architecture of the photoreceptor cells to be used in an imaging mode. These elements of system design can have a large impact on center-surround studies using large diameter stimuli. It also suggests that most signal recording should be done in the S-plan of the retina before the data is applied to the threshold circuits of the ganglion cells. Finally, much of the information concerning the stimuli is carried in the time domain and good temporal discrimination must be designed into the test set.

In recent times, several investigators have extended the definition of the parvocellular and magnocellular pathways between the LGN and area 17 of the cortex to include the paths from the retina to the LGN. Unfortunately, this nomenclature obscures the fact that the parvocellular paths include more than one class of signals. These P-channel and Q-channel signals are not only different, they are fundamentally orthogonal to each other. By definition, they are not antagonistic to each other. In fact, in one quadrant of color space, they parallel each other. Good temporal and chromatic control of the stimuli, along with adequate temporal discrimination in the test set will demonstrate these facts.

3.1.5 Matters of photoreceptor cell classification

One can track the problems of classification as a function of time in the literature for many features in vision. As the state of the art moved forward, definitions changed and were refined. This problem has been particularly chronic regarding the photoreceptors of the eye. It occurs to a lesser extent in the recent discussions regarding the amercine cells of the retina. Here, the surface features (topography) were used to describe both the morphology (shape and structure) and the functional elements of the cell. The results have been less than adequate. Many investigators have extolled the value of morphology in explaining the physiology of parts of the eye, i.e., shape explaining function. At the present level of understanding, this method of inference has become a trap. Without a better theoretical base, using morphological features to infer physiological function at the level of cytology is very difficult. This is particularly true where the signaling function in vision has not been defined precisely. 3.1.5.1 Background--Rods and cones

Because the literature of the retina contains so many references to them, having a definition of what is a rod and what is a cone would be very useful later. This is especially true because so few precise definitions of a rod or cone appear in the literature. Similarly, there are few places that illustrate the range of photoreceptor cells (and possibly non-photoreceptor cells) in the retina. The following discussion is needlessly long but is presented in the interest of completeness. It will be shown later that there is no functional (signaling) distinction between the morphological designations “rods” and “cones.” In fact there is no morphological distinction between the The Retina 3- 13

inadequately defined concept of rods and cones. More specifically, the designation “rod” is a euphemism for a putative photoreceptor exhibiting the spectral sensitivity associated with scotopic vision. This spectral sensitivity is actually the logarithmic summation of the S–channel and M–channel photoreceptors. Only a few serious attempts to provide descriptions of rods and cones could be found by the author. Some of the better attempts include Walls28, Detwiler29, Young30, Rodieck31, Crescitelli32, and Adler33. Many of these authors try to merge the morphology of the cells (or various parts of the cells) with the habitation of the animal to support a classification duality. Summarizing their discussion is impossible because there is no common thread. The penchant of man to employ a dichotomy is the only idea that appears throughout the discussion. Ebrey & Koutalos made an extensive effort to identify rods and cones in 2001 without success34. They reviewed the early history of the concept and the evolution of the “Duplex Theory” of human vision. They noted, “There are at least four points of view one can take in order to classify or group the vertebrate photoreceptors: (i) their visual pig- ments; (ii) the enzymes and other proteins associated with the phototransduction apparatus; (iii) their anatomy, structure, and topology, at both the light and electron microscopic level; and (iv) their electrophysiology. In addition, one could add to this list their psychophysics, which, unlike the first four must take into account not only the photoreceptors themselves, but also processing at the post-receptoral and central levels of the nervous system.” Their analyses concentrated on the earlier writings on chemical and genetic processes. They did not resolve which of the above categories correctly described the putative differences between rods and cones. The emergence of the ability to resolve individual photoreceptors in the topography of the retina using OCT technology, specifically in the form of AOSLO, the demise of the concept of a rod is now confirmed. As noted in Section3.2.3.2, via a private communications, no rods were identified beginning in 1999. With the recent advances in tremor compensation AOSLO, the absence of any rods in the retina of the human eye is now confirmed. The intellectual concept of an achromatic and a chromatic set of photoreceptors, the rods and cones, is now totally archaic.

The ancient and totally archaic Duplex Theory of vision, with rods responding to low light levels and cones responding to high light levels, has been completely falsified and should be purged from all textbooks in a timely manner. The Duplex Theory constitutes a blight on the teaching of biological science.

Walkey et al. have recently discussed the concept of “rod intrusion” in spectral measurements at low light levels35. Their results were generally inconclusive, except to agree with one proposition of this work. “The results of this study suggest that chromatic and luminance contrast signals are also processed separately in the mesopic range.” 3.1.5.1.1 The clinical attempt of Shultze to identify rods in 1860-70

Walls provided a good summary in 1942 starting from the work of Schultze in 1866. Schultze had worked in comparative ocular histology for more than 15 years and propagated a conclusion based on this work. He noted that nocturnal vertebrates had a preponderance of “rods” over “cones” (or no cones at all); and that diurnal species had many “cones,” and might even lack “rods” entirely. He thus suggested the “rod” is the organ of scotopic (dim-light) vision and the “cone” is the receptor for photopic (bright-light) vision. Schultze then added a corollary that the “cone” alone is responsible for color vision; for in dim light colors are no longer discriminable and the world

28Walls, G. (1942) The Vertebrate Eye. Bloomfield Hills, MI: Cranbrook Institute of Science 29Detwiler, S. (1943) Vertebrate photoreceptors. NY: Macmillan pg. 45 30Young, R. (1971) Hypothesis to account for a basic distinction between rods and cones. Vision Res. vol. 11, pp 1-5 31Rodieck, R. (1973) The vertebrate retina. San Francisco, CA: W. H. Freeman pp. 9-10 32Crescitelli, F. (1972) A paradigm: the receptors and visual pigments of an Anuran. In Photochemistry of Vision. Vol. VII/1 Dartnall, H. ed. NY: Springer-Verlag pg. 258 33Hart, W. (1992) Adler’s Physiology of the eye. St. Louis, MO: Mosby Year Book. pg. 586 34Ebrey, T. & Koutalos, Y. (2001) Vertebrate Photoreceptors Prog Ret Eye Res vol 20(1), pp 49-94 35Walkey, H. Barbur, J. Harlow, J. & Makous, W. (2001) Measurements of Chromatic Sensitivity in the Mesopic Range Color Res Appl Suppl vol 26, pp S36-S42 14 Processes in Biological Vision

presents itself only in shades of gray. Rodieck provided a different summary. According to Rodieck, Schultze originally described the retina as containing two types of photoreceptors in 1866. His definition was a static one. It described rods in the peripheral retina as exhibiting a cylindrical Outer Segment and a thin Inner Segment. Cones were described as having a tapered Outer Segment and a thicker Inner Segment. This view has been challenged often. Schultze also originated the relationship between the morphology of photoreceptors and their sensitivity. He found the retinas of nocturnal animals had retinas virtually free of cones. He therefore proposed, based on no other evidence or data, that the rods subserve vision in dim light and the cones subserve vision in bright light. It is not clear whether this definition should be restricted to the peripheral cells but it is the foundation of the Duplicity Theory of animal vision. Schultze was wrong! He neither knew, or claimed to know, the dynamic range of the cells he was discussing. The dynamic range of individual photoreceptors, in collaboration with the iris, is approximately 50,000:1. This range negates the need for two separate sensory channels based on illumination level. 3.1.5.1.2 Attempts at redefining the retina during the 1940-70s

As technology improved, particularly in light and electron microscopy, a whole series of new attempts at defining putative differences between rods and cones were made. Most of these relied upon morphology but additional attempts were made to tie the morphological features to the observed psycho-physical features. Little attempt was made to tie any morphological features to physiological features.

Walls went on to express his own views: “The visual cells of vertebrates . . . were long ago given the names ‘rod” and ‘cone’--though with our superior modern knowledge of their phylogenetic ramifications and physiological characteristics we might wish that a more apt pair of names could be substituted for the traditional ones. In a given retina containing both highly sensitive visual cells (rods) and relatively insensitive ones (cones), the high- and low- threshold cells can always be told apart; but the rod on one retina may resemble structurally the cone of another, or may give evidence of having been recently derived from a cone-type in an ancestor of different habits.” Walls gave no explicit statement about how they were told apart but he did develop several themes: “One of the most noteworthy peculiarities which cones may have is that presented by the cones of the greater portion of the human retina, and also by some other placental mammals: the cone outer segment is a cylinder enclosed by a tubular process of the pigment epithelial cell opposite to it . . . No such arrangement is ever seen in rods. . . .” Unfortunately, Fine & Yanoff36 seem to take a contrary view when speaking about foveal cones: “Their relation to the pigment epithelium here resembles that of rods elsewhere (i.e., they reach to the surface of the pigment cell and are enveloped by extremely delicate apical villi of the pigment epithelial cells).”

36Fine, B. & Yanoff, M. (1972) Ocular Histology, A Text and Atlas. New York: Harper & Row The Retina 3- 15

Fine & Yanoff expanded on their above words in an attempted to redefine the differences between rods and cones in man: “The foveal cones resemble the rods superficially. The foveal cones possess all the cytologic characteristics of cones elsewhere except for their shape. Their outer segments are cylindrical and elongated like rods but possess lamella that are typical of cones. The lamella [as used in discussing photoreceptors] are of the ‘tight’ or closely apposed type, with direct connections to the surface plasma membrane. Longitudinal furrows are absent. Their synaptic expansions are all typical broad cone ‘feet’ containing the characteristic multiple synaptic lamella [as used in neurology]. The last two references only agree on two aspects. First, the outer segments of both cones and rods are cylindrical. Wald (1965) stressed this fact when he said “It has long been recognized that the outer segments of foveal cones are rod-shaped; they are attenuated cylinders about 50 microns long and about 1-2 microns wide.” Dowling wrote with consternation in 1965 that cones were clearly rod shaped in the fovea37. He provided a side by side comparison of a “rod” and a “cone” from the same retina and provided many relevant dimensions. No structural difference can be seen in his pictures and there is no indication of where along the Outer Segment the pictures were taken. Dimensional differences were too small to be quantified at 162,000x. Second, the feet of rods and cones seem different. However, they are careful to suggest there is some overlap in this area, some cones are occasionally seen to synapse with a “summing” type of bipolar cell. At the same time, Walraven and many others in the psychophysical community were dismissing the concept of a rod altogether based on their experiments38. They generally defend block diagrams containing only three types of spectrally sensitive photoreceptors and show the luminance response being calculated from the individual spectral responses. No achromatic, broadband photoreceptor is required.

Note that two other large texts in histology fail to define and/or differentiate the structural features of rods and cones. Neither Hogan et. al.39 in 1971 or Rodieck40 in 1973 are explicit in this area. In 1975, Ohman was unable to define this difference in respect to the river lamprey and finally concludes with: “Ultrastructurally both long and short receptors show characters that favour the impression of rods41.”

Stell and Harosi42 performed a comprehensive study of the goldfish. Following extensive morphological and electrophysiological examinations, they described the structural features of both putative rods and cones but then were unable to find a single photoreceptor exhibiting a broadband spectral response. Instead, their micro spectrophotometer measurements found all of the photoreceptors fell into one of three spectral bands with peaks near 455, 530 and 625 nm.

In addition, Walls said: “The stalk-like portion of the inner segment [of cones] is highly contractile and hence is called the myoid (=muscle-like).” He then went on to illustrate six rods and labeled the myoid structure in three of the six. More specifically, he described the rod labeled d as the “common or ‘red’ (rhodopsin-containing) rod of the leopard frog; dark-adapted (i.e., with myoid contracted).” Next to it was shown the rod labeled e, “‘green’ (Schwalbe’s) rod [of the leopard frog].” This contractile function may be important to note; it could account for the various body shapes of what are frequently reported to be cones. Is it possible, their body shape changes significantly in aspect ratio over a short time like other muscle tissue? Could their shape relate to how tense they are in holding the outer segments in alignment or position during or after phagocytosis?

Bernard (1900) and Cameron (1905) took a dynamic view of the photoreceptors. They believed they saw significant changes in the cells with time. They proposed that cones were developmental stages in the formation of mature visual cells and that there is no such thing as duplicity as proposed by Schultze. This position was reviewed and expanded by Muntz in 1964. Based on more recent experiments measuring the rate of movement of individual disks

37Dowling, J. (1965) Foveal receptors of the monkey retina: fine structure. Science. vol 147, pp. 57-59 38Walraven, P. (1962) On the mechanisms of colour vision. Soesterberg, Netherlands: Institute for Perception. 39Hogan, M. Alvarado, J & Weddell, J. (1971) Histology of the Human Eye, An Atlas and Text. Philadelphia: W. B. Saunders 40Rodieck, R. (1973) The Vertebrate Retina, Principles of Structure and Function. San Francisco: W. H. Freeman 41Ohman, P. (1975) Fine structure of photoreceptors and associated neurons in the retina of lampetra fluviatilis (cyclostomi). Vision Res. vol. 16, pp 659-662 42Stell, W. & Harosi, F. (1976) Cone structure and visual pigment content in the retina of the goldfish. Vision Res. vol. 16, pp. 647-657 16 Processes in Biological Vision

toward the RPE of about 7.2 microns/day, it is clear that an Outer Segment of conical shape is unsustainable in a living retina. It is noteworthy that after more than 100 years, Young was moved in 1971 to offer a new hypothesis to separate rods from cones43. Young addressed the morphological difference between rods and cones from a morphogenic perspective only44. The use of the term “a” in his title is provocative. Using nuclear chemistry, he showed evidence that the cells he described as cones did not continually generate new disks as did the cells he described as rods. Further, the cones did not participate in the phagocytosis process since they did not reach the area of the RPE cells. He also addressed the fact that immature rods exhibited a conical outer segment until they grew to the point where phagocytosis removed the smaller diameter disks. Young did not address any physiological issues such as the spectral response of the cells he enumerated. His recommendation to retain the designation rods and cones and define cones as possessing a conical outer segment because of the large literature can only be relevant for the morphological community. He did not review whether these “cones” were electrophysiologically functional. His last paragraph suggests they might not be.

Weale45 gave the status of morphological cones a different twist while referencing Young, “Cones being conical, presumably because of their arrested development in most species, the mechanical problem is different from that believed to exist in rods.” It appears that morphological cones are not a temporally stable physiological component of a healthy visual system, at least in humans.

Ohman also addressed the question of phagocytosis in a species at the bottom of the vertebrate family. He found the same dynamic situation. “It is known that rod, but not cone, discs are continually renewed at the base of the outer segment, transferred apically and phagocyted by the epithelial cells. . . .”

Additional support for the hypothesis that “cones” are immature or non-functional comes from the observation that only “rods” secrete IRBP, a necessary protein for the transport of chromophore molecules through the IPM. See Section 7.1.2.3.5.

Crescitelli46 was critical of the proposition by Muntz because it was based on indirect evidence; Muntz used an ERG of the b-wave to support his position. Crescitelli said the b-wave was not indicative of the performance of the photoreceptor cells since it originated proximal to those cells. He did not comment on the quality of the evidence presented in 1866 by Schultze.

Many caricatures appear in the literature attempting to illustrate the critical features of rod and cone outer segments. Variations in this ratio may occur due to growth, death or other factors related to the myoid and not yet recognized. The caricatures frequently focus on the apparent differences in lamella structure and their close association with an outer membrane of the outer segment if present. Most drawings show the content of the outer segments as consisting of many disks stacked like coins. Some show the material inside the outer segment being laid in as a blanket would be folded, particularly near the connection to the inner segment where the lamella are presumably formed. As we will see later when discussing the lamella, this difference appears to be a trivial one concerning the visual function. In addition, many drawings show the lamella enclosed by an outer jacket. This jacket takes on many forms, including complete absence. Clearly, the lamella are formed at the point of junction with the inner segment and in this region the inner segment appears to provide a tubular shell from which the lamella are extruded. It is also clear that at the outer end of the outer segment, the lamella are generally enshrouded by the pigment epithelium. What is difficult to determine from the literature is whether there is an outer membrane associated functionally with the outer segment or whether various photomicrographs are showing one of these inner or outer sheaths associated with the adjoining tissue. If there is a substantial outer sheath which includes an end cap, then it must be explained how the lamella are able to get out of the sheath prior to phagocytosis. Alternately, it must be explained how an internal part of a cell is phagocytized without destroying the cell. A different definition delineating a rod versus a cone may be (at least in man) related to the neural connection.

43Young, R. (1971) An hypothesis to account for a basic distinction between rods and cones. Vision Res. vol. 11, pp 1-5 44Young, R. (1971) An hypothesis to account for a basic definition between rods and cones. Vision Res. vol. 11, pp 1-5 45Weale, R. (1974) The Eye. Vol. 6 Davson & Graham ed. NY: Academic Press pg. 76 46Crescitelli, F. (1972) in Photochemistry of vision, vol. VII/1 NY: Springer-Verlag pg. 258 The Retina 3- 17

Unfortunately this distinction only applies to two-dimensional pictures of three-dimensional structures. Based on two dimensional images, investigators frequently claim a cone foot is broad( in at least one plane) and makes a synapse-like junction with one or more dendrites of a single bipolar cell (frequently described as of the midget type). A rod foot is smaller and rounded and makes a junction with the dendrite of a “summing” bipolar (frequently described as of the centrifugal type or the diffuse type). Even this definition may only apply strictly to part of the retina. The more significant problems with this definition are two. The two-dimensional photograph is less than adequate and the definition of the three different bipolar types involved is ambiguous.

An attempt to delineate rods from cones by Nilsson47 takes a different approach again. Based on electron- microscopy, he says the difference between them is related to the proximity of the disks in the Outer Segments. He associated the cones with closely packed double membranes, and rods with double membranes separated by 5-10 nm. Unfortunately this definition is an incomplete one since the distance between disks is not constant over their surface. It is also unclear whether the electron microscope presented the external physical contour of each disk or whether it presented a contour related to an area that was opaque to electrons. We will not discuss here the further complications in Walls and others arising from double cones, twin cones, ophidian double cones, or double rods. It must be pointed out that Wassle and Boycott have not served their audience well by propagating the old idea of Schultze (1866) that the fovea is blind at night48. This position goes against reality (See “Some Reality Checks” in Chapter 1). In summary, a very wide continuum of photoreceptor cell types is found in the animal kingdom ranging from the short and fat to the long and thin and including the double types not reported in man. Even the short types may not be subject to unique classification due to their ability to change their aspect ratio through the contractile abilities of the myoid. All known mature photoreceptors exhibit a common outer segment that is cylindrical in basic form. The predominant current method of differentiating between “rods” and “cones” is by the synaptic connection they make with the neural network of bipolar cells. Even this may not form a definitive situation, especially outside the foveal region. The type of synaptic connection may instead be more indicative of the signal processing function exterior to the photoreceptors, especially if the signal processing is self organizing during early post natal development. 3.1.5.1.3 Attempts at redefinition during the 1980-90s

Attempts have continued during recent times to extend the classification of rods and cones in order to classify the spectral performance of cones based on their morphological characteristics49. These attempts exhibit a surreal quality similar to that discussed earlier. Although there may be some morphological differences related to different spectral types of photoreceptors due their location in an overall trichromatic (tetrachromatic) retinal array, these features are probably secondary and less than definitive. The position of Ahnelt, Kolb & Pflug in 1987 appears to still stand50: “The cones differ in having different photopigments and different neural connectivity, but no morphological differences with which to distinguish the three different spectral types have been reported.” That is, cones can be defined in terms of electrophysiological features, but not by their morphological features.

As recently as 1994, Loew found difficulty in delineating rods and cones in the Tokay Gecko. He noted the difficulty of defining rods and cones at the light microscope level so as to agree with their definition at the ultrastructure level51. In 1996, Packer et al. repeated the protocol of Schultze using modern techniques by excising a piece of primate retina, flattening it and photographing patches of it using transmission microscopy using an incandescent lamp and

47Nilsson, S. (1965) The ultrastructure of the receptor outer segments in the retina of the leopard frog (Rana pipiens) 48Wassel, H. & Boycott, B. (1991) Functional architecture of the mammalian retina. Psychological Reviews, vol. 71, no. 2, pp 447-480 49Kolb, H. (1991) Anatomical pathways for color vision in the human retina. Visual Neurosci. vol. 7, pp. 61-74 50Ahnelt, P. Kolb, H. & Pflug, R. (1987) Identification of a subtype of cone photoreceptor, likely to be blue sensitive, in the human retina. J. Comp. Neurology. vol. 255, pp. 18-34 51Loew, E. (1994) A third, ultraviolet-sensitive, visual pigment in the Tokay Gecko (Gekko gekko). Vision Res. vol. 34, no. 11, pp 1427-1431 18 Processes in Biological Vision

three chromatic filters52. An objective lens collected to emerging light and projected it onto a CCD imager. The filters were centered at 440, 510 and 590 nm. Both human and macaque retina pieces were explored. There are serious questions about what they actually recorded and their discussion develops a variety of problems they were unable to resolve. While noting the photoreceptors were not parallel to the radiation from their light source when the retinal tissue was perpendicular to the incident light, they tilted their custom microscope stage to maximize the image contrast, “Some of the light that leaks out of the photoreceptor inner segments without traversing the photopigment will eventually be caught by the CCD, reducing the signal-to-noise ratio of the spectral measurements and making the distinction of small spectral sensitivity differences among the S, M, and L cones more difficult. To minimize this contrast reduction, bright spots corresponding to light emerging from cone outer segments were brought into focus using IR illumination to prevent photopigment bleaching. The cone optical axes were then aligned with the optical axis of the microscope by tilting the stage until the amount of light being transmitted through the photoreceptors was maximized. Figure 2 shows that when the tips of the outer segments.” “Some of the light that leaks out of the photoreceptor inner segments without traversing the photopigment will eventually be caught by the CCD, reducing the signal-to-noise ratio of the spectral measurements and making the distinction of small spectral sensitivity differences among the S, M, and L cones more difficult. To minimize this contrast reduction, bright spots corresponding to light emerging from cone outer segments were brought into focus using IR illumination to prevent photopigment bleaching. The cone optical axes were then aligned with the optical axis of the microscope by tilting the stage until the amount of light being transmitted through the photoreceptors was maximized. Figure 2 shows that when the tips of the outer segments.”

They did not describe the wavelength of their infra-red source, but it appears to be short wavelength IR in order to be compatible with simple IR image converters. Such a source is effective in bleaching visual receptors, particularly of the M– and L– types. Their calculations of the number of photons applied to their photoreceptors indicates they were also bleached considerably by their exposure levels, since the light adaptation function is very fast acting. The quantum count applied to each photoreceptor and described in the caption to figure 4 (6 or 7 log photons) is very large for a photoreceptor accepted generally to be a quantum counter in the unbleached condition.

Their images, which resemble those of Schultze, show the purported rod outer segments to have a diameter of less than 1.5 microns at their extreme tip while the purported cone outer segments have diameters on the order of 3 microns. As noted in Sections 3.6.2.3.3 & 4.3.4.2.1, a cylindrical waveguide of less than 1.5 microns is a very inefficient projector of light at visual wavelengths. The 1.5 micron diameter bright spots are more likely glia or some other type of biological cells. Packer et al. illustrates the problem in their figure 6.

A subsequent statement requires very careful interpretation,

“Figure 2 shows that when the tips of the outer segments are in sharp focus and the axes of the photoreceptors are aligned, individual photoreceptors glow brightly. In fact, the intensity of the light emerging from the outer-segment tip was always greater than the intensity of the illuminating beam, showing that both rods and cones have optical gains greater than 1 and as high as 3. Thus, it is possible in this preparation to funnel incident light efficiently through the photoreceptors in relatively large patches of peripheral retina.” As noted in Section 4.3.4.2.1, the disk stack forming the outer segment of the individual photoreceptor is a very efficient absorber at its absorption wavelength when unbleached. As a general principle, virtually no light should be reaching the peripheral tip of the outer segment when the photoreceptors are unbleached. In analogy with the current silicon photosensors in cellphones, ipads, etc., the absorption coefficient of the individual biological photoreceptors is greater than 90%. In the absence of bleaching, virtually no light should be emerging from the peripheral tip of the photoreceptors. Their data suggests the photoreceptors were already significantly bleached during their measurements. In addition, if their bright spots in figure 2 (right) are actually rods at their peripheral tip, their size at the input aperture must be large enough to account for the noted optical gains. If approaching 3:1, the entrance apertures would be nominally overlapping. They concluded the discussion related to figure 4 with the statement,

52Packer, O. Williams, D. & Bensinger, D. (1996) Photopigment Transmittance Imaging of the Primate Photoreceptor Mosaic J Neurosci vol 16(7), pp 2251-2260 The Retina 3- 19

Unfortunately, despite some successes, most patches of retina yielded absorptances that did not exceed 0.05, as was the case for axial MSP. We found many locations, as shown in Figure 5, in which a bright cone in a raw image of the retina did not correspond to a high absorptance in the transmittance image. Likewise, areas of high absorptance did not always correspond to bright cones in the raw image. Apparently, the outer segments of these cones were bent, as shown in Figure 6, allowing most of the light captured by the inner segment to leak out before traversing the photopigment. At 5% absorptance, the photoreceptors are hardly more sensitive than photographic film, in clear contradiction to their known performance compared to a silicon CCD. Interestingly, figure 7 of Packard et al.shows photoreceptors that they label with question marks and notes, “The question marks are just to the left of two cones with a yellowish hue similar to that expected of S cones.” These correspond to the UV photoreceptors discussed in this work, documented within the RPE elsewhere in this work, and predicted to occur in the retina.

Those working with Arthropoda have never sought or found a variation in elements of the ommatidia (such as rods and cones) based on spectral absorption or presumptive sensitivity range. Even where such a distinction is necessary, in the case of polarization sensitivity, the distinction is limited to relative orientation between the outer segments of such cells. Recent authors have also found the arrangement of spectrally distinct cells to be random within the overall retinal plane of Arthropoda53. It is striking that all recent photomicrographs of in-vivo human retinas show a uniform array of photoreceptors with no sign of any morphologically identifiable subtypes (see Section 3.2.2.1).

In 2000, Livrea observed, “There has been a major difficulty in studying the cones due to lack of material.” She then discusses the considerable theoretical activity going on under the assumption that such functional elements actually exist54. 3.1.5.1.4 Attempts at locating rods during 2000-11 & culminating in 2016

The recent work of the Williams team in imaging the in-vivo retina through the pupil using active optics has placed new emphasis on the spatial characteristics of the retina.. Using false color photographic techniques (L–chromophores reflect complementary pale bluish light), they have identified the various types of photoreceptors (see Section 3.2.3). In 2006, Wolfing et al. (including Williams) used the most sophisticated retinal imaging techniques available to study the retina of a subject with “cone-rod dystrophy55.” No rods were identified in that retina. After thousands of images using this higher resolution approach in several laboratories, the Williams team made the following comments in 200856. “To our knowledge, there is only one report of in vivo images of rods from the normal human retina.” This report was made by the same team in 200457. The report was only an abstract of a convention talk. It did not include any specifics or graphics. Their subsequent comment in 2008 is less than convincing. “The difficulty in imaging them, and the relative sparseness at retinal locations where they should outnumber the cones nearly 10-fold, is consistent with previous data that rods are less effective waveguides than cones.” They did not suggest that there were large voids among the cones that were occupied by unimaged rods. Finally, they note, “This underscores the importance of imaging the retinae of individual subjects rather than making general assumptions about the achromat retina.”

53Arikawa, K. et. al. (1999) An ultraviolet absorbing pigment causes a narrow-band violet receptor. . . . Vision Res. vol. 39, pp 1-8 54Livrea, M. (2000) Vitamin A and Retinoids. Berlin: Birkhauser Verlag pg 64 55Wolfing, J. Chung, M. Carroll, J. Roorda, A. & Williams, D. (2006) High-resolution retinal imaging of cone- rod dystrophy Ophthalmology vol 113, pp 1014-1019 56Carroll, J. Choi, S. & Williams, D. (2008) In vivo imaging of the photoreceptor mosaic of a rod monochromat Vis Res Epub doi:10.1016/jvisres 2008.04.006 57Choi, S. Dobbel, N. Christou, J. et al. (2004) In vivo imaging of the human rod photoreceptor mosaic Inv Opth Vis Sci vol 45, E-Abstract 2794 20 Processes in Biological Vision

In 2011, the Optical Society of America supported a special journal issue on active optics technology58. It included two articles by the Williams team59,60 along with many other papers, some of which are discussed in greater detail in Chapter 18 of this work. The Williams team asserted they identified “rods” for the first time in-vivo in humans based entirely on the location of their imagery at various eccentricities outside of the foveola. No spectral confirmation of their assertions were provided. The first paper describes the configuration and benefits of the University of Rochester scanning AO ophthalmoscope in considerable detail. The Dubra et al. paper requires very close study. It contains a large amount of empirical data but does not include graphical or detailed descriptions of their test configurations. Figure 2 is a particular problem because it is not emphasized that the left two frames show the light exiting the peripheral end of the photoreceptors after removal of the RPE from the test sample while the right panel shows the light reflected from the entrance aperture of the outer segments of the photoreceptors. Their discussion of the difference in the focal planes of the photoreceptors at the RPE interface is largely extraneous as this has no functional significance. Their discussion of the difference in the focal planes of the photoreceptors at the entrance apertures of the photoreceptors is superficial as it does not distinguish the focal planes of the four spectrally selective photoreceptor types. Nor does it describe the off-axis performance of the optics of the eye (particularly with respect to chromatic aberration of the principle ray). It appears the measurements reported by Packer et al. relating to the two left frames were made with a significantly bleached retina although Packer et al. did not explicitly recognize this fact and intimate the eyes were dark adapted. Their provision of imagery based on “linear” and “logarithmic” brightness data is laudatory as it highlights the major differences associated with these scales. Packer et al. did not obtain any spectral data supporting their designation of rods versus cones in a specific image frame. Their figure 7 does appear to show the location of UV photoreceptors. They designate these cells by question marks and describe their pale color (similar to that of S-cones) under their unspecified illumination (color temperature probably less than 5000K).

The 2016 paper by the Roorda team confirmed beyond a shadow of a doubt at the one-half micron resolution level that there are no broad spectral response achromatic receptors (rods) in the fovea of the in-vivo human retina (Section 3.2.3.6). 3.1.5.2 “Rods” and “cones” are not functional descriptors

Schultze was wrong in 1866! And the subsequent in-vitro histological work of Osterberg, Curcio et al have been superceded by the in-vivo imaging work of the Williams team and the Roorda team.

The concept of dividing photoreceptors into two distinct classes will not be used in this work except in this Chapter for four reasons.

1. It will be shown that the operation of the eye can be completely described without reference to cells exhibiting a broad, achromatic, spectral response (rods).

2. No photoreceptor has ever been displayed that exhibited such a broad spectral response in its operational mode as has been attributed to rods.

3. Recent work (post 1997) in gene therapy has provided new information suggesting morphological “cones” are not functional in vision. See Section 4.6.4.1.2, Bennett and Redmund references.

4. The method of adaptation used in vision negates the need to define two distinct classes of photoreceptors based on their range of stimulus sensitivity. 5. The work of the Roorda team using a state-of-the-art AOSLO during the 21st Century has demonstrated beyond the shadow of a doubt there are no achromatic photoreceptors (rods) in the human retina (Section 3.2.3.6).

58Carroll, J, Pircher, M. & Zawadzki, R. (2011) Introduction: Feature Issue on Cellular Imaging of the Retina Biomedical Optics Express vol 2(6) 59Dubra, A. & Sulai, Y. (2011) Reflective afocal broadband adaptive optics scanning ophthalmoscope Biomedical Optics Express vol 2(6), pp 1757-1768 60Dubra, A. Sulai, Y. Norris, J. et al. (2011) Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope Biomedical Optics Express vol 2(7), 1864-1876 The Retina 3- 21

The rod-cone dichotomy will be supported in this chapter only to present data from other investigators found in the literature. There is no functional distinction between photoreceptor cells that can be ascribed to their morphological shape. Only confusion would be served by editing the work of these other investigators to eliminate the descriptors, rods and cones. The ancient and totally archaic Duplex Theory of vision, with rods responding to low light levels and cones responding to high light levels, has been completely falsified and should be purged from all textbooks in a timely manner. The Duplex Theory constitutes a blight on the teaching of biological science. The spectral performance of a given photoreceptor is sometimes confused because of an unusual feature of its spectral absorption characteristic. It is anisotropic. When employed in an Outer Segment of the photoreceptor, the chromophores exhibit a very high absorption coefficient for light incident along the axis of the Outer Segment. This absorption characteristic is its functional characteristic. The same chromophore in the same configuration will exhibit an alternative absorption characteristic to light incident perpendicular to this axis. This weaker characteristic always exhibits a peak absorption near 500 nm. It is the same characteristic that will be obtained by measuring chromophoric material in a dilute solution in-vitro. This subject will be addressed in detail in Chapter 5. The concept of dividing photoreceptors into three (or four) spectral classes based on morphology will not be used in this work. It will be shown below that, to the first order, all photoreceptor cells are functionally identical. The only difference related to their spectral performance is associated with the chromophore coating the disks of the Outer Segment. This difference is at the molecular level. It consists of a difference in physical location of the oxygen auxochromes found along the conjugated carbon chain of the molecule. No other direct, first order, feature separates the cones into classes relative to their spectral performance. 3.1.5.3 On the subject of “red rods” and “green rods”

Another oddity that occurs occasionally in the literature are the terms red rods and green rods. Ma et. al. have recently provided an explanation of this situation61. They note that Hubbard translated a paper by Boll of 187762. In that paper Boll differentiated the physical appearance of two types of photoreceptors based on their apparent color using a light microscope. While the paper also used the terms rods and cones, in this case he was only speaking of a dichotomy between two intermixed photoreceptor types. He described the majority as red rods and the minority as green rods. In such a case, the observed color is the complement of the absorbed light. It is not clear what light source Boll was using. Depending on the light and optics and his state of adaptation, there are two cases. If he used a sufficiently high color temperature source (prior to the invention of the electric lamp), and his optics were sufficiently good, the functional difference between these cell types would be in their absorption of blue or purple light. Since there are not two distinct chromophores relating to the names blue and purple, it appears that both cells were absorbing the light associated with an S–channel chromophore with some secondary factor causing a shading that resulted in a net reddish or greenish tinge. In the absence of sufficient short wavelength illuminant, he would have observed the functional difference between the M– and L–channel absorbers. The M–channel absorber would look reddish (magenta) and the L–channel absorber would appear a pale greenish blue. In either case, this explanation is in complete agreement with the findings of Ma, et. al. In that paper, the spectrally determined S–channel cones and the morphologically determined “green rods” exhibit the same absorption spectra. They are in fact the same electrophysiological entity. This leaves open the question of the role of the red rods. Are they also in fact M–channel cones? If so, the designation “rod” can be stricken from the glossary of vision. The morphologically identified red rods, would absorb maximally near 532 nm as expected for the M–channel cone. If this is true, Ma, et. al. have documented the successful chemical isolation of all four chromophores of vision, those of the UV–, S–, M– and L–channel, using the salamander, Ambystoma tigrinum.. Chapter 5 and particularly Section 5.5.10.6 will discuss the fact that the actual chromophoric materials are the Rhodonines. In the introduction to Boll’s paper, Hubbard quoted Muller (1851), “an individual rod can look alternately colorless and colored, depending on whether it lies on its side or stands upright.” This observation recognizes the anisotropic properties of the photoreceptor outer segment. 3.2 Morphology of the chordate retina

61Ma, J. Znoiko, S. Othersen, K. et. al. (2001) A visual pigment expressed in both rod and cone photoreceptors Neuron vol. 32, pp 451-461 62Hubbard, R. (1977) On the anatomy and physiology of the retina Vision Res vol. 17, pp 1247-1265 22 Processes in Biological Vision

The review of Wassle and Boycott63 in 1991 is an invaluable source of references for studying the retina. The article itself is involved primarily in the morphology of the retina but it only includes two micrographs among myriads of hand drawn caricatures; many of these caricatures include the expression “putative” in the caption. The object of the review “is to survey current understanding of how the different neurons of the mammalian retina are arrayed and interconnected to form functional units.” Although the article attempts to relate morphology to function, it is based on morphology and does not review or include references to the signaling function. It does present archaic sketches purported to show signal flow at the most basic level. The most valuable picture of the cross-section of the retina remains that of Boycott and Dowling of 196964. It is reproduced in Figure 3.2.1-1 This is a picture from a phase-contrast light microscope. This image is taken approximately 1.25 mm. from the center of the fovea in an unspecified direction. The callouts along the left margin have been widely used in discussing the retina. However, a scale and additional nomenclature has been added on the right. The scale is shown starting from Bruch’s Membrane. Light approaches from the bottom in this figure and is brought to a focus at the Petzval Surface. It is located quite near the junction of the inner and outer segments. The inner nuclear layer is further subdivided into the Outer, Middle and Inner Matrix Layers to reflect the overall architecture developed in this work. The Outer Matrix Layer is populated by the morphologically designated horizontal cells of the first lateral processing matrix. The Middle Matrix Layer is populated by bipolar cells. The Inner Matrix Layer consists of the various cell types generally categorized as amercine cells and forming the second lateral processing matrix. Although these layers may not be clearly subdivided morphologically, they are functionally. The previously designated inner plexiform layer is subdivided into an Inner Fiber Layer (closest to the INL) and consisting of axons, and an Inner Plexiform Layer associated with the dendrites of the ganglion cells.

63Wassle, H. & Boycott, B. (1991) op. cit. 64Boycott, B. & Dowling, J. (1969) Organization of the primate retina: light microscopy. Philos. Trans. R. Soc. Lond. B Biol. Sci. Vol. 255, pp. 109-194 The Retina 3- 23

Figure 3.2.1-1 CR Cross section through a human retina, about 1.25 mm. from the center of the fovea. Vertical scale has been added to quantify the thickness of this retina. Labels on the right have been added to highlight other features and to subdivide the inner nuclear layer. A part of a blood vessel containing erythrocytes shows in the ganglion layer. Photographed by phase contrast microscopy. An arrow point to a “displaced” ganglion cell. Modified from Boycott & Dowling (1969)

The labels on the right are in general agreement with those of Kolb65. Wolken has also provided an alternate set of

65Kolb, H. (2001) Web Vision an internet resource, http://webvision.med.utah.edu 24 Processes in Biological Vision

labels to those of Boycott and Dowling66. His drawing provided specific callouts for some of the details not recognized by them but he did not separate the inner and outer segment layers. There appears to be an editorial inconsistency between his figure and some of his text related to layers seven and eight. He recognizes the presence of both the 1st and 2nd lateral neuron layers along with the bipolar layer within his inner nuclear layer. However, he labels the various interconnecting fiber layers as molecular layers. Finally, the choroid layer is described as consisting of black pigmented cells acting as an antireflection device. This is a species specific designation that may be more appropriately associated with Bruch’s membrane. As Walls did point out in 1942, the number of neurons in the 2nd lateral matrix of birds, the IML, may exceed the number of bipolar neurons found within the inner nuclear layer. This is not the case in humans where the number of neurons in the second lateral matrix is minimal. Brown, Watanabe & Murakami have provided several figures showing the variations in the relative thicknesses and curvatures of the layers for the retina of the cynomolgus monkey, Macaca irus67. The figures represent cross sections of retina along a line from the parafoveal ridge to the optic disc. Their figure 2 is particularly useful. It is reproduced as Figure 3.2.1-2 and describes the profile of the retina in the vicinity of the fovea. The figure stresses the extended length of the Outer Segments in this region. It also shows the curvature of the focal plane defined by the demarcation between the inner and outer segments of the photoreceptors. Finally, it illustrates the variation in thickness of the neural layers in this area. This variation constitutes the field lens discussed in Section 2.4.4. 3.2.1 Anatomical Level

The retina in Chordata is an approximately spherical lining to the posterior two thirds of the ocular globe. The human retina can be taken as typical of the chordates except for scale. Whereas its thickness is measured as a few hundred microns, its surface area can be measured in square centimeters. It can be thought of as a laminate as thick as three to five sheets of copy paper and containing a large amount of structure. The retinal laminate fills the space between the vitreous humor and the membrane of Bruch. In many species, the thickness of the laminate is not uniform and the relative positions of different layers within the laminate are not uniform. This variation makes it difficult to obtain a picture or micrograph of the details of a specific layer of the retina. The figures of Snodderly, et. al68. show how difficult it is to select a layer by embedding and then slicing a retina. Taking a Figure 3.2.1-2 The fovea of monkey, Macaca, irus. The slice near the fovea and parallel to the RPE-choroid space between the dashed lines represents the field lens interface passes into and out of several individual formed by the neural layer. The space between the lower layers within distances of 50-100 microns. This dashed line and the RPE shows the variation in the length of problem has contributed to the shortage of reliable the Outer Segments with position in the retina. Modified micrographs of the face of the Outer Segments or inner from Brown, Watanabe & Murakami, 1965. segments of photoreceptor cells. Ahnelt has recently provided an overview of the retina at the anatomical level69. It must be read with care. This work does not support many of the allegations presented in that paper. He assumes the optical axis of the eye is coincident with the fixation point and perpetuates the morphological definition of rods and cones. Discussing cones, he stresses that “the conical tapering is not a consistent morphological feature of this cell type. A comparison of

66Wolken, J. (1986) Light and life processes. NY: Van Nostrand Reinhold, pg. 154 67Brown, K. Watanabe, K. & Murakami, M. (1965) The early and late receptor potentials of monkey cones and rods. In Cold Spring Harbor Symposia on Quantitative Biology, volume XXX, Sensory Receptors. Cold Spring Harbor NY: Self Published pp 457-482 68Snodderly, D. Auran, J. & Delori, F. (1984) The macular pigment. II. spatial distribution in primate retinas. Invest. Ophthalmol. Vis. Sci. vol. 25, pp. 674- 69Ahnelt, P. (1998) The photoreceptor mosaic. Eye, vol. 12, pp. 531-540 The Retina 3- 25

cones along a meridian of human retina shows considerable variation.” Additional, less than convincing, criteria for determining the function of a photoreceptor cell based on morphological features are included in the discussion. The discussion concludes with “So far no morphological criteria for direct differentiation of L- and M-cones have been reported but there may be differing connectivities along their midget pathways.” A cross-section of the retina is introduced based on the classical figure of Polyak.

3.2.1.1 The brain/blood barrier

The current vision literature typically considers the retina as part of the brain. This is based on the generally similar organization of the layers of neurons in the neural laminate of the retina. However, it only briefly discusses a feature associated with brain tissue, the brain/blood barrier. Section 4.5.1discusses the morphogenesis of the eye from a perspective that suggests the retina is not part of the brain but is a complex portion of the peripheral neural system. This interpretation is due to the specific properties of the neural layers and the RPE layer of the eye and the important space, the Inter-photoreceptor-matrix, between them.. This space is essentially exterior to the epidermis of the animal, although it becomes enclosed within the eye with the formation of the lens group. This external space, the very high rate of opsin production by the photoreceptor cells and the digestive processes associated with the RPE cells show little analogy with the neural tissue of the brain. The concept of a blood-brain barrier is not widely detailed in the vision literature. While some describe the barrier as a layer of astroglial cells, others describe it as a special feature of the endothelial cells forming the walls of the capillaries70.

Chen & Heller describe the cells of the RPE forming one membrane of the brain/blood barrier but they do not discuss any isolation between the neural retina and the vitreal humor and the retinal artery. Heller also discusses the blood/brain barrier in his introduction and discussion from the perspective of the conventional wisdom71. The brain/blood barrier is usually described as preventing both large proteins and many other smaller molecules from entering the brain. As will be seen in this work, the situation is more complex in the eye. The “brain/blood barrier” appears to contain three components in the area of the retina. The cells of the RPE are tightly packed and do constitute a general barrier between the choroid arterial system and the neural portion of the retina. They are particularly efficient at controlling oxygen. However, the RPE cells do pass the chromophores of vision through this barrier and associate them with large binding and transport proteins within the IPM. These proteins and chromophores are prevented from entering the neural tissue of the retina by a secondary membrane, the Outer Limiting Membrane. This membrane may be discrete but it is more likely that it is represented by an closeley packed interdigitated layer of photoreceptor and glial cells. On the opposite surface of the retina is the Inner Limiting Membrane that prevents material from the vitreous from entering the neural tissue. It may also prevent large molecules from the neural vascular system from entering the neural tissue of the retina. There may be an anomaly in that Boycott & Dowling showed a part of a blood vessel within the ganglion layer of their micrograph of the human retina. It is possible that these small vessels are surrounded by their own individual brain/blood barrier.

The three membranes create two separate zones. One zone, the IPM, protects the chromophores from oxidation while they are being transported to the disks of the OS. The second zone, the INM, protects the neural tissue from the blood supply as in any zone of the brain and from both the chromophore material and the transport proteins. As will be seen below, this explanation is still too simple. There are a number of materials conveyed through the IPM in support of the neurological functioning of the photoreceptor cells. These materials may pass through the RPE cells. However, it is more likely they pass through and/or are manufactured by the glial cells that are located between the RPE cells or between the photoreceptor cells. A key role of the membranes surrounding the IPM is to prevent any materials, particularly those containing oxygen, from attacking the highly labile chromophores of vision found within this chamber. 3.2.1.1.1 Membranes separating the laminates

Heckenlively72 discusses the morphological properties of Bruch’s membrane in detail.

70McGeer, P. Eccles, J. & McGeer, E. (1987) Molecular Neurobiology of the Mammalian Brain. NY: Plenum Press. pp 35-38 71Heller, J. (1976) Intracellular retinol-binding proteins from bovine pigment epithelial and photoreceptor cell fractions. J. Biol. Chem. vol. 251, no. 10, pp 2952-2957 72Heckenlively, J. (1988) Retinitis Pigmentosa, NY: J. P. Lippincott, pg 50-51 26 Processes in Biological Vision

3.2.1.2 Layers of the Retina and some statistics

Records has provided an informative description of the morphogenesis of the human retina73. Figure 3.2.1-3 reproduces his figure after deleting the label for rods and cones and introducing Verhoeff’s “membrane.” This membrane is resolved in optical coherent tomography (OCT) at the two micron resolution level (Section 3.2.2). However, higher resolution would show that it consists of a concentration of pigment bodies and terminal bars in the 1/3 of the RPE cells closest to the photoreceptor cells.. Determination of the dimensions of the retinal layers are usually performed in-vitro following preparatory steps that frequently impact the absolute dimensions of the specimens. However, excellent data is available from a number of investigations. A new technique has just appeared, optical coherence tomography74,75. This technique based on light interferometry using extremely short pulses of light, can be used to examine the layers of the retina at different locations without surgical intervention. It may offer more precise in-vivo values in the future but is quite complex at present. Blanks has recently provided a brief discussion of the non uniformities in the layers of the retina that highlight the fact that the overall visual system is divided into a series of zones76. The signals from these various zones must be reconciled in the cortex to achieve a perceived uniform field of view. Before considering her figure 3-15, reproduced from Stone, et. a., the reader is advised to review Section 2.6.1.2 of this work.

73Records, R. (1979) Physiology of the Human Eye and Visual System NY: Harper & Row pg 299 74Leggett, K. (2001) Ultrahigh-resolution OCT detects early-stage eye disease. Biophotonics International, Sept-Oct. pp 60-62 75Drexler, W. et. al. (2001) Ultrahigh resolution ophthalmic optical coherence tomography Nature Medicine, vol. 7, pp 502-507 76Blanks, J. In Ryan, S. ed. (2001) Retina, 3rd ed. Vol. 1, St. Louis, MO: Mosby, Chapter 2, pg 48-49 The Retina 3- 27

Figure 3.2.1-3 Embryogenesis of the retina showing cellular origin of the various layers. Modified from Records, 1979.

3.2.1.2.1 The neural laminate

The neural laminate is the most anterior. It is the region between the inner limiting membrane and the outer limiting membrane. It consists of a hydraulic bed for the exchange of nutrients and waste products. The neural laminate contains the neural arterial network, supporting this hydraulic bed, and all of the soma and Activa associated with the signal neurons of the retina. It also includes the soma of the photoreceptor cells. Sublayers of interconnection circuitry occur within this laminate of the retina. They are usually described as the fiber layer, the outer plexiform layer, the inner plexiform layer and the optic fiber layer. The first three of these layers are complex matrices supporting the interconnection of dendrites and axons on an immense scale. Between these layers of circuitry and the outer limiting membrane are several layers of neurons. Beginning with the outer limiting membrane, these sublayers are the outer nuclear layer containing the soma of the photoreceptor cells, the 1st lateral layer, the bipolar layer containing the bipolar cells, the 2nd lateral layer and the ganglion cell layer. Most, if not all, of the amercine cells are found in the 2nd lateral layer. Occasionally, cells of a given type are reportedly found outside their normal domain (See [Figure 3.2.1-1]). These are rare and may involve unknown functions or misidentification. They are often defined as displaced ganglion cells or Dogiel cells and have been reported to occur in many retinas77. As shown elsewhere in this work, if the cells are being probed electrically and excessive capacitance is added to the circuit of the cell, it may go into oscillation. This may have occurred with a few cells in the inner nuclear layer. An alternate effect of capacitance is also shown in the above paper. The effect of excessive capacitance compared with the resistive impedance level of the cell is evident in the spot waveforms for the horizontal and bipolar cells of figure 3. The waveforms are

77Werblin, F. & Dowling, J. (1968) Organization of the retina of the mudpuppy, necturus maculosus. II. Intracellular recording J. Neurophysiol. Vol. 32, pp. 339-355 28 Processes in Biological Vision

essentially noise free because of the RC time constant of the overall circuit. In the reported experiments, the authors say the test circuit was always band-limited by the high distributed capacitance and resistance at the pipette tip, even with negative feedback used to reduce the apparent input impedance. They were using a field effect transistor at the test set input with an impedance reported by the manufacturer as 1013 ohms and 10-12 farads. The test lead to the specimen probably introduced ten times this amount of shunt capacitance. Both excessively smooth (noise free) waveforms and forced oscillation are signs of an inadequate test set. The neurons found in this laminate can be subdivided into two major classifications. Those principally involved in transmitting an in-line signal from the photoreceptor cells to the optic nerve and those principally involved in lateral connections between the in-line cells. The in-line cells are generally involved in signal manipulation aimed at signal summation and subsequent distribution to a large group of orthodromic cells. The lateral cells are used primarily for data processing involving signal differencing and correlation. Because of the proximity of these circuit elements and the limited dielectric strength of the hydraulic matrix, the voltage between these various circuits is limited to less than 200 millivolts. This laminate is within the optical path of the light approaching the photoreceptors and exhibits optical properties that must be taken into account. It is considered an optical field lens in this work. 3.2.1.2.2 The photoreceptor laminate

The photoreceptor laminate contains both the inner and outer segments of the photoreceptor cells immersed again in a hydraulic bed. This bed is known as the Inter Photoreceptor Matrix (IPM). It is delimited by the barrier pierced by the Inner Segments, the Outer Limiting Membrane (OLM), and the retinal pigment epithelium (RPE). This bed is in intimate contact with and supplied with material from the RPE laminate.

Figure 3.2.1-4 clearly illustrates the structure of both the photoreceptor and neural laminates of a bullfrog78. The figure is of a moment in time and was chosen for its clarity and is similar to those of all chordates, including man. Note the incompatibility of the structure labeled a cone outer segment with growth that proceeds continuously from the inner segment to the location of phagocytosis at the RPE laminate.

It is important to recognize that the neurons known as the photoreceptor cells are neuro-secretory cells, as are most if not all sensory cells. The Inner Segments extrude the protein substrate, generally identified as Opsin, into the IPM and form the uncoated disks of the Outer Segment. Chromophoric material is secreted from the RPE and transported across the IPM by diffusion to the region of the IS/OS interface. At that location, the chromophore condenses onto the substrate as a liquid crystalline film.

Note the structures labeled “cone outer segments” are shorter than the other outer segments. They are not considered mature functional Outer Segments in this work. 3.2.1.2.3 The RPE laminate

The third laminate consists of the retinal pigment epithelium (RPE) and the arteriola network associated with the posterior of the retina. It performs two major functions. A major function is to supply energy to the inner and outer segments of the photoreceptor cells via Figure 3.2.1-4 CR Electron micrograph of the photoreceptor the IPM located in the adjacent laminate. By and nuclear laminates of the bullfrog. Chosen for clarity supplying the active portions of the photoreceptor cells and believed similar to that of all chordates, including from the RPE, the competition for resources is greatly human. From Steinberg (1973 ).

78Steinberg, R. (1973) Scanning electron microscopy of the bullfrog’s retina and pigment epitherlium, Z. Zellforsch, vol. 143, pg. 451 [in Nolte, pg. 404] The Retina 3- 29 reduced compared to the situation in the neural laminate. The diffusion bed of the RPE is not in the optical pathway and it can be uniformly supplied by the capillaries. This minimizes the time constant of the hydraulic path serving the very important accommodation amplifiers. These amplifiers are located along the sides of the outer segments and connect to the inner segments through the cilium collar. A second major function of this arteriola network is to support the continual reconstruction of the Outer Segments of the chordate eye. A similar function may occur in the eye of other phyla if the animal has a long life span. Otherwise, the function may only be one of construction, without involving phagocytosis. In these other phyla, the equivalent of the RPE is not planar but appears to be found in the interstitial spaces between the retinula and the rhabdom, or even the ommatidia as a whole. The equivalent structures are given names like outer or corneal pigment cells. The pigment cells are so named because of their storage of chromophoric material in bulk form. This material is eventually used in building (rebuilding) photoreceptor Outer Segments. The RPE removes old disks from the Outer Segment, recycles their constituents and aids in the recoating of the new disk substrates (protein) extruded by the Inner Segments. The process of removal is normally considered to involve phagocytosis. The details of this processing will be discussed in Chapter 7. 3.2.1.2.4 Laminate dimensions

Scaling from the figure of Boycott and Dowling79, the three layers in an adult human have thicknesses of;

Layer Thickness Relative to sheet of paper

Neural Laminate 310 microns 4 sheets Ganglion layer 60 Inner nuclear 55 1st lateral layer ~5 Bipolar layer var. 2nd lateral layer var. Outer nuclear 25

Intercon. fibers 175 2 1/3 sheets Photoreceptor Laminate 40 microns ½ sheet Inner Segments 15 Outer Segments 22 RPE Laminate 18 microns 1/4 sheet

[Figure 2.2.2-4] shows the minimum thickness of the retina in the foveal area to be 200 microns for a rhesus monkey. For more details regarding the thickness of the retina, see Hogan who reproduces the graph of Straatsma et. al80. for the human eye. Figure 3.2.1-5, also from Hogan, provides the definition of the various regions of the retina. The fovea is a small area (typically 350 microns in diameter) where the photoreceptors are particularly densely packed.

79Boycott, B. & Dowling, J. (1969) op. cit. 80Straatsma, B. Foos, R. & Spencer, L. (1969) The retina-topography and clinical correlations. in Symposium on Retina and Retinal Surgery. St. Louis MO: C. V. Mosby (also in Hogan, et. al. (1971) Histology of the human eye. Philadelphia PA: W. B. Saunders pg. 401 30 Processes in Biological Vision

Figure 3.2.1-5 CR A fundus photograph matched with a meridional light micrograph of the macular region. The fundus photograph shows the foveola (a), fovea (b), parafoveal area (c) and perifoveal region (d). From Hogan (1971) The Retina 3- 31

3.2.1.3 Other anatomical features

Some care must be taken in analyzing features at the anatomical level. What appear to be simple anatomical characteristics may in fact be more complex than first perceived due to a more complex histological architecture. These differences will be highlighted below using material from Stone81. 3.2.1.3.1 The visual streak versus an elongated fovea

Stone stresses a concentration of ganglion cells frequently located along the horizontal axis of a chordate eye. This concentration is frequently described as a “visual streak” at the anatomical level without any consideration of whether it is also present in the photosensing layers of the retina. On the other hand, an area of concentration of photoreceptors within the photosensing layer is frequently described as a fovea, sometimes including an even more concentrated region known as a foveola. Many of the figures in Stone indicate a peak density of ganglion cells coincident with the area centralis or fovea. However, this characteristic is not borne out by micrographs of the area centralis. While the average density of ganglion cells may be high in this general area, the instantaneous density at the immediate location of the fovea is extremely low. Micrographs at the histological level show clearly that there are in fact no ganglion cells overlying the fovea, at least in man and other anthropoids. This is shown clearly in [Figures 3.2.1-2 and 3.2.2-1] of this work. While a visual streak may be indicative (at a gross level) of a degree of spatially oriented signal processing within the neurological laminate of the retina, it suffers two shortcomings. It is not descriptive of the organization of the photoreceptor laminate of the retina and it is not representative of the ganglion layer at the histological level.

An effort should be made to differentiate between a potentially elongated fovea, in a variety of species, located in the photosensing laminate and a functionally separate concentration of ganglion cells along the horizontal meridian of the neural laminate of a retina. The former impacts the performance of the physiological optics of the eye. The latter more likely impacts the neurological signal processing of the eye subsequent to image detection. 3.2.1.3.2 The optical disk (or blind spot)

The most important nonfunctional feature of the retina is the optical disk or blind spot. The dimensions of the optical disk were developed in Chapter 2. The disk is the region where all of the signaling circuits and support functions enter the ocular orb and are separated to serve all functional areas of the eye. Little attention has been given to this feature in the literature. Pirenne has reproduced an early and limited drawing of the cross-section of the disk82. The disk is three-dimensional; some of the elements within the optic nerve separate from it at three different levels. The arterial system separates and subdivides at the level of three different surfaces, the choroid, the RPE laminate and the neural laminate. Similarly, some motor nerves separate from the optic nerve at the choroid level for purposes of controlling the objective optical group at the anterior of the eye. All of the sensory nerves associated with the retina come together as a group within the neural laminate and pass out of the eye through the optical disk. They form the major part of the optic nerve, a somewhat restrictive name for this element. Without photoreceptors in the optical disk, the eye is completely blind in this region, an area larger than that subtended by six moons positioned side by side. This fact forces the adoption of the idea that the visual system involves computational optics consisting of two elements. The first element is a short term memory of significant size. The second element is a “fill program” not unlike a paint program used in modern desk top publishing and other computer programs.

Section 2.2.1 reviews the impact of the blind spots on the overall performance of the visual system. 3.2.1.3.3 The macula or macula lutea

The clinicians tend to describe two distinct regions as the macula or macula lutea. Many academicians do not recognize any structure deserving these names. Clinicians prior to the middle of the 20th Century argued that there was a distinctly colored region of the retina when viewed through the pupil. It was frequently described as from 8 to 14 degrees in diameter (when referred to object space) and of a very slightly different color than the outer retina. Many relatively naive associates claimed they could not see any distinct shading of this diameter. More recently, other clinicians have defined the more distinct ring of dominant vascularization encircling the foveola (1.2 degrees in diameter) at a diameter of 2–3 degrees centered on the point of regard (nominal center of a circular foveola). There

81Stone, J. (1983) Parallel Processing in the Visual System. NY: Plenum pp 291-305 82Pirenne, M. (1967) Vision and the Eye. London: Chapman & Hall. pg. 4 32 Processes in Biological Vision

is very fine vascularization extending from this ring in toward the smaller foveola. However, this finer vascularization does not extend into the 1.2 degree diameter region of the foveola. The foveal area of the retina in humans, and apparently other primates, exhibits a distinct yellow coloration when viewed through the pupil in-vivo. The origin of this coloration is obscure. At mid-century, many considered it an overlay on the vitreal surface of the neural layer of the retina and given the name macula lutea. The work of Polyak at that time and more recent photographs of cross-sections of the retina suggest the coloration is due to physical conditions within the neural layer. Miller83 has provided excellent color images of the area, although he defines it as occurring centered on the posterior pole of the human retina. In this work, the posterior pole is the point where the optical axis penetrates the retina. The point of fixation is not at this point and it is generally agreed that the macula lutea is centered on the point of fixation. In the pictures taken with different colored light sources, the overall color is seen to be due to a variety of spatially separate sublayers. A major question is whether the layers are indicative of the presence of a new localized material or the absence of neuron tissue in this area. This absence is well recognized in the literature. It would be expected to reduce the amount of scatter associated with the light passing through the neural layer on the way to the most spatially sensitive part of the retina, the foveola. In the latter case, the conclusion would be that the color of the macula lutea is merely the basic color of the neural layer as a substrate in the absence of neurons. Wald made spectral difference measurements on the in-vivo retina and calculated the spectrum of a material on the assumption that it overlayed the retina. His conclusion was that the change in spectra was due to a material that was probably similar to retinol. He proposed, in his normal charismatic style, xanthophyll from the limited catalog of chemicals with the desired spectrum available at that time. The assumption was that xanthophyll either reflected more light near XXX or absorbed light at complementary wavelengths.

Snodderly, el. al. have presented two papers exploring the “pigments” of the macular lutea of macaque retinas84. They included excellent pictures of a cross section of the fovea taken with narrow spectral bandwidth lights at 460 and 525 nm, Figure 3.2.1-6. The lights also had very small spatial diameters, 12.5 and 7.5 microns diameter. The results show the retina to consist of various layers of different reflectance. These layers vary in thickness near the fovea and provide an alternate interpretation of the mechanism causing the appearance of the macula lutea. The measurements with the 7.5 micron aperture were also quite noisy, requiring additional smoothing in the data processing. This would suggest the structure of the material of the neural layers was important.

Snodderly, et. al. confirmed that the pigment concentration was centered on the fovea. They also determined that it was dichroic with the major axis of absorption oriented tangential to a circle centered on that fovea. They did not find scattering to be a significant factor in their experiments. Their figure 9 of the second paper shows the pigment only extends to a radius of 750 microns, which corresponds roughly to the area they describe as the foveola. The most significant absorption at 525 nm was in the Outer Segments of the photoreceptors as would be expected. No major absorption could be associated with any individual layer of the neural laminate. At 460 nm, the situation was quite different. The absorption within the outer fiber layer (labeled RA in their figures) and the inner plexiform layer is significant. It is also significant in the outer nuclear area at the center of the foveola. They also found significant differences specimen-to-specimen. They associated most of the absorption with the axons of the photoreceptor neurons. However, there imagery shows a clear association with the inner plexiform layer and the thin layer between the ganglion layer and the inner limiting membrane.

Snodderly’s laboratory has recently released more cross sections of healthy Macaque retinas85 that can be compared to examples of diseased human retinas in Chapter 18. Examining Figure 3.2.1-6xxx and [Figure 3.2.2-1] of the next section, a case can be made that the additional absorption in the region of 460 nm is due to the absence of neural tissue in the INM. This would leave a higher density of matrix material in these areas. The higher absorption would then be related to the material of the INM and not the axons themselves. Based on the generally accepted caricature of Polyak, there is a lower density of photoreceptor cell nuclei at the center of the foveola, there are fewer interconnections in the outer fiber layer and outer plexiform layer in the vicinity of the foveola, and there are virtually no interconnections associated with the

83Miller, D. ed. (1987) Clinical light damage to the eye. NY: Springer-Verlag pp. 97-99 84Snodderly, D. Auran, J. & Delori, F. (1984) The macular pigment. Invest. Opthalmol. Vis. Sci. Vol. 25 pp. 660-673 and 674-685 85Snodderly, D. (2013) http://www.sbs.utexas.edu/SnodderlyLab/gallery.html The Retina 3- 33

Figure 3.2.1-6 Cross-sections of a macaque retina taken in 460 nm (blue) and 525 nm (green) light. The foveola is represented by the flat region of each retina devoid of any vascularization from the layers labeled IN, IP and GC. It has a nominal diameter of 350 microns in Appendix L. The darker regions near the foveola appear to be due to the absence of neural material relative to the material of the inter-neural matrix, IN. See original art for optimum reproduction. From Snodderly, et. al. 1984.

inner plexiform layer and the optic fiber layer in the region of the foveola. The material remaining in these areas is essentially that of the INM itself. This would suggest that the observed phenomena given the name macular lutea results from an absence of neural material and not the introduction of an additional filtering component. It is more due to the increased density of the matrix material than it is to the presence of axons, which exhibit a lower density in these areas.

There has been no confirmation of the xanthophyll hypothesis in the literature subsequent to Wald. The imagery of Snodderly, et. al.does not show any short wavelength absorption in the regions of high axon density beyond the fovea. Axons are normally considered colorless and are generally known as white matter when found in the brain.

This analysis suggests that the macula lutea is due to an optimization designed to minimize loss in resolution due to scattering of the incident light from the pupil and has nothing to do with improving resolution by raising apparent scene contrast. Such an increase in contrast would be obtained at the expense of sensitivity in the short wavelength spectral region. It is further suggested that a macula lutea is an inherent feature of any retina exhibiting a foveola and its presence is not related to evolutionary adaptation. Whether the net absorption associated with the macular lutea is associated with only one chemical constituent of the INM is a subject for further experiment.

3.2.1.3.4 The signal paths on the neural laminate surface

Although it is common in the literature to see maps of the vascular structure on the surface of the retina facing the aperture, it is less common to find maps of the neural paths coursing over that surface. The neural map contains much finer detail than the vascular map. Miller provides a picture but the neural paths are obscured by the large 34 Processes in Biological Vision

number of vasculature included in the same scene86. Fine & Yanoff discuss this optic fiber layer in more detail87 as does Adler88. Most of these materials concentrate on the area near the fovea and do not provide a comprehensive treatment of the organization of the neurons as they approach the Lamina Cribosa at the center of the “blind spot.” The Adler material shows the distinct division of the axonal paths into upper and lower sectors passing through the area of the fovea and also shows the variable length axon lengths complimentary to Henry’s Loops of the optical radiation. They do not discuss the multiple layer nature of the axonal paths. Oyster appears to provide the most complete caricature of the Lamina Cribosa based on earlier data from Hogan, Alvarado and Weddell89. An equally informative caricature appears in Records90. The material presented in Section 15.2.4 suggests that the arrangement of the neurons in the optic fiber layer is as significant as the arrangement of photoreceptors in the Outer Segment Layer. There appear to be a number of neural subsystems overlayed and intertwined within the 100-micron thickness of the optic fiber layer. These subsystems support different functional requirements within the overall visual system. Based on experiments partially dissecting the optic nerve, it appears the nerves of these subsystems are grouped as they pass through the lamina cribosa and maintain a fixed organization for the length of the optic nerve. The optic nerve contains a group of neurons dedicated to the foveola at the center of the fovea. It also contains groups associated with the various quadrants of the visual field surrounding the foveola. There also appears to be a group associated with a coarse mapping of the visual field into vertical and horizontal stripes. These groupings are used in the LGN to derive pointing signals that drive the eye muscles to bring threats to the center of the fovea quickly. By providing a neural map as above, the brain avoids the necessity of computing trigonometric functions in order to derive pointing commands based on polar coordinates relative to the foveola.

3.2.1.3.5 The tapetum

The tapetum is an intriguing layer of cells on the opposite side of Bruch’s membrane from the RPE layer. It provides retroreflection of light back through the photosensitive layer of the retina. The initial absorption coefficient of the retina is typically greater than 80%. Therefore, the utility of this retro-reflection is marginal in terms of overall performance. It may offer an improvement of about 10% at the photodetection level. Such an improvement would only be useful under scotopic conditions. At higher levels, it would be ignored by the adaptation function.

The effect is intriguing because of its apparent importance in the retroreflection from the eyes of animals observed by humans at night. It is important to recognize that just the specular reflection from the vitreous/neural surface of the retina contributes significantly to the observed phenomena. While the light passing through the retina, reflecting from the tapetum, and returning to the eyes of the observer has been reduced to only a few percent of the incident light, a similar percentage can be reflected by the vitreous/neural interface due to a change in index of refraction. The significance of the effect is that the total light leaving the neural layer is retroreflected by the optical system. This causes the effect to be orders of magnitude brighter than the Lambertian light scattered by equivalent areas of the rest of the animals body. The effect has little to do with the tapetum per se. The figure provided by Rodieck in 1973 should be disregarded91. All RPE cells contain mobile concentrations of chromophores that may be of the appropriate size to cause some retro-reflection due to their own spherical shape. Thus, his “retinal tapetum” is generic to all eyes and is actually an incidental feature of the RPE layer. For further details at the histological level, see Section 3.2.2.1.1.

86Miller, D. (1991) Optics and refraction. Vol. 1, edited by Podos, S & Yanoff, M. NY: Gower Medical Publishing. Pg. 3.24 87Fine, B. & Yanoff, M. (1979) Ocular histology: a text and atlas NY: Harper & Row. Chapters 6 & 12 88Hart, W. editor (1992) Adler’s Physiology of the Eye, 9th ed. St. Louis, MO: Mosby Year Book pgs. 495 & 617 89Hogan, M. Alvarado, J. & Weddell, J. (1971) Histology of the Human Eye. Philadelphia, PA: W. B. Saunders 90Records, R. (1979) Physiological Aspects of Clinical Neuro-Ophthalmology Chicago, IL: Year Book Medical Publishers, pg 509 91Rodieck, R. (1973) The Vertebrate Retina San Francisco, CA: W. H. Freeman & Co. pp 253-254 The Retina 3- 35 3.2.1.4 The optic nerve

Blinkov & Glezer have provided the most detailed tabulation of the structure and contents of the optic nerve and compared it with the properties of the auditory and olfactory nerves92.

3.2.2 Gross histology of the retina

Historically, histological studies have not been able to differentiate between cells of different spectral performance93. This is largely true today although a few studies of small areas have begun to suggest at least the statistical distribution of photoreceptors as a function of their spectral class.

Detwiler94 provides a comparative chart of the cross sections of the retinas of a variety of animals to a common scale. The results are worthy of study but are printed at a small scale. The highlight of his discussion concerns the significantly more neurons of the second order, bipolar, horizontal and amercine cells, in the retina of birds. The ratio of inner nuclear layer to outer nuclear layer thickness is five or six in some birds compared to two or less in man. Perry & Cowey have provided a wealth of information on the retinas (as well as information on the physiological optics, although interpreted using Gaussian optics) of several families of monkey95. It is noteworthy that they omit any discussion of the presence and densities of rods in the retinas. A caution is appropriate in their use of the term magnification factor of the retina. This term is used more like a convergence factor in the neural path than an actual magnification factor associated with the physiological optics. Although their work is strictly histological, they conclude that 80% of the ganglion cells are associated with the chrominance channels connecting to the parvocellular layers of the dorsal LGN.

The frequently reproduced caricature of the human fovea by Polyak is shown in modified form in Figure 3.2.2-196. The curved line representing the cross-section of the Petzval surface of the optical system is indicated by the arrow. The original sketch is asymmetrical. The center line marked A represents the center of the foveal pit formed by the Inner Limiting Membrane. The center line marked B represents the center of the curved Petzval cross-section. Also shown is the nominal 350 micron width of the foveola and the approaching rays of the light passing through the pupil. The dashed rectangle is shown centered between the two center lines. The F/8 bundle represents the light adapted pupil and the F/2 bundle represents the dark adapted pupil. From these overlays, it is seen that the optical path to the foveola is essentially free of neurons associated with the INL and the ganglion cell layer under light adapted conditions. Nuclei of the photoreceptor cells remain in the optical path but are quite close to the Petzval surface. Their presence is a compromise between optimum optical resolution and metabolic support to the IS’s. Under low light conditions performance is further compromised by the incursion of INL neurons into the F/2 optical bundle. Additional, although dated, comments by Polyak can be found in his later work97. It is worth noting that neither of his figures exhibits a distinct layer that can be described as a macula lutea.

92Blinkov, S. & Glezer, I. (1968) The Human Brain in figures and Tables. NY: Pergamon Press pp 118-125 93Oyster, C. (1999) The Human Eye. Sunderland, MA: Sinaure Associates, Inc. Chapter 15 94Detwiler, S. (1943) Vertebrate photoreceptors. NY: Macmillan pp.17-25 95Perry, V. & Cowey, A. (1985) The ganglion cell and cone distributions in the monkey retina. Vision Res. vol. 25, no. 12, pp 1795-1810 96Polyak, S. (1941) The Retina. Chicago, IL: Univ. Chicago Press. pg 161 97Polyak, S. (1957) The vertebrate visual system. Chicago, IL: University of Chicago Press pg. 276 36 Processes in Biological Vision

Figure 3.2.2-1 A caricature of the central one-third of the human fovea from Polyak labeled with the conventional morphological terms and overlayed with the optical parameters of the system. See text. The horizontal white dashed line (labeled Osterberg section) will be discussed in Section 3.2.2.3.

A new method of imaging the living retina in cross section has recently appeared and been improving very rapidly. Figure 3.2.2-2 shows an ultrahigh-resolution spectral OCT image of human macula from Wojtkowski et al98. This version employs false color imaging to highlight specific features. The technique employs Fourier domain optical coherence tomography (FD-OCT) with numerical compensation for the dispersion of light within the biological tissue. Alam et al99. and Choi et al100. have provided similar examples of the FD-OCT technique applied to the human retina. The technique views the retina through the pupil and measures the time delay (convertible to distance traveled by the light) associated with each axial layer of the retina. This new technique is the first to “resolve” Verhoeff’s membrane in a living human retina. Verhoeff’s membrane is actually a representation of the average response from a more complex layer containing a variety of individual elements, including terminal bars formed between adjacent RPE cells (Section xxx). The representation represents the 1/3 of the RPE closest to the pupil. The remainder of the RPE and the closely associated Bruch’s membrane (BM) are represented by the feature labeled either choriocapillaris (CC) or RPE/BM by different investigators. Note the longer outer segments directly below the pit of the foveola.

98Wojtkowski, M. Srinivasan, V. Ko, T. et al. (2004) Ultrahigh-resolution, high-speed, Fourier domain optical coherence tomography and methods for dispersion compensation Optics Express vol 12(11), pp 2404-2422 99Alam, S. Zawadzki, R. Choi, S. et al. (2006) Clinical application of rapid serial Fourier-domain optical coherence tomography for macular imaging Ophthalmology vol 113, pp 1425-1431 100Choi, S. Zawadzki, R. Greiner, M. et al. (2008) Fourier-domain optical coherence tomography and adaptive optics reveal nerve fiber layer loss and photoreceptor changes in a patient with optic nerve drusen J Neuroophthalmol vol 28(2), pp 120-125 The Retina 3- 37

Figure 3.2.2-2 Ultrahigh-resolution spectral OCT image of living human macula using 2nd & 3rd order numerical dispersion compensation. Nominal axial (vertical) resolution is 2.1 microns. Lateral resolution is poorer than 5 microns. Illumination was 144 nm wide FWHM centered on 850 nm. Alternate labels are shown as used by various investigators. Note asymmetric magnifications as indicated by the Plimsoll mark at lower right. VM = Verhoeff’s “membrane.” CC = choriocapillaris. BM = base membrane. From Wojtkowski et al., 2004.

3.2.2.1 The sensing laminate

The sensing laminate of the retina provides the best reference for describing the retina at the histological level. It consists of the space between Bruch’s membrane and the OLM. This laminate only includes three sub-laminates and is readily visible through the neural laminate that is sufficiently transparent. The high degree of planarity of these layers and the lack of interconnections in the plane of the layers make them easily identifiable. The layer adjacent to Bruch’s membrane is the RPE laminate. The next laminate is the Outer Segment laminate and the final layer is the Inner Segment laminate.

The sensing laminate is dominated by the large number of individual photoreceptors of the visual system and a variety of additional cells providing physical support, isolation and possibly other functions. Most of these ancillary cell types have received little attention. The laminate is easily subdivided into two layers, the Inner Segment layer and the Outer Segment layer. The Outer Segment layer is very fragile. The Outer Segments are usually sheared in half when attempts are made to separate the photoreceptor laminate from the RPE laminate. This type of shearing is also the dominant form of damage in the pathological condition known as detached retina. Because of the fragility of the Outer Segment layer, it is rare to find experimental histological results that involve the Outer Segment. The results invariably represent sections through the Inner Segment layer. It is also rare to find any discussion of the correlation between the parameters associated with the Inner and Outer layer in order to support the generalizations usually applied to the results relative to the actual Outer Segments. Recently, Williams at the University of Rochester has been perfecting a method of studying the Outer Segments in- vivo using photographic techniques and selective spectral filters in the illumination path. This has provided retinal maps at resolutions of better than 2 microns. These maps have provided the first maps defining the location of individual photoreceptor according to their absorption characteristics. 3.2.2.1.1 The RPE sub-laminate

At the histological level, the RPE consists of two separate planes of cells, the sheet of tapetum cells arranged in close proximity to Bruch’s membrane. This membrane forms the boundary between the RPE laminate and the choroid. The retinal epithelium cells form a thick semipermeable sheet opposite to the tapetum and adjacent to the OLM. 38 Processes in Biological Vision

These two layers form the enclosed IPM which contains both the outer and inner segments of the photoreceptor cells. The IPM is thus isolated from the blood stream behind the RPE and from the vascular matrix of the INM by the OLM. This protected environment is required to support the chemically delicate chromophores of vision. The tapetum sheet can evolve to form a variety of functions depending on the animal. It is generally a passive layer. Normally, it can aid in the absorption of stray light that has passed through the retina. In some cases, it consists of small groups of cells that act as a retro-reflector to direct light back through the retina. As seen in the case of the mollusc, Pecten, the cells can also be used to form an optically coherent sheet of cells that form a reflecting optical element in a catadioptric lens system. The retinal epithelium sheet is much more complex. Besides supporting the metabolic requirements of the accommodation amplifiers of the photoreceptors, the retinal epithelium is an important factory supporting the operation of the disks in the Outer Segment. This function is described in detail in Chapter 7. 3.2.2.1.2 Orientation of photoreceptors in the outer segment sub-laminate

It is usually tacitly assumed that the photoreceptors are arranged perpendicular to the surface of the retina. This is the way they are invariably presented in caricature101. The text by Torrey has inappropriately shown the retinal layer of the eye in cross-section as a matter of artistic license102. In fact, the photoreceptors are sheared over based on their location in the retina so that their optical axis is essentially parallel to the principal ray approaching from the exit aperture of the objective optical group (See Section 2.3.1.2). This has been well documented by Bedell & Enoch103 although their caricature of the eye is mis-drawn. They show that all of the photoreceptors are aligned with the center of the exit aperture of the objective lens group. The caricature assumes paraxial optics for conditions as great as 25 degrees off the axis. Although they have not labeled the rays in the caricature, they imply a ray 25 degrees off axis in object space is also 25 degrees off axis in image space. The fact that all healthy photoreceptors have the axis of their Outer Segment (and if appropriate, the portion of the Inner Segment containing the ellipsoid ) parallel to the incoming light ray should not be dismissed. It has a significant impact on the overall sensitivity of the visual system, particularly at high angles relative to the optical axis.

3.2.2.1.3 Spatial parameters of the mosaics of the outer segment sub-laminate

A recent textbook by Oyster provides useful material related to the mosaic parameters of the retina104. However, the material should be considered in the context of a sparse array of data. Obtaining photomicrographs of specific mosaics in the retina is difficult, particularly with respect to the entrance apertures of the Outer Segments. Very few micrographs of human retinas appear in the literature showing the mosaic of the Outer Segments. Most micrographs show the mosaic of a part of the Inner Segment mosaic. Differentiating between caricatures and photographs and between photographs of the Inner Segments versus the Outer Segments is important in this discussion. Rodieck provides references to retinal micrographs of a variety of animals. The caricature of the human retina shown in Rodieck105 is a modern interpretation, without definitive dimensions, of an ancient sketch by Schultze (1866). It claims to show a cross section of the outer segments. It should be noted in that sketch, Schultze implied that all of the rods and cones were of equal diameter, only their packing density changed due to the size of the inner segments of the cones. A more realistic photomicrograph has been presented by Williams et al106. Part of his figure is reproduced as Figure 3.2.2-3. The white dots are soma and the smaller more prevalent dots are axons, or thin inter- soma parts found between the inner segment (extrusion chamber) and the soma of a cell associated with the multilayer character of the outer nuclear layer, ONL in [Figure 3.2.1-1]. The figure shows the same spatial relationships as Shultze but with a scale. Williams et al. assert it is a tangential section of the retina through the inner segment layer near the fovea of Macaca nemestrina. Thus, the elements shown in the Schultze sketch and the

101Kandel, E. Schwartz, J. & Jessell, T. (2000) Principles of Neural Science, 4th ed. NY: McGraw-Hill pg. 515 102Torrey, XXX Pg. 480 103Bedell, H. & Enoch, J. (1980) An apparent failure of a photoreceptor alignment mechanism in a human observer. Arch. Ophthal. Vol. 98 pp. 2023-2026 has a list of references 104Oyster, C. (1999) The Human Eye. Sunderland, MA: Sinaure Associates, Inc. Chap. 15 105Rodieck, R. (1973) op. cit. Pg. 354 106Williams, D. Collier, R. & Thompson, B. (1983) Spatial resolution of the short-wavelength mechanism In Mollon, J. & Sharpe, L. eds Colour Vision NY: Academic Press pg 485+ The Retina 3- 39

xxx micrograph have nothing to do with the division of the outer segments into two classes of photoreceptors.

Figure 3.2.2-3 Tangential section through inner segment layer of Macaca nemestrina near the fovea stained with Procion yellow. The calibration bar represents 25 microns uncorrected for shirnkage, or about 9.6 minutes of arc. (It is assumed that the shrinkage factor is 0.78 and that 200 microns corresponds to 1 deg visual angle for this species.) From Williams et al., 1983.

Figure 3.2.2-4 reproduces a sketch from Shultze in 1866, reproduced in Pirenne, showing a possible fractal nature of 40 Processes in Biological Vision

the human retinal mosaic107. In this map, the size of the photoreceptors varies by less than a factor of two. Although Schultze used a french curve to create a framework describing what he saw when examining a flattened retina, it is possible that the actual framework is described by a specific fractal form associated with biological growth of spherical forms and based on a form known as a spherical spiral108. Alternately, the fundamental pattern may be of a simpler repeating, and overlapping face-centered pentagonal pattern as discussed below. Nolte continues the common practice in his figure 17-12, incompatible with his figure 17-6, of showing cross- sections of only inner segments by Curio. The functional elements are the mature outer segments extending from the inner segments to the RPE. These elements are all nearly identical in diameter and length. (C) of his figure 17-13 shows the diameter to vary by less than 2:1 over a major part of the retina. This is in contrast to the proposal by Hirsch & Curcio that the variation of center-to-center cone spacing exceeds 2.5:1 within two degrees of center109. However, these authors are actually measuring inner, not outer, segments. Their equation for the variation in this spacing, and its applicable range are clearly inappropriate. It defines the impossible condition of a center-to-center cell spacing of zero at the center of the retina.

Farber, et. al. have shown two micrographs of a human retina at different eccentricities110. While the article is entitled photoreceptors, the figures are clearly labeled inner segments. If the figures actually show an area where the inner segments (or nuclei) are arranged in echelon, the labels become misleading.

There is also the challenge of obtaining in-vivo versus in-vitro mosaic micrographs. At the present time, only in -vivo micrographs, taken by reflection spectrophotometry, offer indisputable data with regard to the chromatic sensitivity of individual classes of photoreceptors. They also promise to provide information as to whether all structures photographed are actually participating in the perception of vision or are associated with other housekeeping functions.

Figure 3.2.2-5 from Miller & Snyder shows what they label a mosaic of Inner Segments from both the Figure 3.2.2-4 The human photoreceptor mosaic in the monkey, Macaca fascicularis and the sharp-shinned fovea centralis. x270, the outer diameter corresponds to Hawk, Accipiter striatus. These may actually be the about 0.35 mm. Schultze states that the disposition of the faces of the outer segments as viewed from the pupil receptors is fully as regular as is shown by the side of the retina as seen in the recent in-vivo figure–provided the retina is in a sufficiently fresh state. bb ophthalmological work of Roorda & Williams (Section represents the bodies or inner segments, arranged in 3.2.3). The scale added post experiment in (B) shows curvilinear rows as a shagreen-like mosaic. In a, the pointed the Inner Segments to be nominally two microns in ends, or outer segments, of the receptors are shown as they diameter with a very small standard deviation in the appear when the microscope is refocused. The principle on right half of the picture. Some smaller cells, nominally which the receptors are arranged is shown by the lines cc. one micron in diameter, are seen in the left part of the The figure is based on drawings made of several fresh picture. There is also a change in nominal spacing retinae. The black part of the drawing shows the retina as between the cells in the left and right half of this Schultze supposed it woud appear if the “pigment” were left picture. The image for the hawk may be taken through in position. Schultze, 1866. the field plate formed by the neural laminate. This

107Pirenne, M. (1967) Vision and the eye. London: Chapman & Hall. Plate 7. 108Lauwerier, H. (1991) Fractals. Princeton, NJ: Princeton Univ. Press. pg 64-66 109Hirsch, J. & Curcio, C. (1989) The spatial resolution capacity of human foveal retina. Vision Res. vol. 29, no.9, pp 1095-1101 110Farber, D. et. al. (1985) Distribution patterns of photoreceptors, protein and cyclic nucleotides in the human retina Invest Ophthal Visual Sci vol. 26, no. 11, pp 1558-1568 The Retina 3- 41 may introduce differential distortion in the micrograph of up to 30%111. In (A), the photoreceptors are as closely packed as possible for cylinders. They appear as hexagonal groups of seven photoreceptors locally. On a larger scale, the array exhibits minor dislocations and specifying the characteristics of the overall pattern is more difficult. Some micrographs of retinas have shown “rods” of much smaller diameter interspersed among “cones.” Snyder & Menzel have discussed the optical properties of Outer Segments from the perspective of light pipes in detail112. It is important to note that an Outer Segment with a diameter less than the wavelength of light to which its chromophore is optimized is a very poor acceptor of light. Structures in these micrographs with a diameter of less than one micron should not be considered photoreceptor structures without overwhelming evidence.

Bowmaker & Kunz113 have provided some local area mosaics of fish as a function of age. They show the pattern of cells changes considerably with age and confirm that trout are tetrachromatic at ages up to about two years. The local pattern is repeated similar to the replication of the retinula in molluscs. When reviewing micrographs of a retina, the investigator usually perceives a pattern to the cells. However, the pattern is usually more a work of art than a geometrical pattern designed for simple data manipulation. It appears that science has not yet discovered the underlying fundamental pattern(s) used to form retinas in animals. 3.2.2.2 Geometrical patterns in retinal arrays

The geometric layout of the retina is occasionally described as based on triangles, more often on hexagons and most frequently on close spaced hexagons containing a center element (groups of seven cells). It is often suggested that the breakdown in orderliness frequently results in close spaced pentagons being interspersed with the close spaced hexagons.

From a geometrical perspective, describing the arrays in terms of triangles, even equilateral triangles, is generally inadequate. A higher order description is needed to avoid ambiguity. The descriptions used in crystallography are probably most appropriate. Coxeter has provided a good, and hard to find, introduction to two-dimensional crystallography and the various faults found in that field114. The title of his book is misleading. It is an introduction to college Figure 3.2.2-5 CR Micrographs of foveal “cone” inner level geometry and requires vector algebra and segments at fixation point of monkey, Macaca fascicularis differential equations as prerequisites to follow the (A) and deep fovea of hawk, Accipiter striatus (B) at the analyses. Hull & Bacon115 have provided an level of the ELM. Arrows added to mark discontinuities. introduction to dislocations in crystallography that From Miller & Snyder (1979) proceeds from Coxeter. Unfortunately, it moves rapidly into three-dimensional rectilinear structures. It

111Walls, G. (1942) The vertebrate eye. Cranbrook Institute of Science pg. 183 112Snyder, A. & Menzel, R. (1975) Photoreceptor optics. NY: Springer-Verlag. pg. 45 113Bowmaker, J. & Kunz, Y. (1987) Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the brown trout Salmo trutta. Vis. Res. vol. 27, pp. 2101-2108 114Coxeter, H. (1961) Introduction to Geometry. NY: Wiley & Sons, Chap. 2, 3 & 4. 115Hull, D. & Bacon, D. (2001) Introduction to Dislocations, 4th ed. Oxford: Butterworth Heinemann 42 Processes in Biological Vision

divides the possible types of imperfections in a regular array (or lattice) into point, line, surface and volume defects. These defects disturb the locally regular arrangement of the atoms. The point defect can consist of a void in the regular lattice or an interstitial insertion. The line defect usually involves the introduction of a short row of elements between two longer rows of a lattice. Alternately, it can consist of the removal of a short segment of a row, causing the adjacent rows to distort. The most common surface defect is the grain boundary between adjacent grains with different intrinsic alignments. The grain boundary can look like a simple slip line only affecting a narrow region of lattice elements or it can appear as a stretching of the adjacent lattices to fill a localized void. This stretching introduces other local distortions as well. Each of the above imperfections is readily recognized in the above micrographs of retinas. It is likely that interstitials are unusual in the morphogenesis of the retina and that voids are masked by the plastic nature of the liquid crystalline structures of the outer segments. While a close-packed hexagon is the most intuitive form of fundamental region and linear axes of translation are to be expected in the retina based on the theory of flat lattices; however, the retina is not flat. It is based on a spherical surface. The actual basic non-overlapping geometric form on the surface of a sphere, consists of intersecting, equal length, segments of great circles called geodesics. These are found to result in a fundamental array based on a close-spaced pentagonal array. The fundamental array consists of five hexagons surrounding a pentagon, all face-centered (Chapter 20 in Coxeter). This fundamental array is shown approximately in Figure 3.2.2-6 as distorted by showing on a flat surface. Each straight line is actually a geodesic. The fundamental pattern consists of five hexagons surrounding a pentagon. The central pentagon is surrounded by three pentagons forming the centers of adjacent overlapping fundamental patterns. The individual photoreceptor outer segments occupy the center of each geometric figure and the points of union of the individual vertices of the figures. The diameter of these individual outer segments are found to be generally round and of the largest diameter possible. However, the plasticity of the outer segments may force some of them into pentagonal or hexagonal shapes. It is also possible for this fundamental pattern to change scale to accommodate larger diameter outer segments as a function of the distance from the center of the overall pattern.

If a retina is flattened for study, one should not be surprised if an apparent hexagonal structure is noted. The human visual system appears quicker to recognize hexagons than pentagons. However, the significance of this hexagonal appearance should not be over- emphasized. The individual elements are still connected (theoretically) by great circles, not straight lines and the fundamental pattern is a face-centered pentagon. This work will take the face centered pentagon as the fundamental pattern of the human retina. Such a pattern contains five face-centered hexagons Figure 3.2.2-6 The proposed fundamental array used in the surrounding a face-centered pentagon as described spherical human retina. The array is necessarily distorted above. This is the fundamental pattern of a when drawn on a flat surface. However, the plasticity of the basketball. outer segments accommodates this distortion in the laboratory. See text. Note that when projected onto a flat surface, the retina does not exhibit clearly defined major axes. While one might associated major axes with the pair of hexagons located side by side in the figure, other similar pairs of hexagons are at angles relative to this pair. This will be important when discussion the Nyquist frequency of the retina as a sampling mechanism in Section 16.6.3. The Retina 3- 43

The use of the optical Fourier Transforms offers a unique and rapid method of studying the array parameters of a retina, even in-vivo. This technique will be discussed further in Section 16.6.3. By varying the size of the illuminated window on the retina, the parameters can be evaluated as a function of position and area on the retina. Recently, Deering has provided a computer generated replica of the spherical human retina that he believes has captured the actual rules employed in morphogenesis116. Because the paper is in an obscure journal, it may be hard to retrieve. A more readily available copy was available on the Internet in 2007117. The synthesized retina is virtually indistinguishable from recent retinal images from Roorda & Williams (Section 3.2.3). Stewart118 has offered an opposing origin of the map of the retina from that of Deering. Figure 3.2.2-7 is based on the biological rules for cell replication (phyllotaxis) developed by Hofmeister in 1868. Hofmeister’s generative spiral was later revealed to be a so-called Fermat spiral based on the “Golden Angle” of 137.5 degrees appearing in derivations related to Fibonacci numbers. While based on projections on a flat surface, it appears to explain the various swirl patterns frequently seen in images of the receptor mosaic. The sensitivity of the patterns to variations from 137.5° are obvious. Dubra and associates reproduced their data on the shapes of retinal elements with the implicit assumption that the cells should be present in hexagonal groupings as frequently assumed119. Their mapping clearly shows the presence of significant numbers of 5-sided as well as 6-sided arrays. Their data was collected using through the pupil imaging of living human retina. However, they did not account for the spatial distortions associated with the anamorphic lens of the real eye at positions off-optical-axis. Figure 3.2.2-8 shows their figure 5 with their caption. Figure 3.2.2-7 Fermat spiral patterns. Left; spacing 137°, just less than the golden angle. Middle; spacing 137.5°, the golden angle. Right; spacing 138°, just greater than the golden angle. From Stewart, 2011.

116Deering, M. (2005) A photon accurate model of the human eye ACM Trans Graphics vol 24(3) 117Deering, M. (2005) http://delivery.acm.org/10.1145/1080000/1073243/p649- deering.pdf?key1=1073243&key2=5477055711&coll=&dl=ACM&CFID=15151515&CFTOKEN=6184618 118Stewart, I. (2011) Mathematics of Life: Unlocking the Secrets of Existence. London: Profile Books pp40-49 119Dubra, A. Sulai, Y. Norris, J. et al. (2011) Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope Biomedical Optics Express vol 2(7), 1864-1876 44 Processes in Biological Vision

A Voroni diagram is defined with respect to a flat plane and the data from the retina is not strictly compatible with the planar requirement. However, the concept is useful. Quoting Wikipedia as a convenience, “In mathematics, a Voronoi diagram is a partitioning of a plane into regions based on distance to points in a specific subset of the plane. That set of points (called seeds, sites, or generators) is specified beforehand, and for each seed there is a corresponding region consisting of all points closer to that seed than to any other. These regions are called Voronoi cells.”

3.2.2.3 Statistical parameters of the complete mosaic of the outer segment sub-laminate RE-OUTLINE

The availability of precise statistics on either the geometrical or chromatic parameters of the retinas of animals is very limited. There are many first order estimates but very few statistically relevant estimates of these statistics. Obtaining the geometrical parameters appears to be limited by the lack of an adequate theoretical base on which to collect the statistics. A fundamental fractal equation that can be modified for different situations is probably needed. This section is complicated by the functional division between the region of the retina called the foveola and the surrounding retina. The statistics of these two areas are not likely to be the same. They should be considered distinct subsets of the mosaic statistics. A following section will discuss the statistics of the foveola particularly.

Another problem associated with evaluating the statistics of the foveola involves the processing of the data. There is

Figure 3.2.2-8 Dubra’s analysis of the regularity of the peripheral photoreceptor mosaic. Shown in a is the 6-hour averaged image (logarithmic display) from subject JC_0138, taken at about 10/ temporal to fixation, collected using 680 nm light and 1.1 Airy disk pinhole size. Color-coded Voronoi domains associated with each cell are shown in panel b), where the color indicates the number of sides on each Voronoi polygon (magenta = 4, cyan = 5, green = 6, yellow = 7, red = 8, dark blue = 9). Regions of six-sided polygons indicate a regular triangular lattice, while other color mark points of disruption of the mosaic. Panel c shows the color-coded Voronoi domains associated with just the cone photoreceptors in the image. From Dubra et al., 2011. evidence that the pretectum attempts to treat all of the photoreceptors of the foveola as spectrally equivalent when evaluating fine detail. It is only when highly selective illumination is used that the signals delivered to the pretectum are limited to those from a specific set of spectral receptors. It also appears that there is little requirement for orderliness in the individual spectral arrays in a system based on edge detection and color estimationg involving nearest neighbors to the edges. As a result, it has been very difficult to quantify the performance of the foveola in terms of the spatial responses and spatial frequency responses as a function of chromaticity. This is particularly true when the chromatic sources used have not been carefully chosen to maximize contrast. Williams addresses the data The Retina 3- 45

in this area120. The reader is cautioned to differentiate between mosaics of the actual outer segments and the much more commonly reported mosaics of the inner segments (which are not photosensitive). There are considerable differences between these two mosaics at the research level. Recently, a Fourier transform technique has appeared that is able to determine the orderliness of the retinal mosaic, irrespective of chromatic content, in-vivo and very rapidly. Pum, Ahnelt & Grasl121 have attempted to define the quality of the lattice found in the humans and primates using a “radarscope” presentation. Starting from the premise that the retinal mosaic should be a perfect close spaced hexagonal array of seven photoreceptors, they have provided first order statistics on deviations from this ideal. They were using stained Inner Segments and were operating in-vitro. The results provide an autocorrelation function along the various axes of the array and are similar to those found in diffraction studies of crystals. The presentations are both technically and artistically interesting. They were able to clearly demonstrate a dislocation between two zones that involved a 12 degree shift in orientation and a definable change in packing density along specific axes. The simultaneous presentation of more than two zones resulted in the expected statistical characteristics that could be read directly from the presentation. They also presented some information attempting to define the distribution of photoreceptors of different absorption within the overall arrays. They make the interesting statement that the Inner Segments display a tubular shape above the external limiting membrane and reference Miller & Bernard, 1983. As discussed in this Section, Williams and his colleagues have frequently recorded a tubular image of Outer Segments in-vivo.

Schultze122 provided a large scale map of the inner segments of a human retina that seems to show a fractal pattern. The collection of statistics about the chromatic sensitivities of the photoreceptors, as an overlay to the basic pattern has not been productive to date.

Because of the irregular arrangement of human photoreceptors, most investigators have attempted to determine the statistical properties of the photoreceptor cells based on either rectilinear grids or by measuring the shortest distance to adjacent cells, the number of cells within a given radius, etc. These statistics are helpful but not definitive.

Lee gives a set of statistics in his Table 1123. related to the topology of the retina but places them in a framework that may complicate their interpretation.

Kageyama & Wong-Riley124 provide a variety of statistical parameters concerning various elements in the retinas of several animals. Their work was based primarily on staining studies related to cytochrome oxidase staining as an indication of activity level in the cells.

Chan et. al125. Provide a set of statistics for horizontal cells in two different primate retinas. 3.2.2.3.1 Statistical parameters of the complete mosaic(s)

Figure 3.2.2-9 illustrates the total number of photoreceptors per square mm in the retina of the rhesus monkey. The data is taken from a composite figure prepared by Wassle & Boycott126 based on earlier data from Wassle, et.al. and from Steinberg, et. al. Total photoreceptor cell densities are remarkably uniform, averaging 100,000-110,000 cells per square millimeter for eccentricities between 1.0 mm and 10 mm from the fovea (without accounting for the optical disk). The density of “cones,” in the original composite was shown as rising to more than 200,000 cells per square millimeter for a small region within 0.175 mm. of the fixation point. Other details on how this data was

120Williams, D. Sekiguchi, N. & Brainard, D. (1993) Color, contrast sensitivity, and the cone mosaic. Proc. Natl. Acad. Sci. USA vol. 90, pp 9770-9777 121Pum, D. Ahnelt, P. & Grasl, M. (1990) Iso-orientation areas in the foveal cone mosaic, Visual Neuroscience, vol. 5, pp. 511-523 122Pirenne, M. (1967) Vision and the eye. London: Chapman & Hall, plate 7 123Lee, B. (1996) op. cit. Pg. 634 124Kageyama, G. & Wong-Riley, M. (1984) The histochemical localization of cytochrome oxidase...with particular reference to retinal mosaics. . . . Jour. Neurosci. Vol. 4, no. 10, pp. 2445-2459 125Chan, T. Goodchild, A. & Martin, P. (1997) Vis. Neurosci. vol. 14. pp. 125-140 126Wassle, H. & Boycott, B. (1991) op. cit. Fig 2 46 Processes in Biological Vision

assembled appear in Wassle & Boycott. It appears that the rod density should be shown approaching an asymptote at 150,000-160,000 instead of rising without a limit. This small region is often called the foveola and is equivalent to about +/- 0.6 degrees of visual angle in object space. Osterberg127 has provided some very old data that is still reprinted. It provides similar data for the human on a linear scale with eccentricities measured in object space angles out to 100 degrees. Unfortunately, the provenance of Osterberg’s data was largely lost when Pirenne plotted it128. It has subsequently been reprinted without provenance in virtually every text on vision including Davson, 1962, Rodieck, 1973 &, Wandell, 1995. The data of Osterberg was obtained by sectioning a human retina at a given depth below the RPE surface of the tissue. The result is a plan-view of the retina taken at a location shown by the dashed white line in Figure 3.2.2-1 xxx. Note that within the foveola, the dashed white line only intersects outer segments which are all cylindrical in shape. Osterberg called these outer segments rods. Note that outside the foveola, the dashed white line intersects the inner segments of only some of the photoreceptors. It also intersects the axons of the photoreceptors where the inner segments are not intercepted. As a result, the plan-view image of inner segments and axons agrees with that drawn by Shultze in 1866, except his so-called rods are actually axons. The common wisdom associated with this figure must be reinterpreted. The result is shown in Figure 3.2.2-9. In the region between plus and minus two degrees, the count represents the density of outer segments intercepted. In the region beyond plus and minus two degrees, the two counts represent the number of axons intercepted and the number of inner segments intercepted. The ratio between the number of axons and the number of inner segments suggests this retina had an outer neural layer (ONL) that consisted of an average depth of 10-20 inner segments. The caricature in Figure 3.2.2-1 xxx suggests the lower number. A reinterpretation of a caricature by Shultze presented in 1866 (and reproduced in Pirenne in 1948) is shown at the lower left. It applies to the peripheral region of the retina on both sides of the foveola, except in the blind spot. The elements he labeled rods are clearly axons in this caricature based on the location of the section. The figure suggests a higher ratio of axons to inner segments, near 30. Modern micrographs may provide more definitive numbers.

127Osterberg, G. (1935 ) Topography of the layer of rods and cones in the human retina. Acta. Ophthal. suppl. 6, pp. 1-103 128Pirenne, M. (1967) Op. Cit. The Retina 3- 47

Figure 3.2.2-9 The reinterpreted photoreceptor and neural densities of Osterberg. The break in the line between the outer segments at zero degrees and plus two degrees is usually drawn to be symmetrical with the region between zero and minus two degrees. Outside of plus and minus two degrees, the same curve represents the fraction of intercepted inner segments. Lower left; a reinterpretation of a caricature by Shultze, 1866, showing a section taken through the outer nuclear layer of the retina. See text.

The data of Osterberg, as interpreted by Pirenne, leads to different conclusions from that of Wassle & Boycott. On the other hand the data as interpreted here is in close agreement with that of Wassle & Boycott. Their data, for both the temporal and nasal meridians, show a peak (nearly constant) total photoreceptor density of about 147,000 cells per square mm. It is clear from the reinterpreted graphs above that the maximum value of rod and/or cone density does not rise without a limit as might be inferred from the logarithmic plot of Wassle & Boycott. Figure 3.2.2-10 also illustrates the ratio of photoreceptor cells in the retina of the rhesus monkey on both a morphological and a functional basis using a logarithmic vertical scale. Whereas Wassle & Boycott show the cone 48 Processes in Biological Vision

to ganglion cell ratio as turning down at small eccentricities, this is a poor assumption. Within the foveola, there are no ganglion cells. A morphological ratio with a denominator approaching zero must approach infinity in the limit. There are ganglion cells supporting the foveola. However, they are displaced laterally within the retina. The result is a ratio that approaches infinity for the morphological case after dipping to a minimum at an eccentricity near 0.5-0.75 mm. For the functional case, Chapter 13 will discuss the “straight through signal path” believed to apply to the photoreceptors of the foveola. Using this concept, the functional ratio of photoreceptors to ganglion cells approaches 1.0 in the limit as the eccentricity approaches zero. As noted in Chapter 2, care must be taken in relating the density of photoreceptors to the spatial resolution of the eye and to the degree of physical convergence relative to the number of neurons in the optic nerve. The morphological concept of convergence, as expressed in the ratios of the above figure, does not account for the use of time-diversity and spatial diversity encoding in the visual system. By employing these concepts, the visual system is able to perform its normal functions without any loss of significant information. The physiological optics of Figure 3.2.2-10 Ratio of rods, cones and total photoreceptor the eye controls the spatial resolution of the system to cells to ganglion cells in a primate retina (rhesus monkey). a much greater degree than does the density of The data is a replot of a composite prepared by Wassle & photoreceptors. Thus, the curves presented as figure 5 Boycott based on morphological experiments. They only by Lee primarily describe the performance of the displayed the cone to ganglion ratio and showed it turning optics of the eye and not of the types of cells down near zero eccentricity. There are no ganglion cells enumerated129. within the foveola and the cone-to- ganglion cell ratio must rise to a morphological asymptote in this area. This work The above graphical relationships between the peak does not recognize the dichotomy of “rods & cones” in a values in human can be evaluated more precisely in functional context. Only the top line represents a functional Figure 3.2.2-11. This figure shows the size and cell relationship. On a functional basis, there is one displaced density near the fixation point of human. ganglion cell for each photoreceptor in the foveola. “A”, in Unfortunately, that figure is labeled as a section the lower left points to this functional asymptote. The through the inner segments of the retina. A section original densities were measured along the temporal through the outer segments would be more definitive. horizontal meridian, zero eccentricity represents the fovea. However, if the section were through the area of the ellipsoids, it would be descriptive of the entrance apertures of the outer segments. One must question whether the figure was obtained from a planar slice obtained using a microtome. If so, the image may include various zones of the retina as can be seen from [Figure 3.2.2-1]. A slice taken approximately 50 microns from the RPE would show Inner Segments in the central portion of the image and soma in the outer portion. Similarly, a slice taken at 25 microns from the RPE would show Outer Segments in the center and Inner Segments in the outer portion. Most of the literature, shows the Inner and Outer Segments to have the same diameter close to the fovea. Osterberg also shows that the first rods appear at approximately 0.16 mm. from the center of the fovea (0.13 mm. in the fixed retina). A more definitive change occurs at 0.05 mm. in the figure. Within this region, the very small inner segments are usually related to a “rod free zone.”

129Lee, B. (1996) Receptive field structure in the primate retina. Minireview. Vision Res. vol. 36, no. 5. pp. 631- 644 The Retina 3- 49

Figure 3.2.2-11 CR Horizontal section through a region of a fixed human retina containing the fovea whose exact center is at the intersection of the straight lines at the left. The section is through the inner segments. The actual dimensions of the unfixed retina were 1.31 larger than indicated. Pirenne (1948) indicated that cones are open circles whose diameter enlarges toward the parafovea; rods are seen as small black dots. Using Hogan’s dimensions, the foveola extends to 0.175 mm. from the center of the circle (0.13 mm on this scale). From Osterberg, 1935.

Roorda et al. have recently provided photoreceptor densities in the human retina using their new adaptive optics equipped scanning laser ophthalmoscope (AOSLO)130. The data is a great improvement over earlier measurements and assertions, Figure 3.2.2-12. No discussion of the role of rods and cones appears in their paper. Note, no measurements are provided from within the radius of the foveola, 0.6 degrees.

130Roorda, A. Romero-Borja, F. Donnelly III, W. Queener, H. Hebert, T. & Campbell, M. (2002) Adaptive optics scanning laser ophthalmoscopy Opt Express vol 10, pp 405-412 50 Processes in Biological Vision

Figure 3.2.2-12 Change in photoreceptor spacing with eccentricity. The circle symbols show the cone photoreceptor spacing as a function of eccentricity from the fovea. The long-dashed line shows anatomical data from Curcio et al. 1990 and the short-dashed line show psychophysical estimations of cone spacing from Williams. From Roorda, 2002.

Correlating these and various other comments in the literature concerning this data on morphological photoreceptor densities with the corresponding statements regarding the sensitivity of the retina is difficult. There are many casual statements in the scientific literature that the human eye is blind at night in the region of the fovea because of the lack of rods in this area. Such a statement is absurd. This area forms a circle with a radius of 3.2 degrees from the fixation point and has a diameter equal to nearly 10 moons. Does the statement include the region of the foveola as well? Is blind the right term? Would a more precise statement be that the sensitivity of the eye in the region of the fovea is limited to that of the cones? Based on the familiar adaptation curves, the sensitivity of “cones”is about two orders of magnitude less than that of “rods,” at night? This is also unrealistic. The photoreceptors of the fovea, and the foveola, provide the same psychophysical threshold for night vision as any other photoreceptors in the same eye, despite location. They may even provide slightly higher sensitivity under certain conditions because of the greater length of their Outer Segments. This greater length is due to the curvature of the Petzval Surface in the fovea.

Although the literature frequently gives the impression that the density of photoreceptors is much higher in the foveola than elsewhere, it is not in the human. The maximum density is restricted by the requirement that the diameter of the photoreceptor cell must be large enough, about 2.0 microns, to accept light without significant loss due to diffraction/refraction effects. This figure also illustrates that the density of cells cannot be calculated with great accuracy when only a few cells are present per unit area. Looking at the figures provided by Wassle & Boycott, Osterberg, and Pirenne as a group, it appears the cells in the human fovea are not less than one quarter the diameter of the peripheral photoreceptors and the density of photoreceptors in the fovea is less than 135 percent ( 147,000 divided by 110,000) of that in the parafovea. Looking at the figure from Osterberg, it does appear there is a slightly higher packing density of cells in the region from 0.1 to 0.175 mm. from the visual fixation point in the chemically fixed retina. This impression may be due to The Retina 3- 51

the printing/reproduction process. Cicerone & Nerger reproduce the Osterberg data in their figure 8131. Their figure confirms the above comment that the density of photoreceptors is near 147,000 per mm2 in the foveola and drops to about 1/3 of that density (cells rising to diametess of 165% of those in the foveola) beyond 0.2 mm from the center of the foveola.. Ahnelt132 has provided a similar black and white micrograph of the human retina in the central foveal region but without notation as to what plane is shown. It appears to agree very well with the image by Osterberg. It shows the foveola extending out to about 0.02 mm. from the center and a rapid increase in photoreceptor size (of about 2:1 in diameter) in the region from 0.02 to 0.05 mm. Dubra, cited above, has provided recent statistics on the in-vivo human retina using adaptive optics techniques based on the archaic rod-cone assumption. 3.2.2.3.3 Statistics of the foveola only

The foveola is a functionally distinct area of the fovea normally centered on the point of fixation. It is the critical area supporting the analysis, interpretation and eventual perception of the scene within the field of view of the eye. The foveola is generally circular with a diamter of 1.18 degrees. This diameter (equivalent to 175 cells) contains about 23,000 photoreceptor outer segments. The outer segments associated with the foveola are connected by individual direct neural circuits to the pretectum of the mid brain and the other elements of the Precision Optical System. The POS plays the critical role of causing fine tremor motions used to analyze a scene within the two- dimensional correlator of the pretectum.

This area also exhibits a significantly different level of performance due to the diameter, spacing and possibly effective absorption length of the outer segments. Figure 4 of Williams, although showing a slightly smaller diameter, illustrates this area under a given set of test conditions133. Putnam et al. have provided very precise information about the foveola and the location of the point of fixation134. They show the point of fixation is not correlated with their center of the foveola calculations. 3.2.2.3.4 Major axes of the foveola mosaic

While many investigators have studied the orderliness of the retinal mosaic and discussed this orderliness in terms of a close packed hexagonal array, results have been marginal. Yellott has used a Fourier transform technique to illustrate that there is no preferred axes of the chromophore containing outer segment mosaic in the foveola135. His data and the Fourier transform technique are discussed in Section 16.6.3. 3.2.2.3.5 Limiting resolution of the foveola of the retinal mosaic

The conventional wisdom has always treated the retina as a static imager made up of an array of individual photoreceptors. The assumption has been that the resolution of such an array is determined by the packing factor (typically close-packed hexagonal array) of that array. In the foveola, this packing is usually defined by a center-to-center cell spacing of three microns (based on an outer segment diameter of about 2.0 microns). The calculation of the limiting response based on this spacing using the Nyquist criterion has always given a calculated value of less than the measured psychophysical performance of the human eye. This has led to the concept of hyper-acuity. Unfortunately, this concept is not well founded. In the recent measurements of Williams shown in Figure 3.2.2-13, the disparity between the calculated Nyquist limit for an assumed outer segment diameter of three microns and the actual measured performance, is more than four to one136. See the original article for the

131Cicerone, C. & Nerger, J. (1989) The relative numbers of long-wavelength-sensitive to middle-wavelength- sensitive cones in the human fovea centralis Vision Res vol 29(1), pp 115-128 132Ahnelt, P. Kolb, H. & Pflug, R. (1987) Identification of a subtype of cone photoreceptor in the human retina. J. Comp. Neurology. Vol. 255, pp. 18-34 133Williams, D. (1985) Aliasing in human foveal vision. Vision Res. vol. 25, no. 2, pp 195-205 134Putnam, N. Hofer, H. Doble, N. Chen, L. Carroll, J. & Williams, D. (2005) The locus of fixation and the foveal cone mosaic J Vision vol 5, pp 632-639 135Yellott, J. (1982) Spectral analysis of sampling by photoreceptors: topological disorder prevents aliasing. Vision Res. vol. 22, pp 1205-1210 136Williams, D. (1985) Op. Cit. 52 Processes in Biological Vision

Figure 3.2.2-13 A measurement of contrast sensitivity versus spatial resolution in the human eye. The contrast sensitivity was determined using a lateral shearing type unequal path interferometer. The data points should not be considered a simple measure of the conventional contrast function. Modified from Williams, 1985. psychophysical conditions related to this data. Using a more precise treatment of the Nyquist criteria, a more reasonable fit between theory and performance can be obtained (see Section 16.6.3). Under this treatment, the diameter of the average outer segment is between 1.5 and 2.0 microns based on this measured data. The value of 2.0 microns has been used as a nominal standard in this work. It is consistent with the available micrographs of the foveola portion of the retina (shown earlier in this section). See Section 17.6.3 for an analysis of the test set used.

In the actual case of vision, the retina is not an array that is sampled periodically as in the case of a television sensor. Each of the photoreceptors in the foveola portion of the array is directly connected to the brain. Furthermore, the image of the scene is scanned across this photoreceptor continuously by the tremor of the eye (during the analysis intervals between the larger saccades). It is this scanning motion that generates the electrical signal that is transmitted to the brain. The motion results in a signal that changes with time. It is this change in the signal from a single photoreceptor that is of interest in determining the resolution of that channel. In this case, the Nyquist criterion is applied to individual outer segments and not to the elements of the mosaic The limiting spatial resolution of the retina depends on the diameter of the photoreceptors. For a nominal two micron diameter photoreceptor, the spatial resolution of the system exhibits a high pass characteristic with a limiting resolution of 250 cycles/degree. These values are for a square wave test pattern not filtered by the physiological optics of the eye. As in the case of any optical system, the response below the limiting resolution varies slightly for a sinusoidal test pattern. The impact of the physiological optics is always to convert the test pattern to a more sinusoidal form due to the response of the optics. Figure 3.2.2-14 shows the limiting resolutions of the human retina calculated under both the imager assumption (added by this author) and the change detector assumption. It is clear that the change detector assumption applied to a change detector model correctly describes the limiting resolution of the human visual system. Pask & Stacey have The Retina 3- 53

calculated a similar value based on a single detector model137. Their value using a 3.2 micron diameter waveguide was between 125 and 165 cycles/degree (see Section 4.3.4.2.1). There is no role for a hyper-acuity concept when using the change detector model and the Nyquist criterion. The data points in the figure are only useful in determining the limiting performance of the system. The test configuration developed by Williams does not provide a meaningful estimate of the contrast (spatial) function (CSF). The data points shown in the above figure are interesting by themselves. They represent the results of a test employing a sub-aperture illuminating test set with special coherent capabilities. See Section 17.6.3 for a discussion of the development of the CSF and the limitations of the Williams test configuration. The performance of the physiological optics of the visual system is discussed in Section 2.4, with particular emphasis on the human optical system. 3.2.2.4 Statistical parameters of the chromatic mosaics of the outer segments

To date, the investigations into the chromatic aspects of retinal arrays must be considered exploratory. There is no direct data connecting the data obtained by staining with the spectral response of the cells stained. Furthermore, all papers on the subject to date have assumed the human retina is trichromatic, in spite of excellent data to the contrary available since the 1970's (see Section 17.2.2). The most successful techniques employ differential staining that is very time and concentration sensitive. Whether the result is a bulk staining of the cell or a surface staining of the cell wall, or a material on the surface of the cell wall, is seldom detailed. In cases where the stain is introduced into the vitreous humor, how the stain penetrates the outer limiting membrane is also of concern. Laties, et. al. have provided the broadest discussion that could be found in this area138. This work takes the general position that all photoreceptors are identical in composition and surface coatings under quiescent conditions, except for the particular molecular structure of the chromophores. These structural differences are only recognizable at the molecular level. The molecules are essentially isomers of Rhodonine and are very difficult to differentiate based on conventional chemical procedures, such as staining. The Rhodonines do not include any amino acid groups in their structure.

Three results stand out from this study. All animals are theoretically capable of ultraviolet vision. All three phyla examined in this study include animals capable of ultraviolet vision. The human retina is capable of detecting ultraviolet light as demonstrated by aphakic subjects. However, the human optical system filters out nearly all of the ultraviolet light projected toward the retina.

Two principal methods of collecting statistics on the chromatic characteristics of the retinal mosaic are common. The physiological method is to employ microspectroradiometry in a reflective mode to observe the chromatic variation in the retina. The psychophysical method is to use short flashes of diffraction limited light sources to attempt to stimulate individual photoreceptors and record the perceived response of the individual. This method generally suffers from at least two complications. First, the method does not control the tremor within the eye. This tremor has a magnitude equivalent to at least the diameter of the typical photoreceptor OS. Second, the method does not recognize the differencing technique employed in the architecture of the visual system or the “paint” program used at the cognitive level of the visual system to determine the most likely chromatic content of an area.

The chemical staining method involves staining a retina either in-vivo or in-vitro and then analyzing it in-vitro. To date, this method has not employed spectroscopic methods to identify the stained photoreceptor cells. This has been generally attributed to the fact the dyes used tend to exhibit spectrums that would interfere with the measurement of the target cells. The method typically involves introduction of a dye into the vitreous humor and the assumption that the dye passes through the outer limiting membrane to dye some portion of the photoreceptors that is related to their chromatic performance. Very little statistics exist on the chromatic characteristics of the photoreceptor population of a retina. It appears no satisfactory criteria have appeared upon which to collect these statistics. So far, only one type of photoreceptor has been isolated by the dye technique. This type has been inferred to be the S-channel photoreceptor based on the putative mean density of S-channel detectors (typically 8-10%) from psychophysical experiments. It is suggested, based on this work, that the prevalence of S– and L–channel photoreceptors are nearly equal. If so, this criteria is

137Pask, C. & Stacey, A. (1988) Optical coherence and Wolf’s theory for electromagnetic waves J Opt Soc Am A vol 5(10), pp. 1688-1693 doi: 10.1364/JOSAA.5.001688 138Laties, A. Bok, D. & Liebman, P. (1976) Procion Yellow: a marker dye for outer segment disc patency and for rod renewal. Exp. Eye Res. vol. 23, pp 139-148 54 Processes in Biological Vision

not useful. As will be shown in the next two Chapters, all of the photoreceptors of vision are functionally the same. This is true for retinas, retinulas and ommatidia. There are, however, at least three operational differences in photoreceptors: + The form of the axon terminal varies depending on the complexity of interconnection with the signal processing neurons. This feature is not directly associated with chromatic performance. + The Outer segments of the photoreceptors vary in diameter with location in the mosaic. This parameter probably varies in response to optimization designed to the meet environmental requirements of the animal. It does not appear to be related to chromatic performance. + The photo/piezo material of, i.e., the chromophoric coating on the disks of the Outer Segments, is spectrally specific. Simple methods of determining the spectral properties of this material would lead to a significant increase in knowledge about eyes of different species and eyes in general.

All eyes can conceptually populate their retina with photoreceptors coated with one of four chromophores. An equal distribution would call for each type to be represented by 25% of the population. This is unlikely for a number of reasons. First, it appears that the population is strongly influenced by temperature during the gestation of the animal. Second, most eyes exhibit a mosaic that is not easily defined in terms of four quartiles.

Because the lens of the eye of humans (and other large chordates) absorbs most of the ultraviolet light available in sunlight, the performance of the overall visual system of humans is considered to be trichromatic. However, as is easily demonstrated in aphakic eyes, the human retina is actually tetrachromatic. It exhibits a high sensitivity in the ultraviolet spectral region between 300 and 400 nm. 3.2.2.4.1 Chromatic mosaics based on trichromatic

The majority of the work on isolating a specific subset of photoreceptors occurred during the 1980's. Subsequent to that time, various investigators have assumed the earlier work was conclusive. This work involved seeking a stain that would isolate the spectral varieties of photoreceptors. The investigations were based on the premise that the trichromatic assumption applied to the retina as it did to the overall performance of the visual system. No attention was given to the possibility that there were ultraviolet photoreceptors in the retina of humans and other primates. The summary of the situation presented in de Monasterio, et. al. is informative139. It can be divided into two areas.

They first discuss the relative density of the labeled cells. In the introduction, they say; “These stained photoreceptors were identified as the blue-sensitive cones because of their characteristic retinal distribution, large angular spacing, and different incidence in mammalian species (see Discussion).” In that discussion, they state; “Taken together, the preceding observations strongly support the admittedly indirect identification of the stained cones as blue-sensitive.” This position is followed by the statement that; “We have not examined the spectral sensitivity of the stained retina to demonstrate a loss of blue cone function [because the dyes themselves absorbed blue light].”

While describing the procedure as involving a differential staining, under carefully controlled stain concentrations, they describe the stain as being introduced into the intravitreal space in front of the neural retina. As they say, “While the trans-retinal transport mechanism was not elucidated, the dye normally did not stain other parts of the cone soma or postreceptoral cells at the concentrations used. . . .” Under the proper conditions, they claimed they achieved extracellular staining of all of the outer segment of all cones. They indicated their stains bind covalently with proteins as well as other compounds containing amino groups. In a slightly earlier paper by the same group, they describe the staining as at low concentrations as staining the extracellular compartment of the outer segment of cones. . . .140” They provided no model or description of “the extracellular compartment.”

139De Monasterio, F. McCrane, E. Newlander, J. & Schein, S. (1985) Density profile of blue-sensitive cones along the horizontal meridian of Macaque retina. Invest. Ophthalmol. Vis. Sci. vol. 26, pp 289-302 140McCrane, E. de Monasterio, F. Schein, S. & Caruso, R. (1983) Non-flourescent dye staining of primate blue cones. Invest. Ophthalmol. Vis. Sci. vol. 24, no. 11, pp 1449-1455 The Retina 3- 55

Kouyama & Marshak141 provide a topographical relationship between two mosaics in the same retina, one of “blue cones” and one of bipolar cells associated with the blue cones. Their isolation of the nuclei of the photoreceptors and the bipolar cells was based on staining techniques similar to those of the above authors. Kouyama & Marshak make the assumption that the inferences of the above authors concerning wavelength sensitivity were correct. They also rely upon the trichromatic assumption. 3.2.2.4.2 Chromatic mosaics based on tetrachromatic assumption

Both De Monasterio, et. al. and McCrane, et. al. were careful, except in the title, to stress the isolated subset of photoreceptors was only tentatively associated with the S–channel of vision. Many, more recent, studies have relied upon both the above inferences and the trichromatic assumption to support their conclusions. However, an alternate interpretation is available. First, any analysis based on statistics must recognize the presence of UV-channel photoreceptors in the human and primate retina (see Sections 5.5.10 & Section 17.2.2) as well as the putative presence of rods (if the rod-cone dichotomy is supported). If the retinas being examined were protected from ultraviolet radiation by the lens, there should be a subset of photoreceptors present that are in a different state of adaptation, and/or operating point. These cells are likely to exhibit a different sensitivity to differential staining than other operational cells. The glutamates used to support the operation of all photoreceptors through electrostenolytic mechanisms at the surfaces of the cells, contain amino acid groups. In general, these electrostenolytic mechanisms occur on the surface of both the inner segments and the dendrites found in the furrows of the outer segments. The aggregations of glutamates in these furrows clearly qualify as “extracellular compartments” of the outer segments. If the UV–sensitive photoreceptors are non functional due to the suppression of UV light by the lens, it is likely that the concentration of the glutamates will be higher in the immediate vicinity of these UV photoreceptors. Under this scenario, the cells isolated by the stains introduced by these authors are more likely UV-channel than S-channel photoreceptors.

There is a great need for conformation of the spectral sensitivity of the stain isolated photoreceptors, to include exploration of the ultraviolet region.

Liang, et. al. make the interesting observation that the various photoreceptors show different absorptions in their retinal recordings that are fixed with time. This would suggest that future laboratory efforts should attempt to map not only the location and size of the various chromophoric photoreceptors, but also the absorption of these cells. This could lead to the correlation of the absorption of these cells to their age or possibly how uniformly straight they are ( a factor in their waveguide characteristics, Section 3.6.2.3.3).

Chapter 12 will show that such variations in absorption, detected by reflectometry, are easily removed by the adaptation amplifiers of the photoreceptors.

3.2.2.4.3 Statistical parameters of the individual spectral channel mosaics

Prior to 2000, no demonstrably precise method of spatially isolating the photoreceptors of a specific spectral sensitivity has appeared. Only one method has shown consistently good isolation of a subset of cells of unknown spectral sensitivity. That technique has involved differential staining of an undetermined feature of certain cells. Beginning in 2000, the use of adaptive optical techniques integrated into an ophthalmoscope have provided exceptionally good data on the spectral channel array parameters of the retina. De Monasterio, et. al. have provided an excellent statistical analysis of the subset of photoreceptors isolated by their staining technique. They also provide a few comments on the crystallography they found and introduce tesselation and the concept of Voronoi (geometrical) regions. From their data they calculate several Nyquist frequencies associated with the arrays. They appear to use the designation “blue cones” in the captions to their figures while avoiding that label in their text. They were careful to caveat the equations they show in their paper. Recently, a number of papers have appeared discussing the distribution of the S-channel photoreceptors in the human retina. These must be carefully examined because they usually rely upon inference to determine what cells are actually sensitive to S-channel radiation. Many of them rely upon staining techniques developed largely independent of any spectrophotometric work. The best results appear to come from microspectroscopy. These will

141Kouyama, G. & Marshak, D. (1997) The topographical relationship between two neuronal mosaics in the short wavelength-sensitive system of primate retina. Vis. Neurosci. vol. 14, pp. 159-167 56 Processes in Biological Vision

be discussed in Section 3.2.3. Cicerone & Nerger have performed psychophysical experiments to determine the density and ratio of L and M cones using the common trichromatic assumption142. They did use test wavelengths of 520 and 640 nm to separate their M– an L– channel photoreceptors. Their model does not provide for either S–channel or UV– channel photoreceptors in the fovea and is therefore of questionable value. A team at the University of California, Irvine, carried on the Cicerone & Nerger work and has determined that, to the first order, the chromatic photoreceptors in human are randomly arranged143,144. Their experiments were psychophysical, based on a Hering model and only examined L and M cones. Their assertion that the distribution of the S cones in the human is well established in the literature appears weak. Their subsequent use of the words “consensus” and “disagreement about the presence or absence of cones in the human central fovea” suggest otherwise. No reference appears in these papers to tremor or its compensation. They do refer to microsaccades and the ability of the subject to suppress these through concentration. They also address the subject of hyperacuity without discussing its origins. One of their assumptions was perfect fixational accuracy. They employed 50 ms exposure times in the periphery experiments (17 degrees temporally from fixation) and 200 ms exposures in the fovea. The stimulus was a square, one minute of arc on a side for the fovea, 0.86 minutes of arc for the periphery. Assuming a photoreceptor diameter equal to 14 seconds of arc and an average tremor excursion of 1.5 photoreceptor diameters at a nominal frequency of 30 Hz, the image projected on the fovea is expected to excite approximately 36 photoreceptors based on the assumptions of paraxial optics, during the foveal exposure interval. Because of the length of the exposure, many cells near the periphery of the test image will be excited multiple times while those in the center remain excited for a majority of the interval. Use of a dim light further complicates the question of perception. The likelyhood of multiple triads of photoreceptors being excited adequately to be perceived during one trial appears to be quite high. The perceived color under such conditions appears to be a matter of chance. In the case of the peripheral experiments, the variable focal length of the optical system was not discussed. Whereas the photoreceptors are somewhat larger at this eccentricity (as they indicated), the focal length is somewhat shorter. The resulting photoreceptor size in object space is approximately the same. By using a much shorter exposure time of 50 ms, the situation is somewhat different. The same large group of photoreceptors will be illuminated. However, the image will only move about one third of a photoreceptor diameter in each orthogonal direction during exposure. The result appears to be the same, the perceived color of the spot will be determined primarily by chance. Their conclusion that the distribution of spectrally different photoreceptors in the retina is random appears based heavily on the test design and instrumentation and provides a different interpretation of the same effect observed by other experimenters. Those experimenters report flash to flash variability of perceived color for a one minute of arc test stimulus, as cited in section 1.2 of the second paper.

Stabell & Stabell have presented information on the relative spectral sensitivity of the retina at 45 degrees temporally from fixation in object space relative to the fovea145. They employed a variety of test methods used by others in an attempt to rationalize the data in the literature. Using a 2800 Kelvin source, the general findings were that the retina showed the same spectral performance at both locations after accounting for the absorption of the macula. This material continues to imply the interdigitation of spectrally selective photoreceptors is uniform over the useful extent of the human retina.

Neitz & Roorda hosted a special issue on Chromatic topography of the retina146. However, the papers are notably lacking in graphic models. In Martin, et. al., as an example, the S-channel photoreceptors are represented by their nuclei layer on the assumption that the nuclei layer is a monolayer. Their statistics are suspect based on this assumption. The micrographs of Sections 3.2.1 & 3.2.2 show clearly that this is not the case. Several papers investigating the ratio of L– to M–channel photoreceptors provide ratios between the two that vary over a range of

142Cicerone, C. & Nerger, J. (1989) The relative numbers of long-wavelength-sensitive to middle-wavelength- sensitive cones in the human fovea centralis Vision Res vol 29(1), pp 115-128 143Gowdy, P. & Cicerone, C. (1998) The spatial arrangement of the L and M cones in the central fovea of the living human eye. Vision Res. vol. 38, pp. 2575-2589 144Otake, S. Gowdy, P. & Cicerone, C. (2000) The spatial arrangement of L and M cones in the peripheral human retina. Vision Res. vol. 40, pp. 677-693 145Stabell, B. & Stabell, U. (1980) Spectral sensitivity in the far peripheral retina. J. Opt. Soc. Am., vol. 70, no. 8, pp. 959-963 146Neitz, J. & Roorda, A. (2000) Chromatic topography of the retina. J. Opt. Soc. Am. A. vol. 17, no. 3, pp 495- 650 The Retina 3- 57

ten or twelve to one. Such a range clearly shows the lack of an understanding of the architecture involved and the resulting problem, as suggested by Brainard, et. al. The papers are clearly exploratory, take no account of the ultraviolet photoreceptors found in human retinas (Section 17.2.2.3) and rely upon an inadequate adaptation protocol (Section xxx curve showing wavelength versus chromatic adaptation level xxx). Because the number of UV–channel photoreceptors may exceed the number for the L–channel, reported ratios of L– to M– may be quite questionable. While some of the papers hint at the return of the S–channel as a contributor to the luminous efficiency function, the tests were still run at very low color temperatures (typically 2850 Kelvin). The Brainard paper did introduce the luminous efficiency function as involving a logarithmic sum of the L– and M–channel signals as a function of wavelength. Alternately, it relied entirely on perceived responses to define a “unique yellow” at 574.7 to 576.8 nm. Their discussion of the relationship between photoreceptor spectra and genetics did not recognize the existence of UV–photoreceptors in the human eye. The use of adaptive optics equipped ophthalmoscopes has provided unprecedented data on the statistics of the human retina. References to the operation of these devices are provided in Section 18.4. Three significant papers have been published by the same basic team147,148,149. As noted in the titles of these papers, the authors did not consider the presence of UV–channel photoreceptors. Their spectral channel isolation protocol assumed that the S–channel photoreceptors could be isolated from the M– and L– channel photoreceptors before these two channels were further isolated. It can be assumed the UV–channel photoreceptors were combined with the S–channel photoreceptors in these studies. The M – and L–channel photoreceptors were separated with an error rate on the order of a few percent. In each of these cases, an area of the retina at one degree eccentricity was explored. Thus the following comments may not apply to the foveola. However, they probably do based on the paper by Calkins et al150. The statistical tests used were described in detail. The results of these studies were clear. “In all eyes, the M and L cones are arranged randomly. This gives rise to patches containing cones of a single type. In humans, . . , the arrangement of S–cones cannot be distinguished from random.” The clumping of photoreceptors of a specific spectral sensitivity can lead to very large differences in the ratio of L– to M– photoreceptors in a given area. The variability of this ratio is significant in confirming the architecture of the stage 2 signal processing within the retina (Section 13.5.3). The ratio varied from 1.1:1 to 16.5:1 in the Hofer et al. paper.

The method of describing the statistical results varies among the above paper. However, in the general situation, the percentage of UV– and S– photoreceptors combined is on the order of 5.5% of the total. After subtracting the UV– and S– photoreceptors, the percentage of L–photoreceptors has a mean of about 60% but varies from 27% to 94% among eight subjects. The remainder were M–photoreceptors.

If the investigators had been aware of the presence of UV–photoreceptors in the human eye, separation of the UV– and S–channel photoreceptors could have easily been accomplished using the same type of differential adaptation used to separate the M– and L–channel photoreceptors.

The fact the four statistically random arrays (including their clumps) are inter-digitated makes the description of the overall arrangement very difficult. It also makes it very clear that the photoreceptors of color vision are not arranged in an organized lattice of triads or tetrads.

3.2.2.5 The neural laminate RE-OUTLINE

This is the most complex laminate at the histological level. The neurons of each sublayer have immensely complex dendritic input and axonal output structures which are difficult to trace in detail. Interpreting the traces is also impossible without a clear understanding of how they interconnect and the characteristics of the signals carried by these connections. Wassle & Boycott151 have provided a large group of drawings showing the morphology of many different types of neurons found in these sublayers. They also provide information on the spatial position and

147Brainard, D. Roorda, A. Yamauchi, Y et al. (2000) Functional consequences of the relative numbers of L and M cones J Opt Soc Am A vol 17(3), pp 607-614 148Roorda, A. Metha, A. Lennie, P. & Williams, D. (2001) Packing arrangement of the three cone classes in primate retina Vision Res vol 41, pp 1291-1306 149Hofer, H. Carroll, J. Neitz, J. Neitz, M. & Williams, D. (2005) Organization of the human trichromatic cone mosaic J Neurosci vol 25(42), pp 9669-9679 150Calkins, D. Schein, S. Tsukamoto, Y & Sterling, P. (1994) M and L cones in macaque fovea connect to midget ganglion cells by different numbers of excitatory synapses Nature vol 371, pp 70-72 151Wassle, H. & Boycott, B. op. cit pp. 449-454 58 Processes in Biological Vision

density of these different types. 3.2.2.5.1 Signal paths through the laminate

Many authors have presented conceptual drawings of the signal flow through the neural laminate without correlating these paths with the characteristics of the signals involved and the capability of the various cells to accommodate these signals. The caricatures of Shepherd in 1974152, 1978, & 1979 are frequently reproduced in other works, including Shepherd of 1988. Hubel in 1988 presented an artistically attractive caricature153. Both the Shepherd version of 1974 and the Hubel version illustrate the apparent synapse of lateral cells, which exhibit no morphologically identifiable axon with the dendrites of bipolar cells or ganglion cells. They explained this situation by defining a new dendro-dendritic synapse. They also define bidirectional synapses at one junction between two cells of different type. These assumptions allowed them to define a typical signal path made up of typical elements to provide plausible solutions to a variety of primarily psychophysical tests. More recently, Daw has presented similar caricatures showing fewer directions of signal flow. Whereas earlier authors attempted to show a composite signal flow diagram, Daw divides his caricatures to define additional signal flow possibilities. His caricatures rely on two different types of cone bipolar cells and two different types of ganglion cells. These can be interconnected in a variety of ways to provide plausible solutions to the results of primarily psychophysical tests. His models depend on the ideas of “ON” and “OFF” responses in an apparent achromatic signal environment. No color specific data is offered in his model. There is little data in the electro- physical literature supporting both a depolarizing bipolar cell and a hyperpolarizing bipolar cell. The methodology used by Daw calls for both sign conserving and sign reversing signal transmission at different synapses. He also implies both sign conserving and sign reversing bipolar cells.

The above caricatures leave many questions unanswered. None of the authors mentioned above offers any explanation about how the signals are amplified, inverted, or otherwise manipulated within or between neurons. Unambiguous electro-physical data supporting the existence of any bipolar cell with a depolarizing output would be most interesting.

There are a variety of papers in the literature assigning the name bipolar cells to cells exhibiting either depolarizing or hyperpolarizing output signals based only on the fact that the cells were located within the inner neural layer. For a variety of reasons, the authors did not confirm that the cells they were reporting on were physiologically bipolar cells and not lateral cells of either the 1st or 2nd lateral matrix. These cells share a common morphological area and great precision is required to separate them on morphological grounds.

By examining the electro-physical data available on all of the neurons of the retina, defining the characteristics of the signals accepted and produced by each of them is possible. This includes the quiescent voltages and currents at each node, the polarity and amplitude of the output signals, and whether the output signals are electrotonic or pulse in nature. By studying the simpler neurons, determining how they operate internally is possible. See PART C.

With specific knowledge of the operation of the neuron in hand, an additional look at the problem of defining both the signal flow and the signal manipulation leads to a more definitive component organization. Defining a “fundamental signal path” as opposed to a typical signal path is the best way to accomplish this. The Fundamental signal path is a theoretical construct found only in the foveola. It is a straight through path involving a single photoreceptor cell, a single bipolar cell and a single ganglion cell. Schmitt154 described this configuration as a through-projection with long axon Golgi Type I neurons. Once this fundamental signal path is understood, defining a second path, a fundamental difference signal path, is possible (using short-axon Golgi Type II neurons). This is a path involving two photoreceptors, two bipolar cells, one lateral cell, and one ganglion cell. Once these signal diagrams are understood, expanding the ideas to any degree required to account for other signal paths in the retina is quite easy. The next level of neural circuit complexity in the retina is yet to be clearly understood. It involves very complex

152Shepherd, G. (1974) The synaptic organization of the brain : an introduction. New York : Oxford University Press 153Hubel, D. (1988) Eye, Brain and Vision. NY: W. H. Freeman pg. 52 154Schmitt, F. (1979) in 4th Study Program in Neurosciences. Cambridge, Mass. : MIT Press, pp. 9-10 The Retina 3- 59 summation and differencing mechanisms involving the signals from hundreds of adjacent photoreceptors. In earlier times, these interconnections were studied in a piece-meal fashion. These early studies employed stimulation patterns based on simple color patterns in a center-surround configuration. While these early color studies tended to isolate the diameter of the foveola, they provided little insight into operation of the visual system. More recent studies exploring the contrast improvement capabilities of the visual system offer additional insight. These studies have generally involved algorithms of the type first developed by Edwin Land in conjunctions with his Retinex Theory of Color Vision (Section 17.3.5.8). They suggest the primary purpose of these broader interconnections are to improve the contrast performance of the system in the presence of large intensity variations in the overall scene. These interconnections may be found primarily among the amercine cells of the retina. However, the minimal presence of these cells in the human retina suggests the interconnections are found mainly among the horizontal cells of the retina. Several underlying principles must be understood before the signal paths can be described. The understanding that is key is that every neuron contains at least one biological semiconductor amplifier, known as an Activa. The Activa is an active three terminal biological semiconductor device. Furthermore, every “gap junction” synapse forms an Activa. These devices can be analyzed using the same tools used for analyzing solid state semiconductor devices, transistors. Furthermore, recognizing that each lateral cell contains a neuritic structure and an axonal structure packaged within a single tubular cell membrane, known morphologically as a dendrite, is necessary. The overall configuration of a lateral cell is developed in Chapter 9. The most important observation is that each lateral cell has two input structures and one output structure. One input structure provides a sign conserving signal path and one input structure provides a sign reversing signal path. The morphological bistratified input structures in lateral cells have been documented by Dacey & Lee155. A similar bistratification has also been reported in ganglion cells by the same authors in 1994 (See Section 13.4.5).

By assuming that all photoreceptor cells provide a hyperpolarizing output signal in response to illumination, that all bipolar cells are sign conserving amplifiers, and that lateral cells can provide both sign conserving and sign reversing signal paths, two different classes of signal are presented to the ganglion cells. One is a monophasic signal of the same sign as that of the photoreceptors. The other is a biphasic signal that goes positive in response to illumination of one of the photoreceptors and goes negative in response to illumination of the other photoreceptor. This situation calls for two types of ganglion cells, one that responds to a monophasic signal and one that responds to a biphasic signal.

Note that the signals defined here do not involve inhibition. The process involves simple subtraction.

The signals from the ganglion cells are collected into the optic nerve and travel to a variety of locations in the brain as illustrated in Section 2.6.1. It will be shown that the ganglion cells that respond to monophasic input from the foveola connect to an area of the Pretectum within the brain. Those from outside the foveola connect to the magnocellular region of the LGN in the brain. The ganglion cells that respond to biphasic inputs from the foveola also connect to an area in the Pretectum within the brain. Those from outside the foveola connect to the parvocellular region of the LGN of the brain. It appears that the Pretectum is optimized for analyzing complex imagery brought to the fixation point of the eye.

Based on the above, the caricature from Hubel can be redrawn as in Figure 3.2.2-14 to illustrate both achromatic and chromatic inputs and to eliminate the need for a dendro-dendritic synapse. Before describing the circuits of this figure, some common points can be summarized;

+ All neuron cells are three terminal biological semiconductor structures. + All gap junctions are three terminal biological semiconductor structures. + All interconnections between cells are feed-forward circuits. + All bipolar cells are functionally alike. + There is no demonstrated need for external feedback circuits.

155Dacey, D. & Lee, B. (1995) Physiological identification of cone inputs to HI and HII horizontal cells in macaque retina. Invest. Ophthalm. & Visual Sci. vol 36, S3 60 Processes in Biological Vision

Figure 3.2.2-14 Caricature of the signal paths found in the retina of the Chordate, with nomenclature associated with the human. See text for explanation of the individual numbered situations. A; axon. D; dendrite. P; poda. These are the external terminals related to the internal terminals of the Activa found in each neuron. Para; parasol type ganglion cells which accept monophasic inputs but only generate a series of action potentials when the input is above a threshold level. Midget; midget type ganglion cells which accept biphasic inputs and generate a series of action potentials when quiescent. A highly modified version of a caricature in Hubel.

This basic configuration can provide, depending on the chromophoric character of the two photoreceptors, both “ON” signals, i.e., action potentials in response to illuminance, and more complex signals. These more complex signals can be described as “ON-Center, OFF-surround” for the achromatic case or “ON-Blue, OFF-Red,” for the chromatic case. By providing additional lateral cells connecting to adjacent photoreceptor channels, describing signals such as “ON-Center-Blue” and “OFF-Surround-Red” is possible. Additional interconnection can produce signals such as “ON-Surround-Parallel lines,” etc.

Every neuron contains at least one 3-terminal Activa. Leads to these terminals appear on the surface of the neuron. The Retina 3- 61

Two of these are the synapses associated with the dendrites and the axons. A lead to the third terminal may appear at the cell surface as a de-minima contact with the surrounding fluid matrix or it may be represented by synapses on a poditic structure that resembles a dendritic structure. For the straight through cell types (photoreceptor cells, bipolar cells and most ganglion cells) the third terminal of the Activa and therefore the cell, is not shown explicitly in caricatures. However, many caricatures in the electro- physiology literature show a current emanating from the Inner Segment of a photoreceptor cell, at some sort of terminal, and re-entering the Outer Segment. For the lateral cells, the horizontal and amercine cells, the third terminal should be shown explicitly since it is used as a signal input point. In the above picture, the dendritic, axonal and poditic structures are indicated by the letters D, A and P respectively. In addition, it is important to recognize that psychophysical experiments involving a center and a surround must specify whether the center is coincident with the fixation point or not. Different signal paths are involved for situations where the center is not at the fixation point. 3.2.2.5.2 Sign conserving amplifiers found in the luminance channels

#1 At the fixation point, there are straight through signal channels such as that labeled #1. The photoreceptor delivers a current to the bipolar cell through a gap junction. The bipolar cell acts as a signal repeater and delivers essentially the same current to the parasol type ganglion cell. Here, there is no sign reversal between the input and the output at either a cell or a gap junction. Although it is the current that is the signal, most investigators prefer to measure the voltage at a circuit node. They prefer to speak of this voltage as hyperpolarizing if it is increasing relative to the quiescent value and depolarizing if it is decreasing relative to the quiescent value. The signal at the gap junction between the photoreceptor and bipolar cells and between the bipolar and ganglion cells are hyperpolarizing. The ganglion cell is normally quiescent, but creates a series of action potentials with a time between pulses that is inversely proportional to the signal intensity, after reaching an initial threshold level. The time before reaching this threshold is usually labeled a latency interval. A parasol cell associated with a photoreceptor in the foveola transmits its signal via the optic nerve to the Pretectum. A parasol cell related to part of the retina remote from the foveola transmits its signal to the magnocellular region of the LGN. In the idealized straight through channel, the signal at every point along the chain will exhibit a spectral characteristic that is the same as that of the individual photoreceptor. This is a true chromatic signal channel.

Signals delivered to the Pretectum from the foveola are used to analyze and identify precise features of the target. This analysis is only possible in conjunction with the motions of the target relative to the line of fixation of the eye. This motion is normally supplied by the small saccades.

#2 Case #2 involves the summation of the signals, in the form of currents, at two points. Signals from many photoreceptors are summed at the dendritic structure of one or more bipolar cells. Signals from many bipolar cells are then summed at the dendritic structure of a parasol cell. Here, there is no sign reversal between the inputs and the outputs at either a cell or a gap junction. This type of circuit is generally found outside the foveola and particularly in the periphery where the ratio of photoreceptor cells to ganglion cells can reach 10 or more. It can provide very high sensitivity to large low contrast, or small high contrast targets in image space. The signal passed along the chain may exhibit spectral characteristics resulting from the summation of currents from photoreceptors of different chromatic types. The weighting given to the different spectral types is generally unknown. Therefore, the spectral performance of these signal channels can vary from that of a pure chromatic channel to that closely resembling the photopic or scotopic spectrums. The parasol type ganglion cell creates a series of action potential pulses and transmits this signal over the optic nerve to the magnocellular region of the LGN. 3.2.2.5.3 Amplifiers for both sign conserving and sign reversing paths

#3 A horizontal cell is used in case #3 to assemble the signals from many photoreceptors. The resultant signal is then passed to a midget ganglion cell that encodes the biphase signal for transmission to the parvocellular region of the LGN. Here, two dendritic structures are shown connecting to a variety of photoreceptor cells in both the surround and the center. In addition, additional signals are being collected at the poditic terminal through the same procedure. Signal currents collected at the dendritic terminal are summed without sign reversal and passed to the non-inverting input terminal of the Activa. Signals summed at the poditic terminal are also summed without sign reversal, but they are passed to the sign inverting input terminal of the Activa. The output signal from the Activa is an algebraic difference between the signals from the two input structures. It is a biphase signal relative to its quiescent value. In addition, the signal applied to the inverting input terminal may be amplified. Therefore, the output signal is of the form: 62 Processes in Biological Vision

A = D -k*P where k is the amplifier gain of that signal path. A is the output signal at the axon D is the input signal applied to the dendritic structure P is the input signal applied to the poditic structure This signal is passed to a midget ganglion cell that can accept such a biphase signal. This type of ganglion cell creates a series of equally spaced action potentials without any input signal. In the presence of an input signal, the spacing between the action potentials is increased or decreased as a function of the biphase input signal. Care should be taken to recognize that the output pulse train passed to the brain is not biphasic. Only the information it carries is biphasic when recovered in the Pretectum or LGN of the brain. #4 Situation #4 is very similar to that of #3 but the signals are summed at the outputs of various bipolar cells. In addition, the morphology of the amercine cells frequently gives no hint of the location of the axonal output. The axon is shown as next to and enclosed within the same cell wall as the dendritic structure, i.e., an internal cell membrane is separating the two structures for functional purposes. As in the previous case, the signals collected at the dendritic structure are passed to the non inverting Activa terminal. The signals collected at the poditic terminal, without sign reversal, are passed to the sign reversing input to the Activa. The signal at the output of the Activa and passed to the axon is biphasic as in the horizontal cell case. This signal is passed to a midget ganglion cell that is able to accept a biphase signal. It is treated the same as in situation #3.

#5 This situation is provided to highlight an additional capability. If a dendritic structure of either a horizontal or amercine cell is formed such that it bypasses certain cells in the surround to reach cells farther removed, it can provide highly tailored inputs to the brain. Thus, a pair of low contrast lines in object space with a width of four pixels each will provide an output signal to the brain that is larger than that from a single line of the same width but higher contrast.

The situations highlighted in #3 to #5 are frequently associated with lateral cells that are described as bistratified. This nomenclature is completely compatible with a cell with two input structures, one connecting to the sign conserving (non-inverting) signal input of the Activa, and the second one connecting to the sign reversing (inverting) signal input of the Activa. Dacey & Lee156 have provided several beautiful caricatures of lateral cells with this form. The types of signals defined in situations 1-5 have all been measured at the S-plane of the retina in a variety of animals. An additional situation may exist if some bipolar cells can accept signals at their signal inverting poda terminals. For that class of bipolar cells, the output signal would be biphasic compared with a quiescent level and would normally be passed to a midget ganglion cell that can handle a biphasic signal. Although the psychophysical community frequently assumes such a cell in their models, the electro-physical literature either does not report or is very ambiguous on the existence of this type of cell.

Only a few of the possible situations are highlighted above. Many other situations can be defined that account for such specializations as the reported capabilities of the cat to differentiate ruled patterns with different orientations. Specifically, it provides a framework for the work of Lee157 and others who are trying to understand the perceived resolution of the visual system based on measurements made at the level of the LGN. Although the title of the above mini-review by Lee implies a concentration on the retina, it is actually concentrated on the computational optics involved in both the retina and the LGN. It does not appear to consider the signal paths between the foveola and the Pretectum. Figure 5 of that paper, originally from Blakemore & Vital-Durand158, is interesting in that it provides a graph of spatial resolution as a function of eccentricity as measured at the LGN level. The graph displays a wide spread in data points in the region of low eccentricity. It should do this if many signals from the foveola are rerouted to the Pretectum. The graph is consistent with the data of Wassle & Boycott and the data of Osterberg presented earlier. The ratio of ganglion cells to photoreceptor cells does track the perceived resolution at the LGN although the horizontal scales are different. One graph used eccentricity measured in mm. across the retina. The other is probably using the eccentricity angle referred to object space. The data of Osterberg used angular eccentricity

156Dacey, D. & Lee, B. (1995) Physiological identification of cone inputs to HI and HII horizontal cells in macaque retina. Invest. Ophthalm & Visual Sci., vol 36, S3 157Lee, B. (1996) Minireview: Receptive field structure in the primate retina. Vision Res. vol. 36, no 5, pp. 631- 644 158Blakemore, C. & Vital-Durand, F. (1986) Organization and post-natal development of the monkey’s lateral geniculate nucleus. Jour. Physiol. vol. 380, pp. 483-486 The Retina 3- 63

referred to object space. Combining all of the above data, the spatial resolution of the sensing layer of the human (primate) retina falls by only about 20% at an object space eccentricity of 30 degrees. It is still 80% or more of the peak value. However, the spatial performance of the complete eye falls much more drastically for two reasons, the optics of the eye has much poorer resolution at this eccentricity and the signal processing within the retina is far less capable beyond the foveola. As a result, the spatial resolution measured in the LGN is reduced to about 10% of the peak value. These statements are consistent with the photoreceptor to ganglion cell ratio presented in [FIG 3.2.2-3] by Wassle & Boycott. Many temporal and chromatic effects noted in Lee’s paper are also compatible with the above framework. One of the important facts highlighted by the differencing equation given above relates to the measurement of temporal frequencies at the LGN level in response to stimuli that are poorly defined spectrally. By holding the illuminance level constant and changing the spectral content, it is possible to change the temporal frequency characteristics measured at the LGN quite drastically. Alternately, it is possible to choose a stimulus with the proper chromatic spectrum so that the difference signal path terminating in the parvocellular region of the LGN will show zero response to significant changes in illumination. This result implies a passband of zero hertz, i.e., no signal transmission due to this stimulus even though the eye is fully functional. Valberg et. al.159 performed a similar experiment involving the magnocellular region of the LGN but using a moving transition between two colored regions in object space. Although the signal paths of the retina are all direct coupled in the electrical sense, great caution must be taken in designing test instrumentation. The highly nonlinear gain characteristic of the initial photoreceptor transduction and translation circuits must be taken into account. These circuits remove nearly all indications of the absolute illumination level applied to the various photoreceptors from the signal path.

Heynen & van Norren160 have provided very detailed probe data on the signal found at different levels within the in- vivo retina of the macaque monkey. They appear to expand the data base provided by Svaetichin, Tomita, and others working in the 1960's, considerably. This data is very helpful in confirming the signal path architecture developed above. However, without a putative architecture, the data is very difficult to reduce. Additional discussion of the signal circuitry of vision will be found in Chapter 11. 3.2.2.5.4 Cell configurations within the laminate

The cell structures found within the neural laminate take on an amazing complexity and variety. Because of this variety, analyzing the operation of these cells in detail is very difficult. Dacey and Lee have provided many beautiful caricatures of these cells that provide fertile material for thought. The minireview by Lee referenced earlier has an excellent bibliography current to 1996. Unfortunately, the review becomes entangled in the old discussion based on whether cells exhibit a difference signal related to blue-yellow or blue-green. Time will not be spent here rationalizing the data in that paper by reinterpreting all comments in terms of S-channel minus M-channel differences. 3.2.2.5.5 Axon sizes within the Optic Fiber Layer

The most exposed surface of the neural laminate is the optic fiber layer. This layer is nominally 100 microns thick over the surface of the neural laminate closest to the vitreous humor. Between this layer and the vitreous humor is the Inner Limiting Membrane. Most of the larger vascular elements supporting the neural layer are located between the Inner Limiting Membrane and the vitreous humor. With axon diameters typically in the 5-12 micron range, the Optic Fiber Layer is able to support many layers of neural axons as they course toward the Lamina Cribosa. Little information could be found that attempted to map the individual subsystem layers defined above. Experimentation is needed to inject dyes into the various groups of neurons in the optic nerve and trace the transport of those dyes into the neural laminate.

159Valberg, A. Lee, B. Kaiser, P. & Kremers, J. (1992) Responses of macaque ganglion cells to movement of chromatic borders. Jour. Physiol. Vol. 458, pp. 579-602 160Heynen, H. & van Norren, D. (1985) Origin of the (L)ERG in the intact macaque eye. Vis. Res. vol. 25, pp. 697-715 64 Processes in Biological Vision

3.2.2.5.4 Cell sizes within the laminate

Collection of reliable data on the size of neurons and neuron elements is difficult. Few investigators observe enough cells of an individual proposed type to gain statistically relevant size information. Frequently, the proposed cell types appear to overlap to a considerable degree. Kolb161 provides a variety of dimensions and ratios that can be illuminating. Of particular interest is the claim that the ratio of ganglion cells to photoreceptors in the fovea is between two and three to one. Further distinction between the foveola and the fovea, as defined herein, would be even more enlightening. Unfortunately, the paper also includes the recitation of a long list of conventional wisdom in the Introduction and a large amount of speculation involving the words must, should, likely to be, etc. In general, these speculations appear to be consistent with the findings of this work when the circuits involved are recognized to be analog in nature. 3.2.2.5.4 Hydraulic elements within the laminate

The neural laminate is a densely packed region of living and signaling tissue, including the soma associated with the photoreceptor cells. It must be adequately supplied with nutrients. If it is not adequately supplied, deterioration in the signaling capabilities of the visual system can be expected to occur rapidly. The neural laminate can be described as a homogeneous hydraulic bed impregnated with a large number and wide variety of individual living cells and supported by a complex network of hydraulic conduits. These conduits all emanate from the main artery entering via the optic nerve. Their size decreases continuously as the lineal distance from the optic nerve increases along the conduit. This leads to a more limited supply capability with distance. Figure 3.2.2-15 illustrates the situation and is taken from a comprehensive description given by Hogan162. The image is in a sense a double exposure because it shows both the arterial and the venous systems overlaid. Although the individual conduits are continuous and cannot truly be modeled as a lumped constant system, using a simplified lumped constant model for study in such a reticulated system is possible. The supply capability at a given point can then be described in terms of an RC time constant associated with the impedance of the channel feeding the nearest reservoir and the capacity of that reservoir. The hydraulic bed also has an intrinsic time constant. Because of this situation, the signaling capability of each neuron of the retina exhibits a temporal response that can be described by at least two time constants describing the hydraulic system in its immediate vicinity.

The competition for nutrients is particularly important in the region of the fovea. Each neuron must compete for nutrients and energy with other neurons in the vicinity. Because of the lack of conduits in the immediate vicinity, the capability of supplying these nutrients is largely controlled by the time constant of the hydraulic bed. This limitation is one of the principal causes of tunnel vision ( a symptom of fatigue) and the after image effects associated with the eye. This is particularly obvious regarding the after images delineating the foveola from the rest of the retina. 3.2.2.6 Subdivisions of the neural laminate

The literature supports the subdivision of the neural Figure 3.2.2-15 CR Capillary bed in the neural laminate laminate into a single series of sub-laminates on both behind the macular region. The area shown corresponds physiological and morphological grounds. Using the closely to the parafovea of the eye. The capillary free zone terminology of Boycott & Dowling, the neural (a) corresponds to the foveola and is the pattern seen by laminate extends from the Outer Limiting Membrane most people in the first afterimage. From Hogan. (OLM) to the Inner Limiting Membrane (ILM). In doing so, it includes the nuclei of the photoreceptor cells found in the outer nuclear layer, all of the signal processing cells of the inner nuclear layer and the signal projection neurons of the ganglion cell layer and all of the interconnections between these cells.

161Kolb, H. (1991) Anatomical pathways for color vision in the human retina. Visual Neurosci. vol 7, pp. 61-74 162Hogan, M. Alvarado, J. & Waddell, J. (1971) Histology of the human eye. Philadelphia, PA: W. B. Saunders pp. 508-522 The Retina 3- 65

The inner nuclear layer can be further expanded. The names suggested for these sub-laminates in this work are descriptive. In order, the 1st lateral laminate supports lateral matrix signal processing based primarily on the class of lateral cells known as horizontal cells. The neurites of neurons in this layer connect directly to the axons of photoreceptor cells. This sublayer consists of multiple arrays of neurons as will be discussed in Section 3.4.2. The data does not show whether these arrays within the sub-laminate are layered. The axons of the most proximal of these arrays probably connect directly to ganglion cells. However, the data is not conclusive in this area. The second sub-laminate contains primarily bipolar cells and is therefore labeled the bipolar laminate. The dendrites of the bipolar cells connect primarily with the axons of photoreceptor cells and their axons connect directly with ganglion cells and probably also neurites within the 2nd lateral laminate. The 2nd lateral laminate supports additional lateral matrix signal processing. As suggested, it appears that most of the neurites of these cells connect to bipolar cell axons. However, the literature is not decisive on this point. This laminate may also contain multiple arrays of neurons that may or may not be arranged as sub-sub-laminates. The axons of at least the most distal of these arrays connect to the neurites of the ganglion cells.

Euler & Wassle have recently provided a set of caricatures describing putative bipolar neurons of two types163. While based on the chemical theory of the neuron and largely exploratory, it is useful in understanding the paths of bipolar neurons through the retina. While they define bipolar cells believed to serve both cone and rod signal paths, it is notable that the resting output potentials of the two types are statistically the same (rod-based, –45 mV ±13 mV; n = 21 and cone-based, –49 mV ±10 mV; n=38). Their method of cell identification relied primarily on the geometry of the photoreceptor axon pedicle. Figure 3.2.2-16 reproduces their figure 1.

Figure 3.2.2-16 Caricatures of bipolar neurons within the retina of rat. The labels are conventional. It is proposed the bipolar neuron labeled RB is actually associated with the R-channel of vision and found in the peripheral retina. Dendritic input (only) from a variety of photoreceptors. It is proposed the bipolar cells labeled CB, serve a multitude of functions associated with the chromatic and spatial signal processing channels of rat vision. The two arrows on the left denote the stratification of the neurites of amecrine cells into their dendritic and poditic ramifications. Scale bar, 60 μm. From Euler, et. al., 1996.

This work has not found any documented case of functional external feedback proceeding toward the distal part of the retina (negative feedback) from any neurons in any of the above layers.

3.2.2.6.1 Spatial parameters of the mosaics of the ganglion cells in cat

Wassle, et. al. have provided good caricatures and statistical data on the mosaic of “alpha ganglion cells” in the retina of the cat164. The data shows the cells aggregate in the location of the fovea (diameter of under three mm) of the cat as they do in human retinas (compare their figures 6A and 6B). They did note a slight aggregation along the horizontal and vertical axes through the fovea. They did not define the spectral performance of these cells but noted

163Euler, T. Schneider, H. & Wassle, H. (1996) Glutamate responses of bipolar cells in a slice preparation of the rat retina J Neurosci vol. 16(9), pp 2934-2944 164Wassle, H. Levick, W. & Cleland, B. (1975) The distribution of the alpha type of ganglion cells in the cat’s retina J Comp Neurol vol. 159, pp 419-438 66 Processes in Biological Vision

their large size relative to other ganglion cells (described as beta and gamma types). Their method of identification was limited to cytological features. 3.2.3 Fine histology of the photoreceptor layer of the retina

There have been a wide variety of experiments performed to determine the spectral performance of the human eye as a function of spatial position. As hinted at above, these have been performed at all levels of spatial resolution, the gross level relating to large areas of the retina, experiments involving regions from 1-2 degrees down to 15 minutes of arc, experiments examining the spatial arrangement of at most 25 chromatically mixed photoreceptors, and attempts to define the location of individual chromatic photoreceptors. There are a variety of spatial maps purporting to show the spectral performance of the retina at spatial resolutions of about one degree in object space. Unfortunately, these have usually been prepared based on psychophysical data taken using wideband spectral filters that are not well correlated to the actual absorption spectra of the eye. More recently, some data has appeared based on the use of specular sources for the test image but these frequently employ a broadband surround lighting that is again poorly correlated to the spectra of the chromophores of the eye. At the next level of spatial resolution, Williams, et. al165. have provided a set of maps of chromatic sensitivity using a flashing spectral target, of 1.1 arc minutes diameter, and a broadband surround. Separate maps were obtained for several individuals under the same conditions. The variability of these maps with location suggests the difficulty of using statistical measures to define the spatial performance of the eye to either achromatic or colored lights. The flashing target was programmed to form an 11 x 11 element array 50 arc minutes on a side and centered on the fixation point. The grid spacing was therefore 5 arc minutes. The maps obtained are very useful but it is not entirely clear what they represent. As discussed in summary in Chapter 1, the combination of the luminance and chrominance signal processing channels of vision and the spatial motion of the line of fixation must be considered when determining what the visual system perceives. Only then is it possible to estimate what the system will recognize and report in a psychophysical experiment. These considerations lead to an entirely different explanation to one situation reported perplexedly by that team. Speaking of the area immediately surrounding the fixation point, they report: “Test flashes at location throughout this region appeared white and sharply defined whereas those falling in the more sensitive outlying regions appeared violet and diffuse . . . ” They concluded that this was evidence of no short wavelength photodetectors in the center of the fixation zone generally defined as the foveola. In the context of this work, the results reported would be highly subject to the difference in intensity between the test target and the background and to the spatial signal encoding by the signal manipulation stage of the retina as well as the spatial encoding of the signal projection stage leading to the visual cortex. In the most obvious example, whenever the signal intensities were such that the logarithmic difference, representing the integral of the flux on each photoreceptor times the adaptation amplifier gain, between them was zero, no chrominance data was transmitted to the visual cortex and the subject would report the test target as white. At other levels, the test target would initially be reported as the same color but less saturated than the background surround, then white and eventually violet.

The above experiments did not attempt to illuminate or resolve individual photoreceptors. Attempts to determine the statistics of the retinal mosaic at the photoreceptor level have remained elusive. The introduction to the paper by Ahnelt, et. al166. suggests the problem and the state of the art. They do not recognize the possibility of UV sensitive photoreceptors in humans (following the archaic pattern of quoting the elementary proposal of Young in 1802-03), although documented in aphakic patients. They also stated in 1987 that: “The cones differ in having different photopigments and different neural connectivity, but no morphological differences with which to distinguish the three different spectral types have been reported yet.” They proceed to postulate that a morphologically definable group of photoreceptors they have studied are likely to represent the blue-cones of vision. The group they propose is unique in several characteristics. However, they are also unique in one critical parameter. As noted, these cells are not long enough to reach the RPE. This fact has several consequences. Because they are not in contact with the RPE, they may not be fully operational. They may be either juvenile or pathological. What is more important, their analysis does not recognize the growth dynamics of normal photoreceptors. Because of the normal growth rate of the disks of the Outer Segment, they cannot exist for more than about 2,000 hours, the time for a normal disk to progress from the extrusion point within the Inner Segment cup to the phagocytosis point within the RPE. As a result, a “short photoreceptor” is not in a stable configuration. If the cells of the proposed subgroup were normal

165Williams, D. MacLeod, D. & Hayhoe, M. (1981) Punctate sensitivity of the blue-sensitive mechanism. Vision Res. vol. 21, pp. 1357-1375 166Ahnelt, P. Kolb, H. & Pflug, R. (1987) Identification of a subtype of cone photoreceptor, likely to be blue sensitive in the human retina. J. Comp. Neurol. Vol. 255, pp. 18-34 The Retina 3- 67

cells in a real eye, they could not be found again or identified after a few weeks.

A collage on page 42 in the introductory text of Rodieck167 is being referenced in primary papers without adequate description. A serious reader should research the underlying papers. This theory proposes that there are no significant differences between photoreceptors at the morphological or cytological level. There is a functionally identifiable difference in the isotropic absorption spectrum of the chromophores and this difference is caused by a difference at the molecular level of the chromophores (only). This identifiable difference is in the length of the conjugated chain between the two auxochromes. Unfortunately, this difference is physically masked by the configuration of the liquid crystal when operational. It is probably only measurable in the crystal via molecular resonance spectroscopy. A variety of microspectrophotometry, MSP, techniques have been used to determine the characteristics of individual photoreceptors in various animal retinas. However, they have also failed to provide conclusive mosaic maps or statistics related to such maps. The task is difficult. For axial MSP, the background for the measurements is not well defined. There may be red blood cells forming the background. Alternately, the chromogens located in the RPE may form an undefined or mixed background. Attempts to mark suspected photoreceptors for subsequent MSP have failed until recently because the illumination was applied to the individual photoreceptor cell transversely. The result was always the isotropic spectrum of the chromophore present. This spectrum, although not used in vision, always has a peak at 500 nm. Very recently, correct anisotropic spectrums have been obtained using axial MSP on individual photoreceptors. However, this has not yet led to a method for characterizing a large piece of or the entire mosaic.

3.2.3.1 The archaic representations of Curcio et al. showing inner segment ellipsoids

This section can be discarded. The recent work of the Roorda team during the 21st Century demonstrates in- vivo at the sub-micron resolution level that there are no rods in the human retina. xxx ] Curcio et al (1990)168 have followed the in-vitro procedures of Shultze and later Osterberg. They have prepared whole mount retinas and then sliced through them parallel to their surface layer (at the exterior limiting membrane) in order to expose various surfaces within a specific lamina (relative to their reference surface and not relative to the Petzval surface). The results are primarily images of inner segment layers which are more stable physically and easier to obtain than cross sections of outer segments. Their figure 1, and their text, also make it clear that the outer segments are generally skewed relative to their reference plane (between 20 and 45 degrees in the figure).

Curcio et al. make no effort to identify their rods and cones by spectral means. They rely entirely on their assumption that cones are larger than rods at the level of the ellipsoids of the cells. No reference is made to the properties of the outer segments. Their images lack detail at the micron level due to their use of Nomarski differential interference contrast microscopy combined with focusing at an intermediate level within the neural matrix at the level of the ellipsoids.

Curcio, et. al (1991)169. addressed the distribution problem differently. They review the previous art and then say in 1991: “Many of these methods are inferential, and cells are presumed to be blue cones by virtue of the similarity of their distribution to that suggested by visual psychophysics (page 611).” Although they were careful to preserve the spatial fidelity of their test retinas, their approach was also inferential. They also note, “morphologically identified cones are 3-fold or more numerous than immuno-cytochemically labeled cones.” (page618). Their conclusion on page 622 is also interesting. They say, “Our results for immuno-cytochemically labeled human blue cones are consistent with a nonrandom distribution outside the rod-free zone.” They lumped all photoreceptors other than those associated with the S–channel under the heading R/G cones (based on the conventional assumption that the retina is only trichromatic. They employed materials that were thought to stain certain cells preferentially. Using a more detailed model, many alternate conclusions can be drawn from their approach. They determined that a circle could be drawn encompassing the central 100 microns of the foveal center that did not contain any cones. However,

167Rodieck, R. (1998) The First Steps in Seeing. Sunderland, MA: Sinauer Associates 168Curcio, C. Sloan, K. Kalina, R. & Hendrickson, A. (1990) Human photoreceptor topography J Comp Neurol vol 292, pp 497-523 169Curcio, C. Allen, K. Sloan, K. Lerea, C. Hurley, J. Klock, I. & Milam, A. (1991) Distribution and morphology of human cone photoreceptors stained with anti-blue opsin. J. Comp. Neurol Vol. 312, pp. 610- 624 68 Processes in Biological Vision

their figure 3 suggests that the “blue-cone” and the “red/green cone” classes they have identified suffer from some of the same problems as those of Ahnelt, et. al. The photoreceptor shown appears either juvenile or pathological. Their paper makes a herculean effort to sort out the conflicting statements, and anomalous situations reported in the literature about various classes of photoreceptors. However, their results are primarily diagrammatic and statistical. Both the Curcio et al (1990) and Curcio et al. (1991) papers note they were not photographing the photoreceptor outer senments. In figure 1 of the 1990 paper, frames B & D are of the ellipsiod within the inner segments. Their frequently referenced figure 1, frames (B) & (C), of the 1991 paper do not show the cross section of the outer segments of the photoreceptors. They are slices through the inner segments of whole mount retinas near the ellipsoid, (B) just vitread to the junction of the outer and inner segments and (C) near junction with the myoid. As a result, the figures are similar to that of Schultze in 1866. The large elements tend to be ellipsoids containing the nucleus of the cell, and the small elements tend to be smaller diameter portions of the inner segment or “axon” segments leading to the nuclear layer. Only their frame (A) actually shows outer segments. Ahnelt170 reiterated the fact, in a 1998 review, that “So far no morphological criteria for direct differentiation of L– and M– [photoreceptors] have been reported but there may be differing connectivities along their midget pathways.” Their work is also based primarily on the location of the “inner segments” of the photoreceptors. It is not necessary for the inner segments of a photoreceptor to be in the optical path associated with the center of the foveola for the outer segments to still be present within this diameter (See Section 4.3.1) . Therefore, there conclusion that there are no blue outer segments within a 0.35 degree diameter area, presumably centered on the point of fixation, may be questioned. It is surprising that they did not reference the work of McCrane, et. al171. McCrane, et. al. provide beautiful pictures of inner segments stained by procion yellow and other dyes. However, they again rely upon inferential evidence that their pictures correlate with a presumed average of 7% blue cones within a retina. Their figures also focus on the inner segments of the photoreceptors although they make the interesting observation that “Procion yellow stains the extracellular compartment of the outer segment of cones, . .”

Curcio, et. al. make a number of observations concerning the orderlines of the S–channel photoreceptor array.

The 1991 paper of Curcio, et. al. and McCrane, et. al., and their references generally, fail to show that the inner segments of short wavelength photoreceptors are different, in any way, from the inner segment of other photoreceptors. The stained inner segments could just as well belong to the UV cells of the retina. This would be especially likely if these cells had degenerated from lack of stimulation by UV light (See Section 17.2.2). 3.2.3.2 The more recent work of Roorda & Williams showing the retinal face

Roorda & Williams172,173 have recently provided small area photomicrographs of in-vivo human retinal mosaics from “normal” trichromats. They used an available test set described in Liang, et. al. It used adaptive optics to compensate for the distortions encountered when attempting to focus a camera through a non spherical optical system174. Although they were aware of the natural tremor, it does not appear that they compensated adequately for it.175 The resulting pictures appear slightly out of focus. Each photoreceptor is imaged as a cylindrical structure. There are a variety of reasons why such an image should arise. One of them is because of the tremor of the eye and the exposure interval employed. There is some concern as to whether they took adequate steps in experimental design to account for any UV sensitive photoreceptor channels (see discussion of the aphakic human eye elsewhere in this work) in the retina and for any chromatic reflection from the retinal epithelium. They published pseudo-color images of a small part of the human retinal mosaic eccentric to the fixation point, within the fovea, and within the

170Ahnelt, P. (1998) The photoreceptor mosaic. Eye, vol. 12, pp 531-540 171McCrane, E. de Monasterio, F. Schein, S. & Caruso, R. (1983) Non-flourescent dye staining of primate blue cones. Invest. Ophthal. Vis. Sci. vol 24, no. 11, pp 1449-1455 172Roorda, A. & Williams, D. (1999) The arrangement of the three cone classes in the living human eye. Nature, vol. 397, Feb 11, pp. 520-522 173Roorda, A. & Williams, D. (1999) Probing the amazing human retina Biophotonics International, May/June, pp. 40-41 174Liang, J. Williams, D. & Miller, D. (1997) Supernormal vision and high resolution retinal imaging through adaptive optics. J. Opt. Soc. Am. A vol. 14, no. 11, pp. 2884-2892 175Roorda, A. (Private communication, June 1999) The Retina 3- 69

macular region. The caption to the black and white pictures in their Figure 1 is informative: “The mosaic was illuminated with a 4-ms flash (1 degree diameter, ~0.3 μJ) through a 2 mm. entrance pupil. Light of wavelength 550 nm was used to maximize absorptance by L- and M-cone photo-pigments. Images were obtained with a 6-mm exit pupil at a retinal eccentricity of one degree nasal from the foveal center, which is located to the left of the image. For each retinal location, about 50 images taken over 5 days were averaged.” The color images presented in two different publications have been cropped differently making comprehensive interpretation more difficult. The 4-ms exposure time is marginal in the presence of a nominally 20-ms period, 50 Hertz (30 to 90 Hertz range in literature), tremor. With a peak amplitude of 20-40 arc sec for the tremor, approximating 1 to 2 photoreceptor diameters, a 4-ms exposure would be expected to result in a blurring of the edges in the photographs of about 1/4 to 1/8 of a photoreceptor diameter. The blur in the imagery presented in Figure 3.2.3-1 is consistent with the above temporal and geometric ratios. The additional blurring associated with averaging of 50 individual recordings was not addressed by this author. In the figure, A & B are subject JW temporal and nasal retina, respectively at one degree of eccentricity from the fixation point. C is subject AN’s nasal retina, at one degree of eccentricity. One of Roorda & Williams obvious conclusions was that the retina in this area did not represent a mosaic of three uniformly spaced and interdigitated photoreceptor types. In fact there is no indication of uniformity in the mosaic associated with a given chromatic channel as depicted in their paper. The authors did not address the presence of any achromatic photoreceptors (“rods”) in their imagery in the paper. However, Roorda stated in the private communication that they did not observe a single “rod.” In a second communications, the authors confirmed that “their measurements were made at a retinal angle of one degree and that all of the color centers are cones and that therefore no rod receptors appear in the views.” It must be pointed out the data presented by Wassle & Boycott and shown in [Figure 3.2.2-3 ] calls for a number of achromatic “rods” equal to the sum of all chromatic “cones” at this position in the primate retina! Their data is apparently based on morphological analyses of in-vitro retinas, probably Inner Segments, and not functional analyses of in-vivo retinas. Roorda & Williams did not address the question of any UV sensitive photoreceptors in the human retina.

It should be noted that the experiments of Roorda & Williams would not require compensation for tremor if performed on many animals that do not employ tremor. It would also be useful to perform these experiments on some animals, such as cats, who appear to be able to control the muscles generating the tremor.

Roorda & Williams176 have employed a retro-reflective spectrophotometric approach (70 nm wide filters for the 550 nm source) to the mapping of a small portion of the in-vivo human retina and have provided some limited statistics on the distribution of some photoreceptor Outer Segments that are useful. As discussed above, a redefinition of their experimental procedure following their exploratory effort could lead to better and more complete data. Until such a technique is perfected, the statistical parameters of all retinas will remain elusive.

The Williams team have continued to be very active during the early 2000's and considerable improvement in their techniques have resulted177. The work of Christou, Roorda & Williams is of particular relevance178. Although their ability to record the mosaic of photoreceptors has improved immensely, they still have not recorded the presence of any “rods” in these mosaics. Their recent paper employed off-line processing to cancel out eye tremor179.

It appears clear from the exploratory work of Roorda & Williams, and all of the above statistical work, that the retina is not organized in an orderly array of interdigitated photoreceptor arrays based on chromatic sensitivity. The brain uses a different method of determining color rendition than the concept generally used by man in the arts, creating of three separate images related to the colors RGB or CYMK. This method will be discussed in more detail later in this work.

At the sub-array level, Bowmaker & Kunz180 have studied small groups of photoreceptors, primarily in fish, morphologically in order to determine if there is a fundamental array in the photoreceptors of Chordata similar to the

176Roorda & Williams, Op. Cit. 177Pallikaris, A. Williams, D. & Hofer, H. (2003) The reflectance of single cones in the living human eye IOVS vol 44(10), pp 4580-4592 178Christou, J. Roorda, A. & Williams, D. (2004) Deconvolution of adaptive optics retinal images J Opt Soc Am A vol 21(8), pp 1393-1401 179Yang, J-Z. Nozato, K. Saito, K. Williams, D. & Roorda, A. (2014) Closed loop optical stabilization and digital image registration in Adaptive Optics Scanning Laser Ophthalmoscopy Biomed Optics Exp vol 5(9), pp. 3174-3191 https://doi.org/10.1364/BOE.5.003174 180Bowmaker, J. & Kunz, Y. (1987) Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the brown trout (Salmo trutta): age-dependent changes. Vision Res. vol. 27, no. 12, pp. 2102-2108 70 Processes in Biological Vision

arrays found in the individual ommatidia of Arthropoda. Although their results do not concentrate on the lattice structure for the chromatic photoreceptors present, they did find a deterioration in the lattices with aging in young trout which they attribute to the loss of UV-sensitivity of these animals with age. Whereas, their caricatures generally show eight photoreceptors, of varying sizes grouped about a center photoreceptor, most other authors tend to describe a fundamental mosaic of six nearly equal sized elements grouped around a central element–a hexagonal unit. The difficulty with many of these caricatures is that they are generally based on micrographs of sections through the inner segments of the photoreceptor cells and not sections through the outer segments. 3.2.3.3 Putative arrangement of photoreceptors in the human eye

If the same architectural concept used in Arthropoda, and probably Mollusca, is extended to Chordata, it would be expected that the photoreceptors would be arranged in a capsular arrangement based on a close- packed hexagonal array of seven cells each. Under this assumption, there are two challenges. One is to identify, or specify, the spectral characteristic of each cell by location in the array. The other is to establish some estimate of the regularity of the resulting capsules. There is also the associated question of how these various cell arrangements support the subsequent signal processing used to support the perceptions of lightness and chromatic content.

In consonance with this work, and the majority of the work summarized above, no space will be reserved in the putative human retina for “rods.”

Some of the above investigators have made estimates of the ratios of spectral photoreceptors, but most have not. The reason is the apparent large variation with position in the retina (and possibly with the technique used to identify different cells). None of the previous investigations have considered the presence of UV- sensitive cells in the human retina. Table 3.2.3-1 summarizes some of the available estimates, along with those from this work.

Figure 3.2.3-1 CR [Color] Pseudo-color images of the human retina taken by reflective spectrophotometry following selective bleaching. See text for details. From Roorda & Williams, 1999. The Retina 3- 71

TABLE 3.2.3-1 Estimates of photoreceptor density by spectral type

McCrane, 83 Ahnelt, 87 Curcio, 91 Roorda, 99 This Theory Without UV With UV L–channel 50.6- -75.8 6% 5% M–channel 44.2- -20.0 88% 50% S–channel 7% 3- -5% 4.2- -7.6 5.2- - 4.2 6% 5% UV–channel ------40% Ahneldt gave a range of densities for likely blue-sensitive photoreceptors based on light microscopy. Their numbers varied from 3-5% in the foveolar center to 15% near the foveolar slope to 7-10% in the peripheral retina. They did note “The cones differ in having different photopigments and different neural connectivity, but no morphological differences with which to distinguish the three different spectral types have been reported [prior to their paper].” Their criteria was unusual, the defined big-cones and regular-cones and inferred the big-cones were S-channel sensitive. They did not describe an absence of big-cones from the center of the foveola. The Roorda data is for two separate subjects and was collected under the trichromatic assumption. No explanation was given for the very high percentage of L–channel photoreceptors although they noted “The proportion of L to M cones is strikingly different in two male subjects, each of whom has normal color vision. The mosaics of both subjects have large patches in which either M or L cones are missing.” These subjects did not report any loss in vision over significant (though small) portions of their retinas. Their data was collected by microspectrometry in- vivo. It is possible that the empty areas are those associated with the UV–channel photoreceptors (See below). It is also possible that some of the cells listed as L–channel might in fact be transparent UV–channel cells reflecting light from the RPE. In a larger study, they found the ratio of L– to M– channel cells varied between 0.6:1 and 10:1181. This is an astounding variation that suggests more exploration is necessary using this technique. The technique they used to isolate the L– and M–photoreceptors may lead to the problem.

The estimate under the “This work” column is shown based on two databases. Without considering the UV absorption demonstrated by Tan and by Stark, the ratios can be determined from the best theoretical fit to the CIE (1924) luminosity function as corrected by Judd & others. Elsewhere in this work, these proportions have been used on the assumption that the signals at the output of the photoreceptor array were all of equal amplitude and a gain factor was introduced that weighted the different spectral signals used to form the luminous efficiency function. The weighting could be performed by adding in different numbers of signals from different groups of spectrally specific photoreceptors or the weighting could be achieved by adding the signals from all photreceptors and depending on their relative abundance as a function of spectral absorption to create the luminosity function. It is this latter approach that will be explored in th remainder of this section.

When the data of Tan and of Stark is considered, the array factors must be adjusted to show a UV sensitivity greater than that of either the S– or L–channel and approaching (if not surpassing, see Section 17.2.5) the sensitivity of the M–channel.

3.2.3.4 Candidate photoreceptor groupinng based on this work

181Roorda, A. (1999) Op. Cit. 72 Processes in Biological Vision

Figure 3.2.3-2 shows a candidate arrangement lacking any UV–channel receptors. This array provides seven percent S–channel and seven percent L–channel photoreceptors, with 86% M–channel cells. The distribution of the S–channel receptors in this map of the outer segments, in both density and distribution, is not grossly different from the map of the inner segments presented by De Monasterio, et. al182. However, it begs the question if there are in fact UV–channel photoreceptors in the human retina (whether used effectively or not, see Section 17.2.2). Section 16.3 and Sections 17.2 & 17.3 of this work develop the equations of luminance and chrominance performance for the human visual system. These sections establish that the luminance response is described by a simple equation. The equation sums the logarithms of the photocurrent sensed by each spectral class of photoreceptors (with an appropriate scaling coefficient in front of each logarithm). These sections also establish that there are multiple parallel chrominance channels in vision. Each is described by a simple equation. The equation takes the difference between the logarithms of the photocurrent for pairs of spectrally different photoreceptors (with an appropriate scaling coefficient in front of each logarithm). Figure 3.2.3-2 Potential mosaic of photoreceptors based on The above equations accept and account for the limited a trichromatic approach to vision. The M–channel dynamic range of the neural system. In their complete photoreceptors are not colored. See text for the ratios. The form, they describe the luminance and chrominance blue cells represent S-channel photoreceptors. The red cells performance of the human visual system with a represent the L-channel receptors. When reproduced in precision of better than a factor of two at all black and white, every other dark cell is a L-channel wavelengths. photoreceptor. The alternates are S-channel photoreceptors. The physical arrangements shown in this and the following figure suggest the simplicity of the signal processing associated with this arrangement. Summing (logarithmically as described above) the signals from all of the cells in two adjacent S– and L–centered capsules would provide a theoretical luminous efficiency function well matched to the observed function. Differencing (logarithmically as described above) the signal from a center cell of a capsule and the signal from anyone of its surrounding cells would provide the correct chrominance channel information. This arrangement would remove any need for complex interconnections between the photoreceptor array and the subsequent signal processing of stage 2.

182De Monasterio, xxxx (1985) in Wandel, pg 52. The Retina 3- 73

Figure 3.2.3-3 shows an alternate candidate arrangement that includes UV–channel photoreceptors in roughly the proportions required to meet the extended luminous efficiency function. The UV–channel cells make up 28% of the array compared to the M–channel cells 57%. The S– and L–channels each remain at 7.1%. This configuration is still compatible with that of De Monasterio. It is also compatible with the images of Roorda, et. al. (recognizing that the UV-channel photoreceptors are photographically transparent and appear as “blood” red in their figures). As above, summing (logarithmically) the signals from all of the cells in two adjacent S– and L–centered capsules would provide a theoretical luminous efficiency function well matched to the observed function, even when extended into the ultraviolet (see Section 17.3.XXX). Differencing the signal from a center cell of a capsule and the signal from anyone of its surrounding M–channel cells would provide the correct chrominance channel information. There is no current information concerning the composition of signals related to the chrominance channel(s) defined by either UV– minus M– or UV– minus S–. But, differencing the signals from either one or two of the UV–channel cells and one, two or all of the associated M–channel cells in a capsule would provide an appropriate chrominance signal. As above, this arrangement would remove any need for complex interconnections between the photoreceptor array and the subsequent signal processing of stage 2. The placement of the UV–cells in opposition within the capsule may cause a directional aspect to the spatial frequency resolution of the eye. A solution would be to have the UV–cells at the 1 & 3 positions within the capsule instead of the 1 & 4 positions. They could also be randomized further, either within the capsule or by randomizing the capsule orientations.

Both of the above arrays of capsules (hexagonal groups of seven photoreceptors) are probably too regular to match the available experimental data. However, if the capsules were to be rotated randomly, or even arranged according to a higher set of rules, groupings similar to those of Roorda & Williams could be achieved. This would be particularly true if the lack of absorption by the UV–sensitive cells resulted in their location Figure 3.2.3-3 Potential photoreceptor array based on a appearing red due to the RPE behind the retina. These tetrachromatic human retina. The M–channels are randomizing steps would also insure that the spatial uncolored. The UV–channels are hatched. When frequency response of the eye was not seriously reproduced in black and white, every other dark cell is a L- impacted by the presence of different spectrally channel photoreceptor. The alternates are S-channel sensitive photoreceptors in an orderly arrangement. photoreceptors. See text. Both of the above arrays appear indistinguishable from the arrays, with the blue cones highlighted, of Curcio, et. al.

3.2.3.5 Putative arrangement of glia cells acting as light pipes

Labin & Rabik have recently modeled the potential for glia to operate as light pipes through the neural layers of the retina outside the fovea (in the parafovea)183. This area of the retina exhibits substantially less resolution than the foveola and fovea. There is considerable evidence that the resolution in this area is limited substantially by the summation of signals from multiple sensory neurons by the stage 2 circuits in order to obtain change sensitivity at the expense of resolution. The authors did not provide a detailed physiological model to justify their assumptions concerning the need to preserve resolution in the parafovea in the face of the neural layer between the pupil and the sensory receptors possibly degrading the resolution of the imaged light. Their cartoons of figure 1 do not explain how the broadly spaced Muller cells contribute to the resolution performance of the much finer array of rods and cones shown. Strasburger provided a physiological model of a retina in 2007-08 but it does not show the foveola situation, only the parafovea184. This research activity does not appear to be based on a solid physiological model, or

183Labin, A. & Ribak, E. (2012) Retinal glial cells enhance human vision acuity. http://physics.technion.ac.il/~eribak/LabinRibakGlialCells.pdf

184Strasburger, H. (2008) A model of Muller cells in a retina www.hans.strasburger.de/materials/muellerzellen.ppt 74 Processes in Biological Vision

more importantly, a solid photomicrograph of a real human retina.

3.2.3.6 The “text rewriting” work of the Roorda team with optimized AOSLO

The subject matter of this subsection involves the intimate interaction of the human retina, the optics of the eye, a very state-of-the-art test set and the changing of a variety of long held intellectual concepts. As a result, it may be appropriate to move part or all of the following discussion to a later section of this work at a later date. Many of the most subtle mechanisms associated with the visual modality developed within the context of this theory have been confirmed during 2016. The Roorda team, based at the University of California, Berkeley has expanded during the 21st Century. Their work has introduced and qualified a new AOSLO with a form of real time tremor compensation to provide a totally new degree of precision in in-vivo human retinal topographic analysis. Sabesan et al185. reported on this very sophisticated test set augmented with adaptive (active) optics, tremor cancelling features and additional features associated with optical coherent tomography (OCT) . This test set allowed them to stimulate individual sensory neuron photoreceptors of the human retina in-vivo for extended periods of time (up to 500 ms.). With this capability, they were able to demonstrate that the M – and L–channel photoreceptors mediated the intensity signaling R–channel of the visual modality as well as mediated their individual M – and L–signaling channels (Sections 1.2.1.2.2 & 1.3.3 & 1.6.1) . They accomplished this psychophysical experiment by stimulating individual photoreceptors and interrogating the fully aware subject as to their perceived response. The major paper is that of Sabesan et al186. but consists of a set of papers including the following major components. Lead author Date Focus

Arathorn 2007 Method and performance of a retinally stabilized stimulus directed at cones Benson** 2014 Algorithmic simulations of unsupervised learning of cone spectral classes Brainard 2008 Reconstruction of a static SML mosaic using a Bayesian model (& no tremor) Curcio 1990 Human photoreceptor topography based on in-vitro cell geometry, not absorption–Archaic* Field 2010 Functional connectivity of macaque SML photoreceptors using multielectrodes Harmening 2014 Physiological features of human retina observed photopically through the lens Roorda 2002 Detailed optical description & performance of one of the AOSLO ca., 20002 sabesan 2015 Mosaic characterization via through-lens dynamic photopigment densitometry Yang 2014 Full time closed-loop optical stabilization reduced residual motion by 10-15x * Other than the Curcio paper examining inner segments of photoreceptors in-vitro, rods were not found to be present in the imagery of the photoreceptors of the retina. ** Paper not critical to the operation of the tremor compensated AOSLO

Saey187 reported on the Sabesan paper which she described as “The textbook-rewriting discovery could change scientists’ thinking about how color vision works.” However, this assertion requires tempering. The suggestion by Saey that, “Red and green cones each come in two types: One type signals “white” and another signals color, vision researcher Sabesan and colleagues at the University of California, Berkeley discovered.” is not supported by any physiological model. By merging the theoretical models of this work (drawn from analyses of empirical data in the literature) with the empirical findings of the Roorda team, a clear case for “textbook-rewriting can easily be made, especially after addressing the challenges in the Roorda team’s reporting in a number of areas enumerated below. - - - - Sabesan et al, 2016 asserted in their Abstract, “We unraveled behavior at the elementary level of single input units—the visual sensation generated by stimulating individual long (L), middle (M), and short (S) wavelength–sensitive cones with light. Spectrally identified cones near the fovea of human observers were targeted

185Sabesan, R. Schmidt, B. Tuten, W. & Roorda, A. (2016) The elementary representation of spatial and color vision in the human retina Sci Adv vol 2:e160079 186Sabesan, R. Schmidt, B. Tuten, W. & Roorda, A. (2016) The elementary representation of spatial and color vision in the human retina Sci Adv vol 2:e1600797 187Saey, T. (2016) Color vision strategy defies textbooks Science News 15 October, pg 10 The Retina 3- 75 with small spots of light, and the type, proportion, and repeatability of the elicited sensations were recorded.” Their stimulation was through the human lens and did not attempt to stimulate the UV-wavelength sensitive photoreceptors found in the retina of all chordates, most insects and many molluscs. Their elicited sensations constituted perceptions reported following cognition by the stage 5 engines of the CNS. They reported, Two distinct populations of “cones” were observed: a smaller group predominantly associated with signaling chromatic sensations and a second, more numerous population linked to achromatic percepts.” This wording should be reinterpreted to indicate the experimenters received two different classes of reports from their subjects: a smaller group predominantly associated with signaling chromatic sensations and a second, more numerous population linked to achromatic percepts. This wording better supports their conclusion in the abstract, “Overall, the results are consistent with the idea that the nervous system encodes high-resolution achromatic information and lower- resolution color signals in separate pathways that emerge as early as the first synapse.” The first synapse occurs at the output of every stage 1 sensory receptor and prior to the stage 2 signal processing within the retina. This signal processing is more complex than suggested by their closing remarks as identified in this work by the high-resolution PGN-pulvinar pathway and the low-resolution LGN-occipital pathways, both of which process chromatic and achromatic information (generically in Section 1.6.1 and more explicitly in Section 1.7.5). The false-color fundus photographs show a preponderance of L–channel photoreceptors apparently due to the method of identification employed. They made a specific effort to identify M –channel receptors and a lessor effort to identify S–channel receptors. They made no effort to identify UV–channel receptors and did not encounter any achromatic (rod) receptors in the fields of view provided. It is likely that any UV–channel receptors and in many cases, the S–channel receptors were lumped in with the L–channel receptors and shown in bright red. This position is supported by their Table 1 which demonstrates that a majority of their L–cones did not report a “red” response but instead reported a “white” response. The white response is indicative of a photoreceptor contributing to an R–channel (brightness) response without contributing to the expected Q–channel response associated with the chromatic signaling channels.

Figure 3.2.3-4 reproduces figure 5 from Yang et al., 2014. It shows the excellent tremor cancellation achieved by their frame-to-frame matching system operating at 30 frames per second on the Rochester AOSLO. The inset shows the fine “hunting” associated with the 30 frames per second cycle time. Yang et al188, 2010 described the design of the hardware and software added to the basic AOSLO, Table 3 of the Yang paper compares the performance of this technique with a variety of previous eye trackers.

188Yang, Q. Arathorn, D. Tiruveedhula, P. et al. (2010) Design of an integrated hardware interface for AOSLO image capture and cone-targeted stimulus delivery Opt Express vol 18(17), pp 17841-17858 76 Processes in Biological Vision

Figure 3.2.3-4 Eye motion trace computed from an image sequence showing vertical (y) component of eye motion before (red) and after optical stabilization alone (blue) and optical stabilization combined with digital registration (green) providing tremor cancellation. Inset is an expansion of the 11 to 15 second interval. Asterisks denote spurious motion measurements during blinks or large saccades. From Yang, 2014.

- - - -

[xxx extract and edit into other paragraphs of this work ] The experiments involved stimulating 273 individual photoreceptors in two male subjects. When stimulating an M –channel photoreceptor, the subject responded 21percent (21 samples) of the time that he perceived a greenish response indicative of a color sensing channel. The other 77 percent of the time (77 samples) , he perceived a whitish response indicative of brightness rather than color. When stimulating an L –channel photoreceptor, the subject responded 29 percent (48 samples) of the time that he perceived a reddish response indicative of a color sensing channel. The other 71 percent of the time (119 samples) , he perceived a whitish response indicative of brightness rather than color. ; the investigators did not investigate any S–channel photoreceptors. Not having an aphakic subject available, and probably not being aware of the significance of such a subject, they did not investigate the putative UV–channel of biological vision either. Saey189 reported on these experiments which she described as “The textbook-rewriting discovery could change scientists’ thinking about how color vision works.” The inset in the figure of her article should be understood to be based on false-color for purposes of illustration. The photoreceptors do not reflect significant light indicative of

189Saey, T. (2016) Color vision strategy defies textbooks Science News 15 October, pg 10 The Retina 3- 77 their spectral absorption performance. From the Sabesan et al. article, “Each cone tested psychophysically was identified with a specific spectral type by aligning the 842-nm reflectance image and the corresponding trichromatic cone mosaic map obtained from densitometry. The alignment was guided by the pattern of blood vessels in the subjects’ retina, and individual cone locations remained stable across days.” The suggestion by Saey that, “Red and green cones each come in two types: One type signals “white” and another signals color, vision researcher Sabesan and colleagues at the University of California, Berkeley discovered.” is not supported by any physiological model. The situation can be interpreted quite differently in the presence of a viable model of the visual modality that processes the sensory neuron signals into both chromatic and achromatic channels following the first synapse (within stage 2) as suggested by the investigators in their Abstract. To achieve the remarkable stability required with this test apparatus, the two subjects used dental impressions to assure constant head position and pointing. Their abstract includes several important statements. They noted, “Spectrally identified cones near the fovea of human observers were targeted with small spots of light, and the type, proportion, and repeatability of the elicited sensations were recorded.” “Sensations generated by cones were rarely stochastic; rather, they were consistent over many months and were dominated by one specific perceptual category.” “ Overall, the results are consistent with the idea that the nervous system encodes high-resolution achromatic information and lower-resolution color signals in separate pathways that emerge as early as the first synapse.” Their introduction adds more detail to their experimental goal. “There are two fundamental impediments to linking visual perception to the activity of individual retinal neurons. First, the retina is situated inside the eyeball and thus can be neither visualized nor stimulated at cellular resolution due to the eye’s aberrated optics. Second, even while steadily fixating, the retina moves over spatial scales far greater than the size of a single cone, impeding the repeated and reliable stimulation of the same cell.”

The section labeled “Brief Methods” adds additional protocol information. “ Cone-sized spots (0.45 arc min; 543-nm wavelength; 25-nm bandwidth) were targeted on a mosaic of spectrally identified long (L), medium (M), and short (S) wavelength-sensitive cones in [only] two male, color-normal subjects — S10001R and S20076R. The subjects reported the color of each flash. The stimuli appeared for 500 ms on a dim background subjectively adjusted to appear approximately achromatic at the start of each session. A gray background was chosen to roughly equate the resting-state activities of L- and M-cones to minimize biases in downstream neurons. In the case of a large stimulus, chromatic contrast from a gray background that increases only L-cone activity produces a reddish sensation in most subjects, whereas excursions from gray that increase only M-cone activity appear bluish green.” These perceptions are totally in accordance with the perceptions expected from the theoretical analyses of this work. The “reddish” sensation is compatible with the L–channels peak response at 610 nm along the spectral locus of the New Chromaticity Diagram of this work. The “bluish green” at threshold is compatible with the M –channels peak response at 532 nm along the spectral locus. Some of their tests were repeated using a 511 nm stimulus according to their supplemental material. However, the difference between 543 and 511 nm is relatively small and not likely to change their results significantly according to this work. They utilized a narrow range of intensities in these experiements (described as 0.5, 0.75 and 1.0 a.u.). They did note with regard to their figure S2, “Frequency of seeing decreased with decreasing intensity.”

It is also important to note that the perceived response of humans is a function of the exposure time to the specific stimulus and to the precise state of adaptation of the individual eye at the time of stimulation. This is particularly true in the case of the Sabesan et al. paper where no data was provided to demonstrate their 543 nm stimulant did not spill over and cause the stimulation of nearby photoreceptors. They relied upon predicting the location of their stimulation on the surface of the retina several milliseconds in the future based on their calculation of the dynamic properties of the tremor encountered and the ability of their tremor cancellation technique. It is important to note the necessity of providing continuity in their discussion where the names of colors are concerned. “Reddish” does not equate to “red” and “Greenish blue” does not equate to “green” in the pantheon of color names associated with either the Munsell Color Space190 or the “color names dictionary” propounded by the United States NBS (now NIST)191. The precise significance of the regression analyses shown in their figure S4 is difficult to ascertain in the absence of a graphic model of their visual modality, and the specific wording in the caption. They noted, “These regressions, while statistically significant, do not fully capture the variance in our data.”

190Nickerson, D. & Newhall, S. (1943) A psychological color solid J Opt Soc Am vol 33(7), pp 419-421 191Kelly, K. & Judd, D. (1955) The ISCC-NBS method of designating colors and a dictionary of color names; National Bureau of Standards Circular 553. Washington, DC:US Government Printing Office. 78 Processes in Biological Vision

Sabesan et al. also explore in concept the ganglion arrangement within the retina but without any graphic model of the signaling involved. “There is little doubt that the long-duration suprathreshold stimulation of individual cones here influences the firing of a number of different ganglion cell types. In particular, a multielectrode study demonstrated that the activation of a single cone simultaneously evoked responses in both midget (parvocellular) and parasol (magnocellular) ganglion cells. The latter has been posited to contribute to achromatic percepts. However, at the eccentricity studied here (1.5/ temporal retina), midgets make up more than 90% of the ganglion cells sampling the cone mosaic. Moreover, the midget ganglion cells respond best to high-contrast, low-temporal frequency stimuli. A 500-ms suprathreshold illumination of the center cone of a midget ganglion cell receptive field, with minimal light from the spot falling on surrounding cones, represents an ideal stimulus for these cells. Therefore, our results may be particularly informative in differentiating proposals about the role of parvocellular neurons in achromatic spatial and color vision.” They briefly discuss the common wisdom and then conclude, “. . .but our results do not align with this expectation (Fig. 4 and fig. S4). They go on to speculate about the prior common wisdom but without any schematic diagram of the visual modality.

- - - - Tentative conclusions relative to the Roorda team approach/results. 1. They did not investigate the UV–channel photoreceptors of the human eye (Section 17.2.2). The result is their use of a simplified schematic (Figure 1.7.5-3 rather than that of Figure 1.6.1-1 of this work) and topography of the human retina (Figure 3.2.3-2 rather than Figure 3.2.3-3 of this work). 2. They intentionally, omitted investigations related to the S–channel photoreceptors but did note the presence of S- channel photoreceptors in the fovea and foveola of the human retina. 3. They described the fine motion of the eyes below the level of saccades but did not relate it to the tremor well documented by Yarbus and other contemporaries during the 1960's (Section 7.3.3). 4. They recognize the waveguide character of the outer segments of the photoreceptors, but at one point assign the responsibility for this condition to the inner segments (their xxx). This operation of the outer segments as waveguides places their effective absorption area to a fraction of their physical cross section. See Stiles-Crawford Effect of the 1st kind, Section xxx. 5. They continue to implicitly assume the peak sensitivity of the L–channel is near 564 nm based on their assumption that a 543 nm laser stimulates both the M –channel and L–channel photoreceptors nearly equally based on individual normalization of their sensitivity responses and based on linear arithmetic to establish their crossover point. The 543 nm value was promulgated in the 1940's based on a conceptual error in a mathematic analysis. The correct peak sensitivity of the L–channel is near 610 nm based on Thornton, and universally accepted within the lighting and display technology areas. 6. They correctly note the L–channel response is perceived as a “reddish” color and the M–channel response is perceived as a “bluish green.” These perceptions are totally in accordance with the perceptions expected from the theoretical analyses of this work. The “reddish” sensation is compatible with the L–channels peak response at 610 nm along the spectral locus of the New Chromaticity Diagram of this work. It corresponds to 8YR in Munsell Color Space. The “bluish green” at threshold is compatible with the M –channels peak response at 532 nm along the spectral locus. It corresponds to 2.5G in Munsell Color Space. 7. Following identification of the principle perceptions of the M –channel and L–channel as “bluish green” and “reddish,” they revert to the colloquial forms green and red in their discussion. Red and green are defined as explicitly different colors than reddish and bluish green in the technical literature (Section 17.3.8). As noted in the section cited, red is a non-spectral color in perceptual space best defined as either 494c or 494,610 nm in the New (Perceptual) Chromaticity Diagram (Section 17.3.8) underlying the CIE 1976 UCS Color Spaces. There is a concern whether the two (experienced) subjects used by the Roorda team should have identified the perception of the M –channel as yellowish-green rather than bluish-green based on Section 17.3.9 of this work. A larger group of naive subjects would clarify this question. 8. On multiple occasions, the authors of the various reports of the Roorda team limit their investigations to the photopic irradiance regime, thereby avoiding direct consideration of the putative “rod” photoreceptors. However, on occasions, they report activities under threshold conditions that imply conditions requiring rod participation(xxx cite paper). More broadly, their reports do not identify the presence of any rods within the field of their ophthalmological images of the retina. [xxx cite at least one paper ] 9. - - - - - The Retina 3- 79 3.2.4 Electronic architectural level

The volume of signal processing carried out within the retina places significant constraints on the electrical topology and topography of the retina. These architectural constraints are familiar to an engineer experienced in the design of electronic microcircuits. For those interested at the very detailed level, many of the comments by Garrett, although focused on phasic applications in man-made circuits, are worth reviewing192. From his figures, one can easily recognize the similarity of the visual circuits to those known by the names DTL (Diode-Transistor Logic) and TTL (Transistor-Transistor Logic). In the retina, the synapses are generally employed as highly efficient diodes. The simple neurons of the bipolar and lateral types employ individual Activas. The resulting circuits are virtually identical, architecturally, to DTL circuits used in an analog mode. The projection neurons consist of a series of Activas easily compared to the architecture of TTL circuits. The complexity of the photoreceptor cells is more closely related to analog operational amplifiers than to standardized TTL circuits. The constraints on the diameter, length and placement of the electrical conduits connecting these circuit elements are discussed in Garrett. The higher impedance level of the neurons must be considered when interpreting these constraints and guides. Whereas a TTL circuit operates near the 10,000 Ohm level, neurons operate at levels 103-104 higher. Where a TTL circuit might support output conduits 3-5 cm, long, a signal sensing or manipulation neuron can only support output conduit lengths of a few microns. The specialized, higher capability, signal projection neurons operate like TTL line drivers. They can support conduit lengths up to 2 mm. 3.3 Metabolism of the chordate retina 3.3.1 Static considerations related to a cell

Before discussing the metabolic system serving the eye, reviewing the elements and constituents involved in the process for a single cell is useful. The conventional wisdom193 is shown in the upper portion of Figure 3.3.1-1. Most introductory material develops the analogy between a living cell and a manufacturing plant. It presents the idea that metabolic fuel, oxygen and various chemicals are taken up by the cell and the output consists of work, carbon dioxide, heat and various secretions and excretions. This is adequate for the retinal cells of the third laminate, the RPE. It is not an adequate depiction of the neurological cells of the photoreceptor and neural laminates. The neuron also involves the output of a signal current and the input of a signal flux that will be defined more precisely below. 3.3.1.1 The Neuron

Neurons exhibit an additional set of functions. These are shown at the bottom of the figure. They accept an input electrical signal, channel that signal to an electrical amplifier and distribute the output of that amplifier to subsequent neurons. The channels are formed by additional bilayer membranes formed within the cell. These membranes act as insulating partitions when the electrical potential across them is appropriate. In accomplishing the channeling function and establishing the insulating properties of the additional membranes, the neuron requires the support of several electrostenolytic sites to convert metabolic energy sources into electrical potential. These specialized sites appear to be functionally identical to a similar site that polarizes the prototypical biological cell, whether it is a neuron or not. Figure 3.3.1-1 Operation of the typical living cell with the Neurological cells accept an afferent flux and input and output signals added that are peculiar to a neuron. discharge an efferent flux. The efferent flux of a Pull on the lariat to squeeze this morphological bipolar cell sensory neuron (combined electrically with a synapse) in to a monopolar cell. The cell may contain one more is a current. In most neurological cells, the input flux intrinsic electrostenolytic site that polarizes the soma of all cells. Compare to Rushmer (1970).

192Garrett, L. (1970) Integrated circuit digital logic families. IEEE Spectrum, Vol. 7, Oct. pp. 46-58, Nov. pp. 63-72, & Dec. pp. 30-42 193Rushmer, R. (1970) Cardiovascular dynamics. 3rd. Ed. Philadelphia PA: W. B. Saunders pg. 2. 80 Processes in Biological Vision

also consists of a current. For a sensory neuron, it may consist of any flux that can cause a current to be generated within the cell. In vision, the photoreceptor cell receives an exciton flux created by the photo/piezo effect occurring in the transduction material of the Outer Segment. In neurons, work is not a significant constituent of the output. Surprisingly, heat is not a significant output product of a sensory neuron either. This is because of the unique reversible thermodynamic process used to operate the neuron. Secretions are significant output constituents for the photoreceptor cells. The Inner Segment of these cells secretes the protein material used to form the disks of the Outer Segment. The figure illustrates a problem with the terminology of morphology. Neural cells have traditionally been named based on the assumption that the cell nucleus, and the elements surrounding it, were the most important elements of the cell. The cell shown would normally be described as a bipolar cell if the neural channels are significantly longer than the diameter of the cell body. The designation is associated with the two long channels ostensibly emanating from the cell body. However, if the lariat shown by the dotted line is pulled and the cell body becomes more isolated from the neural components, the cell becomes known as a monopolar cell because under this condition, there is only one channel emanating from the cell body. In neurons, the Activa or electronic amplifier is actually the most important part of the cell. The nucleus and other elements within the cell body play only a supporting role. 3.3.1.2 The RPE cell

The upper portion of the above figure is nominally adequate to describe the functions of the retinal pigmented epithelium cells. However, here again, the soma and nucleus of the cell only perform a supporting role. The major activity of the RPE cells is the re-manufacturing of the non-protein parts of the disks of the Outer Segments as shown in Figure 3.3.1-2.

The RPE cells obtain the necessary chemical components from two sources, the blood stream and phagocytosis of the Outer Segments originally produced by the photoreceptor cells and coated with chromophoric material from within the IPM. As a reminder, phagocytosis can involve the ingestion of both cellular and extracellular material by phagocytes. Phagocytosis does not refer to the nature of the material ingested. The cells of the RPE re- manufacture, to a large extent, the chromophoric material required by new disks formed by the photoreceptor cells (Section 7.1.3). They also break down the salvaged protein material and return it to the blood stream as an excretion. Any damaged chromophoric material is also returned to the blood stream for disposal.

3.3.2 The vascular supply to the retina Figure 3.3.1-2 EMPTY Operation of the typical RPE cell. The functions shown above the blood/brain barrier are The blood supply to the chordate eye is quite limited primarily in support of the functions performed below the due to many operational constraints. Oyster provides a barrier. These functions involve the salvage (phagocytosis) description of the vascular supply at several levels of 194 of the disks of the Outer Segments. Serviceable detail . When combined, the figures can be used to chromophore material is recovered, stored and eventually describe the hydraulic capacity of the visual system. 195 secreted back into the IPM. Damaged chromophore and the Rodieck shows a simplified view , similar to Oyster. protein materials are excreted to the blood stream. The All of the vascular flow must enter and leave the globe blood/brain barrier is sometimes called Verhoeff’s as near the optic nerve as feasible. This is necessary to membrane or described as built of terminal bars by the achieve the desired ability to rotate the eye in histologist. Chordata. Figure 3.3.1-3 diagrams the vascular network of the eye. At least part of the blood flow

194Oyster, C. (1999) The human eye. Sunderland, MA: Sinauer Associates pp 258-260 & 274 195Rodieck, R. (1998) The First Steps in Seeing. Sunderland, MA: Sinauer Associates pg 29 The Retina 3- 81 must occur on the surface of the retina, in the optical path of the incoming photons. The artery enters the eye within the optic nerve. It branches into two main portions, the portion supporting the sclera and choroid, and the portion supporting the retina. The choroid circulation also supports the muscles and tissue at the front of the ocular as well. The retinal portion further divides into a portion supporting the RPE laminate, including the Inner and Outer Segments of the photoreceptor cells, and a portion serving the neural laminate. The vascular circulation of the eye begins in the cranium and branches at the optic disk of the eye. One portion of the circulation serves the RPE and other tissue on one side of the Outer Limiting Membrane of the retina and the other portion serves the bulk of the neural tissue within the retina on the other side of the OLM. This circulation is volume limited. It is also required to provide a variety of functions. Its ability to satisfy these functional requirements can be described using the conventional nomenclature of hydraulics. As each branch further subdivides, until it terminates in a vascular or diffusion bed, the individual hydraulic paths becomes less capable of supporting significant hydraulic flow. However, the total capability remains significant.

The vascular network related to the sclera/choroid laminate will not be discussed further. Hogan has provided an overall discussion of retinal circulation196. The retina is a portion of the brain. It is protected from the bulk vascular system by a so-called blood/brain barrier. The vascular network related to the retina must pass the necessary nutrients, energy sources and construction materials across this barrier. How this is accomplished varies between the two sides of the retina.

Note the role of the Outer Limiting Membrane in the figure. It plays a major chemical role and a major electrical role. It chemically isolates the IPM from the INM. It also effectively isolates the signaling portions of the retina from the photo-sensing portions. 3.3.2.1 Blood flow to the INM and most neural laminates

The vitreal side of the retina is protected by the Inner Limiting Membrane from the vitreous humor of the ocular. Whether this membrane also forms the blood/brain barrier is less clear. The blood/brain Figure 3.3.1-3 Vascular circulation within the retina. barrier may be more ephemeral and be formed by the Rectangular boxes within the INM and optic nerve represent surface of the vascular walls, or an additional neurons. RPE cells exhibit notches to receive Outer membrane associated with these walls. By whatever Segments of photoreceptor cells. The flow from and to the means, the necessary nutrients are passed into the optic nerve is idealized. It divides approximately as shown. matrix of the INM and they diffuse to the surface of the The hydraulic path length within the INM diffusion bed is individual neurons. At the same time, any materials typically 0.1 mm except for the foveola where it reaches deleterious to the neural system are excluded. 0.25 mm. The path length within the choroid space is much shorter. The RPE layer/Bruch’s membrane and the OLM The neural diffusion bed is approximately 310 microns effectively isolate the IPM chemically from the blood thick as shown on the right. It serves all of the neurons stream. They also may isolate most of the electrical of the retina, including the somas of the photoreceptor potentials of the photoreceptors from the INM. cells which are included between the Outer Limiting Membrane and the Inner Limiting Membrane. Most of the capillaries of this laminate are found close to the Inner Limiting Membrane. They cause less degradation to the optical image when placed there. However, the diffusion path to the soma of the photoreceptor cells is lengthened. The vascular network of the neural laminate is seen to branch profusely in the above figure, eventually reaching the level of the capillaries. The capillary walls are porous and allow the diffusion of materials into and out of the

196Hogan, M. Alvarado, J. & Weddell, J. (1971) Histology of the Human Eye. Phila. PA: W. B. Saunders in detail pg 403 & 508-522 82 Processes in Biological Vision

diffusion bed with little difficulty. Similarly, the cells of the visual system can exchange material with the diffusion bed. As indicated in the figure, the typical distance between a visual cell and a capillary is about 0.1 mm. However, the distance between the nearest capillary and a cell at the center of the foveola in human is 0.25 mm. As will be seen below, 0.25 mm. is approaching a “lethal corner” in terms of the distance between an active cell and its supply capillary. This longer path may play an important role in the after images perceived after looking from a bright light into a dim area. 3.3.2.2 Blood flow to the IPM, RPE and photoreceptor cells

The vascular matrix serving the RPE laminate is shown at the top of the figure. This laminate is thin, about 20 microns thick in humans. It is able to support the entire surface of the retina equally well because it is not in the optical path of vision. There is no degradation in hydraulic performance related to the foveal area. The blood/brain barrier between the vascular matrix and the IPM is formed by a combination of Bruch’s membrane, the close packing of the RPE cells, and (in some works) Verhoeff’s membrane197. This structure plays a complex role in that it must not only allow the nutrients required by any neural cell to pass, it must also extract the chromogenic material from the blood stream. The blood stream itself is hostile to the chromogenic material. Therefore special steps are required. These steps will be discussed in detail in Chapter 6. The photoreceptor cells are seen to be in a unique position with regard to the Outer Limiting Membrane, the IPM and the INM. While the Outer Segment and much of the Inner Segment are within the IPM, part of the Inner Segment and the soma are located within the INM. This is important. As shown by the electrical components and electrical paths associated with the Inner Segment, the electrostenolytic supplies to both the Outer Segment and the Inner Segment are generally supplied from the RPE vascular matrix. However, some materials required by the Inner Segment are hostile to the chromophoric material within the IPM. These materials are provided to the Inner Segment via the soma from the INM. 3.3.2.3 Block Diagram of the Metabolic Components

The hydraulic block diagram of the retina is a restatement of the morphology of the vascular network in hydraulic engineering terms. The diagram begins by recognizing that the arterial and venous systems form a balanced system with the various capillary systems shunted between them. Since working with unbalanced systems is mathematically easier, the effective impedance of each section of the balanced network is usually replaced by an equivalent impedance in an unbalanced network. Figure 3.3.1-4 provides a schematic network of the metabolic system of the eye using this unbalanced notation. It is shown using electrical instead of hydraulic symbols on the assumption that most readers are more familiar with this notation. Specific labels and values will be assigned to the various circuit elements in later chapters. The diagram can be used to describe both the RPE or the neural vascular networks. Five sections are shown along the bottom of the figure beginning with the flow through the optic nerve. It is assumed that the optic nerve section is supplied by an infinite capacity source within the head as shown.

The optic nerve section divides into three sections at the optic disk, the choroid/sclera section, the RPE section and the neural section. Each of these sections continues to ramify until it develops a diffusion bed in the immediate vicinity of its end user cells. Only the RPE laminate is shown in full detail. The vascula of the optic nerve subdivides into a series of vascuolas, then into a larger number of capillaries, and then into a diffusion bed of very great area. The individual cell can be considered a terminating section of this network. The hydraulic characteristics of each of these sections, except for the terminating section, can be described by a serial resistance and a shunt capacitance. Each of these resistances will be considered fixed although each of the vascular sections involves a variable impedance due to the muscular capabilities of the conduit wall. Each terminating section consists of a shunt resistance and capacitance. The shunt resistance is a variable reflecting the energy requirements of the cell when transmitting a signal. Fortunately, the adaptation amplifier of the photoreceptor cell stabilizes the signal levels within a majority of the signaling channels of the eye so that these impedances are also fixed. It is a unique property of the adaptation amplifiers that they also can be represented by a fixed resistance over a majority of the illumination range of the eye.

197Dreher, B & Robinson, S. (1991) Neuroanatomy of the Visual Pathways and their Development. In Vision and visual dysfunction, Vol. 3. Boston: CRC Press Fig. 1.2 Also in Rodieck, R. (1988) The Retina 3- 83

All of the vascular sections involve conductive flow of constituents of the blood and are represented by generally low serial impedances and high shunt capacitances compared with the diffusion section. Thus, the diffusion beds represent the major constriction in the blood supply to a neuron of the retina, and particularly to a neuron in the foveola. This is not always an adequate representation of the situation as will be seen in Chapter 7. Particularly under laboratory conditions, it is necessary to consider the hydraulic system a second order hydraulic system. This requirement is particularly evident in both the dark and light “adaptation characteristic” of vision. In a worst case, adjacent diffusion beds are depleted and they begin to draw down the level of resources in the capillaries. These can, in turn, draw down the vascuolas, etc. Each of the circuits contributes an additional time constant in the above scenario. The Figure 3.3.1-4 The equivalent circuit of the vascular system appropriate mathematics for computing the transient of the eye. The optic nerve artery divides into three response of the system is messy but manageable. It is branches, arteriola, serving different laminates of the eye. also presented in Chapter 7. These further divide into capillaries serving the various diffusion beds. Individual neural cells draw resources from An additional complication is that many adjacent these beds. Only the RPE circuit is shown in detail. neural cells draw upon the same region of the diffusion bed for nutrients and excretion. These are shown as parallel sections in the above network. The effect of their drawing on a common diffusion bed is to cause many of the edge effects associated with vision under high contrast conditions. 3.3.3 Dynamic considerations

The retina is clearly an organ with strict limitations on its ability to draw energy and building materials from the rest of the body. With only one principal artery feeding the entire eye, the different regions of the eye are subject to relatively severe rationing. This rationing concerns what the region can obtain from the system, and what is more important, the rate at which it can obtain it. This becomes even more serious in the central areas of the retina where a high rate of metabolism is needed to process the detected visual signals. The nutrient flow within the actual retina is by diffusion from the nearest capillary. This results in a given signal channel drawing its needs from a pool shared with its neighbors. If one channel is called upon to draw down an excessive amount of nutrients, this will lower the level of nutrients available to its neighbors. In the normal operation of the eye observing natural scenes, this only happens rarely, when looking at the sun for instance. However, in the psychophysical laboratory, it frequently happens when the experimentalist introduces a bright, and/or pulsating light source. It also occurs when the light introduced is of sharply defined geometry. Many “halo” effects have been assigned a cause involving signal matrixing when in fact they have been due to depletion of the metabolic reserves of the photoreceptors and signaling circuitry in a given localized zone of the retina. Furthermore, it will be shown that this depletion of reserves is a principal factor in the so-called dark adaptation of the eye. The process of adaptation is a continuous one, involving both light and dark adaptation. It also involves a number of state-variables. This makes any measured effects highly dependent on both the test conditions and the coordinates of the retinal location used. These coordinates can aid in determining the level of nutrient reserves available in that region. 3.3.3.1 Bulk characteristics

Many approaches have been tried to quantify the consumption of energy by the brain and/or the visual system of the brain. These have been less than totally adequate for a number of reasons. The primary reason has again been a lack of an adequate model to describe forms of energy are involved. The conventional wisdom has been that the primary gauges of energy consumption are the consumption of oxygen and the release of heat. However, to a large extent, the first order operation of the visual system is independent of these two parameters. In the second order, oxygen does play a major role in forming the primary metabolic reactants. The release of heat is not a major first order factor in the operation of the neurons of the visual system. The primary gauge of the energy consumed by the neural system is the consumption of chemical energy associated with the components of the glutamate nutrition cycle used in the electrostenolytic mechanism supporting vision. It appears that a better approach to determining the energy consumption of the visual system, the neural system, the retina or the brain would be to divide the task into an aerobic and an anaerobic components. These could then be evaluated separately and summed. 84 Processes in Biological Vision

There is virtually no heat released as part of the glutamate cycle. Thus a primary measure of the energy consumption of the visual system, and the neural system in general, is a measure of the difference in chemical energy between the materials entering the blood stream of the system and the materials leaving it. Much of the material consumed directly in the electrostenolytic process is reconstituted within the environs of the neural system, a probable role for many of the glial cells closely associated with the neural system. This reconstitution appears to be the mechanism consuming glycogen within the system. Glycogen consumption is probably a good measure of the anaerobic energy consumed within the major elements of the neural system. The literature contains a number of references to the consumption of power by the human body as approximately 20 Watts198. This number is most likely based on calorimetry. 3.3.3.1.1 Studies in heat generation within the retina

The remarkable fact about the energy consumption of the retina is that it is not converted into heat. Kageyama & Wong-Riley199 attempted to measure the heat generated in the retina as an indicator of which neurons were the hardest working. Much to their surprise, little heat could be detected. Energy is frequently juggled within a group of molecules in chemistry without any significant generation of heat. The chemical energy consumed in the retina was used to cause reversible chemical reactions and to support the recycling of photoreceptor materials. Little heat was generated by neural signaling activity. Thus, while energy consumption is high in the retina, heat generation is remarkably low! 3.3.3.1.2 Studies in Oxygen consumption

The following material is paraphrased from the original sources without conforming the language to the terminology of this work. This is to avoid mis-interpretation and confusion.

Guyton200 says 14% of the entire vascular flow goes to the brain, approximately 700 ml./min. This 700 ml. carries about 20 per cent by volume of oxygen. On the average, only about 25% of the available oxygen is extracted during one circuit of the vascular blood supply, i.e., the utilization coefficient is one-fourth. During heavy exercise, the utilization coefficient can reach 75-85% in muscle. However, in local tissue areas where the blood flow is very slow or the metabolic rate very high, utilization coefficients approaching 100 per cent have been recorded.

White201 said: “The retina has the highest known rate of oxygen consumption of any tissue in the body, per unit of weight, with an active phosphogluconate oxidative pathway, a high rate of aerobic glycolysis, and an RQ of one. Lactate is the major endogenous substrate contributing to retinal respiration.” This terminology needs to be compared to that of the present day in the same field.

Miller202 reports the blood flow in the human retina to be in the range of 75 micro-liters per minute. This flow is shared between the choroid artery supporting the outer retina, and the retinal vasculature, supplying the inner retina. He indicates the choroid is controlled by the autonomic nervous system and has “leaky” capillaries. The retinal circulation has no autonomic nervous control, shows auto-regulation of blood flow, and its capillaries have tight junctional complexes. In the cat, the choroid blood flow, supporting the RPE complex is approximately 20 times greater than the retinal blood flow.

The last statement is interesting because it hints at the relative consumption rates for both energy and the materials consumed in disk fabrication and coating. It would suggest the process of final chromophore fabrication is much more energy consuming than the operation of the signaling system and the fabrication of the opsin substrates

198Habib, M. & Bockris, J. (1986) in Gutmann, F. & Keyzer, H. Modern Bioelectrochemistry. NY: Plenum Press pg 78 199Kageyama, G. & Wong-Riley, M. (1984) The histochemical localization of cytochrome oxidase...with particular reference to retinal mosaics. . . . Jour. Neurosci. Vol. 4, no. 10, pp. 2445-2459 200 Guyton, A. (1976) Textbook of medical physiology. Philadelphia, PA: W. B. Saunders pg. 251 201White, M. (1973) Animal cytology and evolution, 3d ed. Cambridge, [Eng.] University Press, pg. 1009 202Miller, D. ed. (1987) Clinical light damage to the eye. NY: Springer-Verlag pg. 83 The Retina 3- 85

combined.

3.3.3.2 Detailed characteristics

Capillaries are typically one millimeter long in humans203. At the level of the capillaries, the velocity of the blood is 0.5-0.7 mm./sec. These numbers lead to a time constant of the capillary section not exceeding two seconds. Although this calculation is crude, it does generate a flag. The investigator should anticipate a power supply time constant in this region when evaluating transient effects. The nominal impedance characteristics of the diffusion bed are not known to this author. They can only be inferred from the perceived effects of tests performed at different locations in the retina. These tests can be of many different types; flash (or flicker) tests, grating tests, etc. There is considerable data in the literature that can be used to determine the diffusion bed characteristics. However, the data was not collected with that goal in mind. It frequently involves many other parameters as variables. Only care and correlation can lead to unambiguous parametric values in the absence of specifically designed tests. 3.3.3.3 Steady state characteristics

By virtue of the accommodation amplifier in the photoreceptor cell, the majority of the circuitry in the retina operates over a quite limited dynamic range. The vascular system of the eye and the properties of the RPE and neural diffusion beds are designed to satisfy the requirements of the neural circuitry in their respective regions. Under normal conditions, these requirements are satisfied very well. The animal perceives a uniform sensitivity over its field of view and does not perceive any unusual shading or hue changes that relate to a specific area of the retina that is undernourished.

There are exceptions to this situation, mostly related to man-made objects in the field of view and/or man made laboratory test conditions. Most of these special situations involve edge effects in high contrast images.

A high contrast edge imaged upon the retina places significantly different requirements on the vascular supply than normal. Those photoreceptors associated with the high illuminance scene must accept and process a great many more photons per unit area than the similar elements in the low illuminance portion of the scene. For those elements sharing a common diffusion bed and/or capillary, competition for nutrients and oxygen will cause a distortion in the perceived image. This distortion will generally be perceived as a sharpening of the edge. The cells in the area slightly to the higher luminance side of the image transition will find it easier to obtain supplies from the diffusion bed than neurons in the center of the high luminance area. These neurons will tend to generate a slightly higher signal level than their colleagues. The neurons in the area slightly to the lower luminance side of the image will experience difficulty in obtaining adequate supplies compared to neurons farther from the transition and will report a slightly lower illuminance than their colleagues in the center of the lower illuminance area. The result is an apparent edge sharpening as illustrated in Figure 3.3.2-1. This effect is achieved without any temporal filtering in the signal channels, of the inhibition or any other type. Although this effect looks to the casual observer like a Gibbs (electrical) phenomena, or other high frequency emphasis in the signal channels, it is merely the artifact of an inadequate power supply under high signal conditions. A similar artifact, due to secondary electron redistribution, was widely encountered in early television camera tubes.

203 Burton, A. (1972) Physiology and biophysics of the circulation. 2nd ed. Chicago, IL: Year Book Medical Publishers 86 Processes in Biological Vision

3.3.3.4 Transient characteristics

There are two distinct categories of transients related to vision. The first, the slower transients are directly related to the hydraulic environment of the retina. The familiar adaptation curves of the human eye are actually slow transient response characteristics. They are only part of a larger characteristic to be presented in Chapter 12. The second and faster class of transients (sampling intervals of less than 0.1 seconds, are actually due to the method of signal encoding by the signal projection neurons of vision. These will be discussed in Chapters 14 and 18. 3.3.3.4.1 Slow transients

A particular problem relates to the accommodation amplifier in each photoreceptor cell. This amplifier appears to obtain its operating biases from the IPM of Figure 3.3.2-1 Perceived brightness in response to a high the photoreceptor laminate which is probably supplied contrast image transition. The effect is due to a competition from the RPE laminate. This amplifier exhibits very for metabolic supplies by elements of the Photoreceptor large changes in amplifier gain with respect to both laminate. prior and present signal level. These changes are related to very large changes in input signal flux and resulting significant changes in electrical voltage and current at the collector terminal of the Activa forming the accommodation amplifier.

The properties of both the RPE and neural diffusion beds clearly affect the slow transient effects known as afterimages. Afterimages are fundamentally a transient response to abnormal signal conditions applied to the eye. Unfortunately they can include individual transient responses related to each stage in the signal chain. These transient responses may be related to the inner and outer segments of the photoreceptor cells. In this case, the transient is associated with the RPE diffusion bed and the IPM. For the other neurons of vision, the transient is associated with the neural laminate diffusion bed. Afterimages frequently display color effects related to the differencing amplifiers of the signal chains. Overall, afterimages show very complex transient characteristics which are difficult to delineate. Careful test design focused on the use of S-, M- and L-channel light sources, instead of the common red, blue, green, yellow and “white,” can aid in the separation of these transient effects and the proper identification of their source.

It is difficult to separate the effect of the neural diffusion bed on each type and subtype of neuron. It is also likely that the dominant transient effect under large signal conditions will be due to the elements supplied from the RPE diffusion bed. Both the transducers and the accommodation amplifiers are located within the photoreceptor laminate. The transducers formed by the disks of the Outer Segment are not active tissue and do not require power for their operation. Thus, the transient responses related to the RPE are dominated by the demands of the accommodation amplifiers.

As a point of departure for later analysis, Rushton204 has co-authored several papers describing the transient response of what are described as in--vivo human photoreceptors. Measurements were taken both during illumination and during recovery. A more appropriate description for the recovery data would be the transient response of the in-vivo photoreceptor/RPE system. He describes the time constant of the recovery transient as 120 seconds for the normal fovea (cone), 130 seconds for two color defective fovea and 360 seconds for a normal peripheral photoreceptor (rod). These values were reported for various levels of illumination and are based on reflex densitometry. 3.4 Functions of the Chordate Retina

204Rushton, W. & Henry, G. (1968) Bleaching and regeneration of cone pigments in man. Vis. Res. vol. 8, pp. 617-631 The Retina 3- 87

The previous sections have discussed the morphological characteristics of the retina. It is equally important to discuss its physiological characteristics. The retina functions in a highly integrated mode that has not been addressed in the literature. This has been a major impediment to the understanding of subtleties of vision. There is little comprehensive discussion of the primary function of the retina as an integral unit in the literature. It is usually discussed in very global terms or at the level of individual elements. Most texts limit their discussion of the function of the retina and other neural subsystems to a metabolic perspective. The primary function of the retina is to support the acquisition and efficient processing in an optimal manner, of electrical signals resulting from the imaging of light. The retina is an integral and major portion of the Central Nervous System (CNS) of the animal. It has the very important function of both sensing and processing the visual information available to the animal. The signal processing function involves a great amount of signal data convergence, and reduction, without significant loss of useful information. It is important to define useful information carefully, as opposed to all information. The visual system does not face the same requirements as a television system or film camera. The eye of the system is not an imaging device. It is a change detector operating in close conjunction with a full frame memory in the brain. However, the full frame memory is in saliency (vector) space and does not represent a spatial image of the outside world. An investigator must determine what information is important enough to include in this saliency space. That determination is required before one can speculate as to whether the visual system is an efficient system or not. Later in this work, the retina will be discussed in terms of an information extraction engine of the CNS. Within the retina, all signal processing is in analog signal space involving electrotonic signals. Because of the distances normally involved, larger than a mm, the output signals from the retina are encoded for transmission to other extraction engines of the CNS. This is the same situation found in the rest of the brain. Information is processed in analog form and transmitted to other centers in pulse coded form. 3.4.1 Functional levels

The primary, or first order, function of the retina can be described entirely in electrical terms. This function is to perform a series of manipulations with electrical signals. These manipulations can be most easily grasped if the manipulations are subdivided into three distinct categories, signal generation, signal processing and signal projection. These signal manipulations are all performed in an electrical environment which includes all of the functions carried out within the individual morphologically defined neurons as well as the all of the manipulations performed in transferring the electrical signals between neurons. Chemical mechanisms are only employed in vision in secondary roles supporting the electrical functions. As will be discussed later, pharmacology has little direct impact on the primary functional role of the retina. However, it can impact this function role by impacting the secondary processes supporting this role. 3.4.1.1 The morphological level

There will always be attempts to understand the function of the retina based on morphological studies. It is better to circumscribe than to denigrate these studies. From the morphological perspective, the function of the retina can be examined from the gross level, the inter-neuron connection level and the cellular or intra-neuron level. The gross level usually involves visual microscopy and current levels of Magnetic Resonance Imaging, MRI. To achieve a greater degree of detailed understanding, the electron microscope is required at magnifications up to about x100,000. This is true at both the inter-neuron and intra-neuron level. For truly detailed understanding of the functional mechanisms of the retina, electron microscopy is needed at magnifications of about x250,000 and above.

Contrary to a few scattered comments in the literature, form always follows function and the available topography within the visual system. In the case of the difference between rods and cones, the difference is entirely the result of the available topography. This difference has no specular or functional cause. 3.4.1.2 The physiological or signal function level

The signaling function hierarchy can be divided into three major stages; signal detection, signal processing and signal transmission. The signal transmission function is actually shared between the transmitting portion in the retina and the receiving portion in the brain. The initial function of the signal detection stage is the sensing of the incident photons passing through and imaged by the outer optical system of the eye. The details of this process will be developed in Chapters 4 and 5. This process is an entirely passive one involving a photon/phonon transducer formed of a coated proteinaceous material similar to a finger nail. After this initial photon sensing, the resulting exciton-based signal is transferred to the neural system and converted into a conventional electrical signal. This occurs in the outer reaches of the 88 Processes in Biological Vision

microtubules emanating from the Inner Segment of each photoreceptor cell and in intimate contact with the extracellular Outer Segment associated with that cell. As noted above, the Outer Segments are supported chemically by the RPE/IPM environment and provide signal data to the photoreceptor cells in a very highly integrated process. The IPM is a very complex chemical environment205. To protect the chromophores, it is intrinsically oxygen and alkali free. The microtubules associated with the Inner Segments, and the Inner Segments are also supported electrostenolytically by the RPE/IPM environment. However, the Inner Segments being integral parts of the photoreceptor cells must also be supported metabolically. This support is provided from the INM as discussed above. The Outer Segments are capable of operating over a very large dynamic range of photon input flux as will be demonstrated in Chapter 4. It is the initial amplifiers in the Inner Segment of the photoreceptor cell that provide the critical ability to vary their amplification factor in order to stabilize the electrical signal within the more limited dynamic range of the electrical signaling system. They receive an electrical signal that can vary over a range of over 1015. Over a narrower range of approximately 106 in amplitude, they are able to deliver an output signal that varies over an amplitude of less than 100:1 in amplitude. This reduction in signal variation greatly simplifies the requirements placed on the subsequent signal processing function. A majority of the signal processing within the retina occurs between the output terminals, the pedicles, of the photoreceptor cells, and the input terminals of the ganglion cells. In some animals, a small amount of signal processing occurs between the input terminals of the ganglion cells and the actual input terminal of the Activa forming the amplifier within that cell. The area between the pedicles and the ganglion input terminals has become known as the S-Plane due to the pioneering electrophysiological work of Svaetichin in the early 1950's. The exploratory data obtained at that time was very good. However, it was difficult to specify the exact morphological or electrical source of the signals. They were usually composite signals obtained with a probe that was an order of magnitude, or more, larger than the circuits under examination. The result was a group of recorded waveforms exhibiting a variety of averaged signals. Some of the more graphically pleasing waveforms were published. The total thickness of the S-Plane in human is approximately 300 microns, 0.3 mm. and it contains on the order of 100 million neurons. Using a probe with a diameter of even 0.1 mm. diameter does not lead to discrete waveforms except on a statistically infrequent basis.

Within the S-Plane, there are at least three recognizable areas of significant signal processing activity, the plane of horizontal cells, the plane of amercine cells and the plane of ganglion input structures. The exact function of each of these planes is difficult, if not impossible, to specify precisely at this time. The individual signal processing functions may be spatially commingled and there is no requirement that the topology of the circuits exhibit a uniform direction of signal flow. However, certain assumptions from signaling theory can be relied upon. It is highly likely that the horizontal cells are heavily involved in extracting chromatic signal information, from the output of the individual chromatic photodetection channels, and forming the chromatic signals transmitted to the brain. This function should be performed prior to, or simultaneous with, the formation of the luminance signal. This is the function of the bipolar cells. It is likely that one of the major tasks of the amercine cells is the extraction of first order spatial relationships between signals from adjacent areas in object space. It is also likely that the input structures of some classes of ganglion cells are involved in more complex spatial signal processing involving larger areas in object space.

In this work, the various signal function levels are designated as follows: Stage 1 The photodetection stage, includes Stage 1a, transduction in the Outer Segments and Stage 1b, translation and amplification in the photoreceptor cells. Stage 2 The signal processing stage, includes Stage 2a, the 1st lateral matrix involving primarily horizontal cells, Stage 2b, the “straight through” matrix involving primarily bipolar cells, Stage 2c, the 2nd lateral matrix involving primarily amercine cells, and Stage 2d involving the input structures of the ganglion cells.

205Hageman, G. & Kuehn, M. (1998) Biology of the IPM-RPE-retina interface In Marmor, M. & Wolfensberger, T. eds. The Retinal Pigment Epithelium NY: Oxford Press pg 363 The Retina 3- 89

Stage 3 The signal transmission, or signal projection stage. Stage 3a involves the output of the ganglion cells. Stage 3b is physically located within the Cerebellum. Although these stages are not marked explicitly, they can be recognized, along with the hydraulic mechanisms of the retina in the “Overall Schematic Diagram of the Processes in Animal Vision” shown in Figure 3.4.1-1. This figure will be subdivided and examined in detail in subsequent Chapters of this work.

3.4.1.2.1 The spectral signal level

There is wide support in the electrophysiological and microspectroradiometry literature for the spectral absorption characteristics of the animal visual process proposed in this work. Unfortunately, a large part of the psychophysical literature conflicts with this work because of an undocumented artifact in those experiments. This artifact, related to one of the Purkinje Effects and the Brezold-Bruche Effect, will be discussed in detail in Chapter 17. There are four specific chromophores of vision used throughout the animal kingdom. These chromophores are found in individual structures related to the photoreceptor cells that vary in morphology greatly within the animal kingdom. These chromophores are classed as the Rhodonines and are derived from retinol in the transition from the blood stream to the IPM via the RPE of the retina. They exhibit equally spaced peak absorptions, based on their stereo-chemistry, at 342, 437, 532 and 625 nm. Some animals only exhibit sensitivity to a subset of these chromophores for a variety of reasons. Humans, and most large terrestrial chordates, cannot sense 342 nm radiation effectively because of the thickness of the outer lens group, consisting of the cornea and “lens.”

The dispersion of these chromophores and their host Outer Segments throughout the mosaic of the retina has been discussed in [Section 3.2.2.1.3] above. They are shown in the upper left of the overall schematic diagram where they are labeled, UV, S, M and L. It is the ensemble of signals from these Outer Segments, grouped according to spectral response, that are described as the spectral channels in this work. 3.4.1.2.2 The signal channel level

These signals emanate from the signal detection stage and are seen to proceed to the signal manipulation stage. Following processing in the three matrices of this stage, the signals are now grouped, according to their information content, into a luminance, a chrominance and an appearance channel.

The luminance information is shown arriving at the magnocellular area of the LGN marked [S] to label the summing path. This information is projected as a monophase signal. There is a sub-group of the spectral signals that appear to be treated differently in the signal manipulation stage. The chrominance information is shown arriving at the parvocellular area of the LGN marked [D] to label the differencing path. The signals along this path are biphase in character.

There appears to be a separate “straight through signal path” for the spectral signals from the foveola that is passed through the signal processing stage directly to the signal projection stage without alteration. This path is shown arriving at the brain marked [S’]. Although neither summing or differencing is involved in this path, the signals are monophase. In some chordates, there is a significant appearance path from the 2nd lateral matrix of the signal processing stage to the Pretectum of the brain and possibly other areas of the brain. As a minimum, this path exists in all chordates to support the control of the line of fixation of the eyes.

Although there is a large convergence in actual neuron count in the signal paths of the retina, there is no similar convergence in the channels of the signal path. The photodetection stage includes no more than four individual chromatic signal channels. The output of the signal processing stage may involve a different number of channels depending on the evolutionary development of the animal. For humans, it typically includes five signal channels, the luminance channel, three chrominance channels and at least one appearance channel. In other animals, particularly hunters, there may be additional highly-developed appearance channels. The signal transmission channels essentially replicate the channels of the signal processing stage, although it is possible that there are appearance channels that originate in the ganglion neurites. 90 Processes in Biological Vision

Figure 3.4.1-1 Overall Schematic Diagram of the Processes in Animal Vision.

A caricature presented as figure 1.2 in Dreher & Robinson206, based on the work of Rodieck in 1988, provides a comprehensive view of the general layout and the complexity of the retina. Unfortunately, it is only a caricature. Interestingly, it defines a blue-cone bipolar, but no red or green cone bipolar. It also seems to define artistically cells with fat inner segments and correspondingly short and chubby outer segments as opposed to other photoreceptors with slim inner segments and slim outer segments. In both cases, the inner ends of all of the outer segments do seem to lie in the focal plane of the optical system (taken as a straight line at this scale). The chubbiness of the Outer Segments of cones, noted above, is not displayed in figure 1.8, taken from the same paper and attributed to Borwein, although the lengthened microvilli of each RPE cell is shown in both. In this figure, the inner ends of the photoreceptors are not shown in the same focal plane, one of the cells is surely out of focus.

Figure 3.4.1-2 illustrates the signaling aspects of the fundamental summing and differencing path of the retina in block diagram form. The actual paths associated with any particular cell are much more complicated. The OS and IS of a single photoreceptor always work as a pair but the output of the pair may be shared between any number of summing/matrixing circuits. Similarly, the ganglion cells act as signal modulators for individual nerve fibers emanating from the retina but they may receive their input signal from one or more summing/matrixing circuits.

206Dreher, B & Robinson, S. (1991) Neuroanatomy of the Visual Pathways and their Development. In Vision and visual dysfunction, Vol. 3. Boston: CRC Press The Retina 3- 91

Note the difference signal formed from photoreceptor 1 & 2. This difference is typically associated with horizontal cells and represents chrominance information. However, the difference signal between 2 & 3 is typically associated with amercine cells and represents appearance information. Both of these signals are biphase and their encoded action potential streams are marked [D]. However, it is very unlikely that they both go to the same area of the LGN. Similarly, the straight through signal from 2, the summed signal from 4,5 & 6, and the signal from the foveola all create monophase signals. However, they do not all go to the same area of the LGN. Chapter 13 will provide complete details concerning the actual circuits of the signal processing stage and their properties. In the more highly developed eyes, particularly those having a fovea, the size of the photodetectors, the presence of certain types of cells and the degree of matrixing in a given zone of the retina are functions of the radius of the zone from the fovea. Thus, in any experimental work, the zone in which the work is being done should be specified by its coordinates relative to the fovea if present. Otherwise, the data cannot be related properly to the overall operation of the eye or to the work of other experimentallists. This Figure 3.4.1-2 Block diagram of principal signal paths of leads to many unneeded contradictions and occasional the eye. The OS of each photoreceptor cell may contain one arguments in the literature. In a few animals, more of four chromophores. The straight through signal paths than one fovea is found in a single eye and it becomes maintain monophasic signals and connect to parasol type doubly important to describe by specific coordinates ganglion cells. Initially, these paths only transmit the zone being investigated. monophase signals from the foveola . At the next level of complexity, the signal processing neurons of the straight By using the above two figures as guides, and through signal paths may collect signals from multiple incorporating the concepts of time-diversity and spectral paths in order to form luminance channel signals spatial-diversity encoding into the signaling plan of the (4,5,6). The differencing paths create biphase signals that visual system, the material provided by Lee in a recent connect to midget type ganglion cells (1,2,3). mini-review is brought into better perspective207, including the conflict between the fourth paragraph and first sentence of the fifth on page 637. The result may provide answers to some of the six open questions he presented in his conclusions. 3.4.1.2.3 The signal projection level

A feature of the retina that has caused considerable confusion in the understanding of the retina involves the nature of the signals projected along the optic nerve. While all of these signals involve pulse waveforms, they carry information by two fundamentally different modes. While the signals passed to the brain along the [S] and [S’] paths involve pulses that appear to be proportional to the monophase luminance of the initial scene, the remaining signals passed to the brain along paths such as that labeled [D] do not exhibit this property. These signals employ a different form of encoding that represents the biphase character of the information transmitted. There are two classes of these signals. Those signals representing the chrominance of the scene and those signals representing the (nominally monochromatic) appearance of the scene. It is likely that some of these biphase signals do not go to the parvocellular area of the LGN but to the Pretectum or other areas of the midbrain. As seen above, the [S’] signals do not go to the magnocellular portion of the LGN. Because of the above variation in signal projection paths between the retina and the brain, the designations parvocellular and magnocellular pathways between the retina and the non-cortical portions of the brain should be avoided. In the above reference, Lee has also provided a tabulation of both the proportion of different types of ganglion cells

207Lee, B. (1996) Op. Cit. 92 Processes in Biological Vision

and their apparent physiological connections. The tabulation contains considerable material that is conceptually related to the conventional wisdom concerning the magnocellular and parvocellular pathways and the center- surround phenomenon which is discussed in the next section. It also defines a broader range of ganglion cell types than outlined above based on morphology. The test methodology used did not separate difference signals related to chrominance information from that related to appearance information. 3.4.2 The center-surround phenomenon (temporary home)

There is a large amount of data related to the response of the visual system to stimuli of a uniform color. This has been extended to concentric stimuli of various colors. However, there is very little theoretical information to explain the exploratory results obtained. Boynton has provided an introductory discussion of these phenomena208. The first electrophysiological experiments related to the receptive fields of vision are attributed to Hartline in 1938. DeValois subsequently studied this area intensely209. More recently, Hubel has provided additional experimental results. Recently, Dacey has presented a variety of papers on this subject. In 1996, he provided a discussion highlighting some of the difficulties and stating: “Although this spectral opponency has been studied for more than 30 years, the underlying retinal circuitry remains unclear210.” This paper did highlight the bistratification of ganglion cells as well as that of the more commonly reported bistratification of lateral cells. The bistratification of the arborization between the dendritic and poditic terminals of the neuron is a feature of the theoretical foundation of this work.

Major difficulties in all of these studies have included;

+ the lack of an adequate theoretical model of the visual system, + failure to correlate the spectrum of the stimuli with that of the chromophores of vision (resulting in cross products in the matrix algebra of the system), and + failure to recognize the limited orthogonality of the chrominance channels of vision.

These problems will be addressed in later chapters. The subject of interest here is the relationship between the arborization of individual neurons of the signal processing stage of the retina and the center-surround phenomenon.

Individual neurons are known to have extensive neuritic arborization supporting interconnections with numerous other neurons. The number of these interconnections mentioned in the literature usually start at around 80-100 and rise from there. Even this number would suggest that a single signal processing neuron could easily exhibit a span of stimulation much larger than the cross section of soma of the neuron. Spans of 250-500 microns are common in the literature211. 3.4.2.1 Types of experiments

Center-surround experiments have been performed based on non-invasive visual evoked potentials and electroretinograms. However, these have given very coarse data. Finer data has been obtained through invasive electrophysiology probing the cortex, the LGN and the retina. A major problem with the probing techniques is the limited scope of the cell sampling that can be achieved in a given experiment. Frequently, about one hundred cells will be probed but only ten to twenty will be found that display the characteristic that was sought. The performance of these cells will be reported on in detail but the others will be ignored. Most of the experiments have involved the lower chordates. Only since the mid-1970's have significant invasive experiments been performed on primates. As the studies evolved, a wide range of spot diameters have been used to evaluate the center-surround phenomenon. Unfortunately, most of these experiments have employed large, generally circular spots of stimulation. When not centered on the fovea, their definition relative to location on the retina has been poor.

208Boynton, R. (1979) Human color vision. NY: Holt, Rinehart & Winston pp. 238-250 209DeValois, R. & DeValois, K. (1975) Neural coding of color. In Carterette, E. & Friedman, M. eds. Handbook of perception, vol. 5. NY: Academic Press 210Dacey, D. (1996) Circuitry for color coding in the primate retina. Proc. Natl. Acad. Sci. USA vol. 93, ppp. 582-588 211Wassle, H. & Boycott, B. (1991) Op. Cit. The Retina 3- 93 3.4.2.2 Span of stimuli versus span of neurons

Most of the center-surround data available is based on the use of a few arbitrary size circular stimuli. Only a few experiments involved a space between the outside of the inner stimulus and the inner diameter of the outer stimulus. This type of data only provides an integral of the response from the underlying mechanism as a function of the diameter of the circle. Analyzing this type of data requires the assumption that the phenomenon is symmetrical with respect to the center point of the field of the stimuli. This assumption is a poor one. By reviewing a large amount of data, some involving long edges, some defendable conclusions can be drawn but a more extensive mapping, using small diameter stimuli at large radii from the center of stimulation, would provide better understanding. Kaneko & Tachibana have provided some data on the response of a cell to various size circular stimuli for in-vitro carp retina212. A related paper provides additional valuable data in other areas213. The original data was provided using a logarithmic abscissa. Although compacting the data, this display does not highlight the underlying relationships. Figure 3.4.2-1 presents their data in both the original and linear form. The two dashed lines on the logarithmic graphs suggest a common functional relationship for the data. Their paper did not present a model of the process they were exploring and the experiment design involved a large number of uncontrolled and/or unmeasured variables. This work would also suggest that 600 and 500 nm were particularly poor choices of wavelength for this type of experiment. Because of these matters, attention will only be called to the spatial aspects of the waveforms. The graphs on the right plot the same data points as on the left. If the data points are assumed to result from an integrative process as a function of stimulus diameter, one could differentiate the smooth curve connecting these points and, ignoring several details, plot the probable signal input from each photoreceptor ultimately connected to this cell. This process suggests that there was little input to the cell from a distance beyond about 500 microns from the center of the stimulus irradiance. In the upper right frame, most of the input appears to occur within 350 microns as suggested by the authors and all inputs lead to an increase in signal amplitude. In the lower right frame, a different result is obtained. A majority of the photoreceptors within a 100-micron diameter of the center of the cell arborization lead to an increase in signal amplitude while a majority of the photoreceptor input from beyond the 100micron diameter appears to cause a decrease in signal amplitude. The net signal amplitude approaches an average of zero in this case after a possible third order input or some transient overshoot due to the procedure employed.

Much of the recent data regarding the dimensions of the arborization of neurons is obtained using very sophisticated dye techniques at the cytological level. A considerable volume of this work is associated with Dacey and his associates214,215. Of particular interest is what they describe as bifurcated dendritic trees. In this work, these trees are functionally separate. One is associated with the dendrite and one is associated with the podite of the neuron. The signal input through the poditic tree is inverted at the axon of the neuron.

Unfortunately, most of the center-surround and arborization data has been interpreted under the assumption that a set of orthogonal Hering axes provide the appropriate foundation. Later Chapters of this work will address these matters. Here it is remains appropriate to look only at the spatial dimensions involved. As Kaneko & Tachibana suggest, the nominal diameter of carp dendritic arbors is usually 60-120 microns with a maximum reported value of 184 microns. Taking 60 microns as the standard deviation of the carp arborization, a scale has been provided between the two right frames calibrated in standard deviations.

212Kaneko, A. & Tachibana, M. (1983b) Op. Cit. 213Yang, X-L. Tauchi, M. & Kaneko, A. (1983) Convergence of signals from red-sensitive and green-sensitive cones onto L-type external horizontal cells of the goldfish retina. Vision Res. vol. 23, no. 4, pp. 371-380 214Dacey, D. (1996) Circuitry for color coding in the primate retina. Proc. Natl. Acad. Sci. USA, vol. 93, pp.582-588 215Lee, B. (1996) Receptive filed structure in the primate retina: A minireview. Vision Res. vol. 36, no. 5, pp. 631-644 94 Processes in Biological Vision

Figure 3.4.2-1 Amplitude versus stimulus size of double opponent cell of carp. Left data using logarithmic abscissa. Dashed lines added for discussion. Right, same data using linear abscissa. Shaded areas suggesting input density from individual photoreceptors. Central scale on right suggesting distance from center of cell to individual photoreceptors in standard deviations. Data points from Kaneko & Tahibana, 1983.

3.4.2.3 Interpretation of experiments

Kaneko & Tachibana indicate in their paper that of 85 “bipolar cells” they examined, only one-quarter (15 on-center and 3 off-center cells) exhibited the double opponent receptive fields they sought. Since their work was in-vitro on a lower chordate, there was no way to determine if the signals from the cells they examined were actually used within The Retina 3- 95

the chrominance channels of the visual process. It also becomes difficult to insure the irradiance was projected onto the retina in a sufficiently precise manner. One of the hallmarks of this work is the fact that there are no achromatic photoreceptors in vision. Therefore, any spatial signal processing must process chromatic signals from photoreceptor cells. This fact complicates the analysis and interpretation of these types of center-surround recordings. They did not indicate how they determined all of the above cells were “bipolar cells.” They did not indicate how they insured the cells were from the bipolar sublayer of the inner nuclear layer and not from the 1st or 2nd lateral sublayers which consist primarily of horizontal and amercine cells. A review of the above statistics, the data of Kaneko & Tachibana and the many arborizations provided by Dacey et. al., suggests that much of the signal information recorded for these cells did not originate at the junction of these cells with an immediately adjacent photoreceptor cell. There is strong reason to believe these waveforms were obtained from a cell at the apex of a much wider signal collection environment. Considering a simple two- dimensional pyramid with one output cell and two input cells, it would be expected that Kaneko & Tachibana would have found about 30% of the cells exhibiting the waveforms they sought. If the pyramid consisted of one output and two layers in the pyramid with each node accepting signals from two other signal processing nodes, one would expect 14% of the neurons examined to exhibit the sought after waveform. However, even this number of stages in the pyramid would not span more than about 240 microns (one sigma). This is about the value Kaneko & Tachibana reported (a peak in their graphs at about 350 microns with no stated tolerance). The waveforms in the upper frames are basically monopolar. However, this may not be the true nature of the signals processed by this cell since it was acquired at a wavelength well beyond the nominal differencing range of this type of cell. The tail also could be the result of the summing of signals from a broad range of distal cells. Alternately, the small negative component of the derivative shown in gray could be caused by a variety of instrumental problems. On the other hand, the waveforms in the lower frames are clearly bipolar. Whereas the upper waveforms could be generated by a bipolar cell that normally only processes monopolar electrical signals, the lower waveforms are electrically bipolar. This type of electrical signal is normally associated with lateral cells within the 1st or 2nd lateral matrices.

Looking at the problem in two dimensions changes the percentages slightly but several reasonable conclusions can be drawn. First, the recorded waveforms were generated as a result of a pyramiding process probably occurring within the 1st lateral matrix of the retina. Second, the pyramiding probably involved a three level pyramid. Third, the recorded signals were probably from the axons of some horizontal cells. Fourth, there is no way to determine whether the recorded waveforms were ultimately used to extract chrominance or appearance information within the brain of the host animal.

Since there is no way to determine the ultimate purpose of the recorded waveforms (their ultimate use could be either for chromatic or appearance purposes), it appears inappropriate to label these waveforms and the presumed receptive fields of the signals from which they were processed to be color related. 3.5 Electrophysiology, morphology & function of the eyes of Mollusca

The eyes and retina of the molluscs are extremely diverse and many show specific ecological adaptations. Messenger reviewed the available data in 1991216 and noted the immense morphological diversity in the photosensitive elements in the phylum. This varies from a variety of simple photosensitive cells, through simple eyes, to the highly-developed complex eyes of the cephalopods. The terminology of the histologist and anatomist who are focused on Mollusca continues to differ significantly from that of those investigating Chordata. The same assertion applies generally to those investigating Insecta (Section 3.6). 3.5.1 The compound eye of Mollusca

As noted in Section 1.7.2.2, the eyes of Mollusca are unbelievably diverse, extending from photo-sensitive spots, to simple eyes to compound eyes. Even the compound eyes exhibit a wide range of specializations adapted to their

216Messenger, J. (1991) Photoreception and vision in Molluscs. In Evolution of the eye and visual system. Cronly-Dillon, J. & Gregory, R. ed. Vol. 2 of Vision and Visual Dysfunction. Boca Raton, FL: CRC Press, pp. 364-397 96 Processes in Biological Vision

environment. 3.5.1.1 Multispectral mollusc retina

As a phylum, it contains all of the expected chromophoric spectral bands. The literature specifically reports the presence of the UV-, S-, & M-channels as expected due to anisotropic absorption by the rhabdom. It also reports a frequently recorded isotropic peak at 500 nm. The instrumentation used to measure this peak must be examined in each case. The arrangement of the chromophoric material in the retina of the higher Mollusca seems to facilitate the measurement of an isotropic (nonfunctional) peak at 500 nm. Limited detailed information is available on the retina of the more advanced members of Mollusca. It obviously consists of multiple individual photoreceptor cells in a two-dimensional array lining the inside of an enclosure opposite the aperture. The retina is of the direct type. The photoreceptors are illuminated at their distal end. The cartoons of Eaken, Wolken and others are relatively simple and difficult to correlate to a two- dimensional array. Both spectral and behavioral data show that the retina is sensitive to at least the S- and M-chromophores217. The spectral response recorded by Hamasaki,218 using electroretinographic techniques, also suggests sensitivity in the ultraviolet spectrum. The animal is sensitive to the polarization of light in at least one spectral region. Young has provided the most details on a retina of Mollusca219 in Figure 3.5.1- 1. This hand drawn caricature is apparently based on visual microscopy. Many finer details expected from an electron micrograph are missing. The identification of the views is also questionable from a draftsman’s perspective. The lower view was not originally aligned to the upper view. It has been realigned in this figure. The lower view appears to be a side view along a fractured surface rather than a true section view. The supporting cells are not as prominent in the upper view as in the lower view. The abbreviations are: bas., basement, ce., cell, co., collateral, eff., efferent, ep., epithelium, f., fiber, in., inner, lim., limiting, mem., membrane, nuc., nucleus, out., outer, pig., pigment, pl., plexus, ret., retina, rh., rhabdom of retina, and su., supporting.

There are two important aspects of the tangential section. First, the individual cells In the tangential section are seen to be symmetrical with the retinula in the center and rhabdomere extending from opposite sides. The heavy black lines, representing four adjacent rhabdomins, appear to form a box like unit similar to that of the crustacean eye. This group could be called a rhabdom if one ignores the rhabdomere Figure 3.5.1-1 Diagram of the retina of Octopus; above as extending outward from the cells into adjacent seen in tangential section and below in radial section. See rhabdoms. On the other hand, a diamond shaped text for interpretation and abbreviations From Young (1971)

217Cronly-Dillon, J. (1966) Spectral sensitivity of the scallop Pecten maximus, Science, vol. 151, pg. 345-346 218Hamasaki, D. (1968) The electroretinogram of the intact anaesthetized octopus. Vision Res. Vol. 8, pp. 247- 258 219Young, J. (1971) The anatomy of the nervous system of Octopus Vulgaris London: Oxford University Press Chap. 16 The Retina 3- 97

rhabdom can be defined by four rhabdomin arranged in a cross. This rhabdom consists of four complete rhabdomins, including both rhabdomere of each cell. This assumes the rhabdomeres are not interdigitated. Assuming this latter arrangement forms a rhabdom, it is interesting to note the presence of two pairs of cells arranged similarly to that of the crustacean eye shown in the previous figure. The caricature of Octopus does not show the interdigitating of rhabdomere although higher resolution work might. It does show rhabdomere arranged orthogonally which is usually associated with sensitivity to light polarization. If this assumption is correct, each rhabdom would include two pairs of rhabdomins. It is quite possible that one pair incorporates the S- and one pair the M- chromophore. The result is an individual rhabdom that is both polarization and color sensitive at two wavelengths. If this interpretation is reasonable, the locations at the center of the rhabdomeres of a given rhabdom, and marked pig. in the upper figure, would be the equivalent of the IPM of the chordate eye. An inset has been added to the figure focusing on the proposed unit rhabdom [xxx ?]. It is consistent with one of the supporting cells providing structural support to, and with pigment material placed around the periphery of, each of these groups. The material surrounding each of these rhabdoms could also act as the wall of a light pipe. Saibil & Hewat220 have provided an alternate configuration and excellent electron micrographs. It shows the OS of the retinula to be 200-300 microns long with the orthogonal microvilli (coated dendrites) about one micron long and 60 nm in diameter. The diameter is consistent with the diameter of the microtubules along the disk stack in Chordata and with the dimensions required to form a distributed Activa. Their characterization of the detailed microvilli arrangement is based on a single retinula and varies slightly from the above interpretation because of their attempt to maintain a cell configuration similar to the Young template for Chordata. Second, the orientation of this figure is unknown with respect to the axis of symmetry found in the Octopus eye. As shown above, the Octopus retina exhibits an axis of symmetry formed by the projection onto the retina of the plane formed by the pivotal axis of the eye and the center of its aperture. The angle between the retinal array and the axis of symmetry could be important when discussing the effect of tremor on the performance of the total visual system.

Valuable insight is also available from the side view of the fractured retina (radial section). The most important feature is that there is essentially no signal processing performed within the retina of Mollusca221. The axons of the photoreceptor cells go directly to the adjacent optic lobe of the brain. There are no lateral processing and no encoding by projection neurons. The figure shows a few efferent fibers entering the retina and contacting the photoreceptor cells through collateral fibers of unspecified function.

Under the above interpretation, the rhabdom of Mollusca is less well defined than the rhabdom in Arthropoda but better defined than the rhabdom (if any) in Chordata. The output of the photoreceptor cells goes directly to the brain without using projection neurons as in Chordata. This is the same configuration as in Arthropoda. 3.5.1.2 Details of the rhabdom

Having electron microscopy of the Octopus retina would be useful. Lacking that data, Figure 3.5.1-2 illustrates the basic geometry of a single mollusc photoreceptor based on Young and using his hierarchal notation222. Although the Young figure showed the rhabdomere of each cell forming triangular collection surfaces, it is proposed that they are more likely to form rectangular surfaces interdigitated with adjacent photoreceptors both across from and perpendicular to this cell. It is also proposed that the features he labeled as “pig” are the Golgi apparatus or mitochondria. In this interpretation, the chromophoric material would be provided from another cell nearby.

A more advanced caricature of a generic individual ommatidium is shown in Section 1.7.2.1.2.

220Saibil, H. & Hewat, E. (1987) Ordered transmembrane and extracellular structure in squid photoreceptor microvilli J. Cell Biol. vol. 105, pp 19-28 221Young, J. (1971) Op. Cit. Chap 17 222Young, J. *1971) Op. Cit. pg 420 98 Processes in Biological Vision

It is proposed that: the photoreceptor cell consists of a nucleus placed proximal to the basal membrane of the retina (as shown) but with an Inner Segment located distal to that membrane. The primary purpose of the Inner Segment is to secrete and extrude multiple cilia of the protein material, opsin, in a direction perpendicular to the length of the Inner Segment. These cilia (rods of protein) are similar to those in Arthropoda and the equivalent of the disks formed in the eyes of Chordata. Upon coating with a liquid crystalline chromophore and contacting by a dendrite of the Inner Segment, these structures become the photosensitive rhabdomere of the cell. The dendrites would be expected to be approximately 250 nm in diameter and can only be identified through electron microscopy. The primary role of the supporting cells of the retina is to produce the chromophoric material and transfer it to the IPM. In this role, they are analogous to the RPE cells of Chordata. 3.5.1.2.xxx The retina of Pecten maximus

More data is available, at a gross level, for the unusual case of Pecten maximus223. Figure 3.5.1-3 shows that Pecten has two separate retina that appear to be arranged back to back. This would imply that one retina must be of the reverse type. However, as seen in the section on mollusc optical systems above, this Figure 3.5.1-2 Early prototypical photoreceptor of conclusion is incorrect. Although the two retina are Mollusca. The form caricaturized by Young in 1971 is back to back in a physical sense, they are not in the shown on the left and upper right (in cross section). The optical path sense. If this cartoon of Land is correct lower right shows an alternate cross section providing and if the generic eye of Mollusca contains a single higher sensitivity and polarization sensitivity through inter- direct retina, Pecten took advantage of an opportunity. digitization. It pushed its original retina away from the aperture, and backfilled with a new retina near the argentea. The result is a mollusc with two reverse retinas in each eye. More careful measurements might locate the two focal surfaces differently. This result would be consistent with two direct retinas in each eye. As indicated earlier, the transmission efficiency of this eye is poor because the unfocussed light must pass through one retina before imaging at the proper image surface. A probable 50% of the light is lost in this process.

223Land, M. (1965) Image formation by a concave reflector in the eye of the scallop, Pecten maximus. J. Physiol. (London) vol. 179, pp. 138-153 The Retina 3- 99

The individual photoreceptors are similar in structure to those of Arthropoda, i.e., the chromophoric material is found in rods exuded from the sides of the photoreceptor cells. This similarity leads some authors to use the same element names as in Arthropoda. However, this is not done consistently and confusion is the result. Major differences appear at the next higher level of organization. Whereas the rhabdom of Arthropoda exhibits a circular symmetry with resp10ect to the centerline of the assembly, this is much less evident or nonexistent in Mollusca. The limited data available indicates an orthogonal grouping of photoreceptor cells to achieve a higher sensitivity to the polarization of the incident light. It is likely that these groupings are repeated across the retina with different groups employing different chromophores to achieve spectral diversity. As indicated in the figure, it is possible that a single group of orthogonal cells could employ more than one chromophore. Further research will be needed to learn the true organization of the retina. Figure 3.5.1-3 Structure of the dual retina of Pecten Menzel224 opened a discussion in 1979 with the maximus and the location of the two focal surfaces. The statement “A mollusc eye containing more than one scale is of axial distance from the argentea. (Modified from photopigment has yet to be found.” He then goes on to Land, 1965) mention the meager amount of available data, the fact that virtually no intracellular recordings had been published, and the ERG data available tended to emphasize the dominant photopigment present. He then reviews the data, pointing out that no measurements in the ultraviolet were available and that two peaks were frequently measured at 475 nm and 540 nm, including those by Cronly-Dillon in 1966 225. Based on the model used here and the location of these two peaks, the data strongly suggests the presence of at least an M-channel and an S-channel chromophore in the mollusc eye. The peak at 540 nm. caused by the M-channel chromophore with a theoretical peak at 532 nm. The 475 nm. peak is due to the Bezold-Brucke Effect in the presence of both an S-channel chromophore with a peak at 437 nm. and the M-channel chromophore. The Bezold-Brucke Effect is normally reported at the psychophysical level. It is observable at the electro-physical level, especially when using a very high impedance (current) probe. A current probe introduced into the IPM is a low impedance device relative to its surroundings. As a result, it will sample and sum the currents from a group of nearby photoreceptors in an uncontrolled manner.

McReynolds & Gorman provide a comprehensive study of the signals emanating from the two retinas of Pecten irradians226. Lacking a credible model, they were unaware of how the eye actually operated. They suggest a 2-3 log unit difference in sensitivity between the two retinas while their imaging light was focused at the entrance aperture of only one of them. They presented considerable electrophysiological data but it is not at all clear which part of the various photoreceptor cells were probed. This leaves much of their data in conflict with other literature and the model presented here. The data in their papers are worth re-analyzing. However, the use of a high impedance probe must be accounted for and their imprecise specification of the probe location recognized.

3.6 Electrophysiology, morphology & function of visual modality of Insecta

224Menzel, R. (1979) Spectral sensitivity and color vision in invertebrates. In Comparative Physiology and Evolution of vision in invertebrates. Autrum, A. ed. NY: Springer-Verlag pp. 537-540 225Cronly-Dillon, J. (1966) Spectral sensitivity of the scallop Pecten maximus. Science vol. 151, pg. 345 226McReynolds, J. & Gorman, A. (1970) Photoreceptor potentials of opposite polarity in the eye of scallop, Pecten irradians. J. Gen. Physiol. vol. 56, pp. 376-406 (Two papers) 100 Processes in Biological Vision

The material in this section is broader than just the retina of Insecta. It is a larger representation of the visual modality of Insecta to show how this modality is a dual of the visual modality of Chordata but employs significantly different anatomical morphological and some histological features. However, except for stage 3, defined below, the electrophysiology of the insect eye and the chordate eye are analogous. The material below is reproduced from Appendix E of this work with the same title as above. The term “process” is used by the authors of the papers in this section as a noun and is used to describe a variety of physical elements with separate functions but not further identified. This is a difficulty that appears to live on to this day among experimentalists. An alternate for process found in some papers is “collateral.” It is meant to include axons and various neurites. In this work, the term process will only be used as a label describing a higher order or broader mechanism. When appropriate, the term element, or a more specific name for a functional element will be used to replace “process” in this work. To orient the reader, Ball has provided an excellent view of the head of a member of Insecta, Figure 3.6.1-1, showing the dominant character of its two compound eyes. The individual hexagonal cornea of each ommatidium is small enough to allow a very orderly array of hexagonal cornea on the otherwise curved surface of the eye.

The terminology of the histologist and anatomist who are focused on Insecta continues to differ significantly from that of those investigating Chordata. The same assertion applies generally to those investigating Mollusca (Section 3.5). This section will review and compare this terminology problem. For a good source of data in this area (although not always professionally edited) appears to be the “Invertebrate Brain Platform227.

Two putative types of compound eyes, consisting of fused ommatidia are found in the literature; the apposition eye (sometimes labeled the photopic eye) employs a one-to-one relationship between a single cornea and a single retinula. A distinctly different superposition eye (sometimes labeled the scotopic eye) is described where the light from a single source is gathered by multiple independent cornea and directed to a single retinula. The superposition eye, originally described in 1891 by Exner, encounters many difficulties when pursued beyond the conceptual stage. Goldsmith & Bernard (pp 221-229) have summarized these discussions. A detailed optical ray tracing activity would be needed to draw firm conclusions regarding this eye. If Nilsson et al. are correct that the cone forms an afocal telescope, the discussions of a superposition Figure 3.6.1-1 Anatomy of the head of an insect. Note the eye are seriously undermined. Only the apposition large number of individual ommatidium fused together to eye will be considered below. form each compound eye. A hair is frequently found emanating from the junction between the hexagonal cornea There appear to also be two major forms of the of adjacent ommatidia. In many species, each cornea ommatidium supporting both the simple and exhibits a very low reflective component due to a dense, but compound eyes of Insecta. The fundamental low average volumetric density, array of nipples protruding ommatidium includes a set of retinula that generally from the bulk structure of the cornea. This array acts as an extend the entire length of the ommatidium. There anti-reflection coating. From Ball, 2016. is also a complex ommatidium involving two sets of retinula extending only about one-half the length of the ommatidium.

227Invertebrate Brain Platform (as of 2016) https://invbrain.neuroinf.jp/modules/htmldocs/IVBPF/Top/index.html A more specific although not totally edited page is https://invbrain.neuroinf.jp/modules/htmldocs/test/General/optic_lobe.html The Retina 3- 101

3.6.1 Background

Much work was done concerning the electrophysiology and histology of the visual modality of Insecta in the middle of the 20th Century. Rockstein in 1974228, Zettler & Weiler229 in 1976 and Land et al230 in 1981 published significant volumes on these subjects. Both the Rockstein and Land et al. volumes were parts of much larger sets of books covering a wide field of vision among many species. Although dated, and in many areas archaic, these volumes remain excellent reference works today. Goldsmith & Bernard presented the broadest summation of the literature (105 pages) within Rockstein231. While they touched on a great many mechanisms, they focused on histology and provided very little electrophysiological information. More recent work has been less than consistent from the functional perspective due to the wide variety of species being explored. The result has been limited new functional data to support the large scale anatomical and histological investigations described in the above volumes. Miller has provided a now dated but broad discussion of ocular optical filtering as it applies to a variety of animals232. Form does follow function, and relies upon function to explain the potentials of different forms to support an ecological niche for the parent organism. Snyder and colleagues produced a large number of papers during the 1970's attempting to model the visual modality of Insecta. However, the modeling was very limited in scope and attempted to begin with a series of assumptions that appeared logical at the time but which now seem quaint233. The paper entitled “Structure & Function of the fused Rhabdom” is cited particularly in this regard. Their firmest foundation was a set of templates originally “from Dartnall's (1953) nomogram which was extended into the UV.” Dartnall did not claim any theoretical or even extensive measurements to support his original nomogram. The peak sensitivities of Snyder’s figure 3 are given as 340 nm, 430 nm and 530 nm for his trichromatic nomogram. After producing at least five papers in 1973, the broadest paper has only been cited about 160 times in the subsequent 40 years. One useful paper from the psychology community was from Chittka & Menzel234. The majority of the papers have provides histograms suggesting peak sensitivity at wavelength within 300-350 nm (UV), 430-450 (S), 520-540 nm (M –) and 600-650 nm (L–). A complicating factor is noted by Chittka & Menzel, “A further accumulation of slopes (of spectral sensitivity functions) can not be exploited for discrimination by animals without red-receptors. It thus appears that pollinators with tetrachromatic systems (such as beetles and butterflies and very few species of Hymenoptera) that possess such receptors. . . .” The variation in the spectral, and polarization, performance, and logically in the histological form of the related sensory neuron receptors of Insecta is very great. It is noteworthy that equation (1) in Chittka & Menzel is labeled the “relative quantum flux P but the equation is in conventional photometry units and does not recognize the 2:1 difference in quantum flux associated with a given narrowband intensity signal with a 2:1 change in wavelength (such as between 300 and 600 nm). This factor is significant when calculating the broad overall spectrum associated with both photopic and scotopic vision.

228Rockstein, M. ed. (1974) The Physiology of Insecta, 2nd Ed. Vol II. NY: Academic Press 229Zettler, F. & Weiler, R. (1976) Neural Principles in Vision. NY: Springer-Verlag 230Land, M. Laughlin, S. et al. (1981) handbook of Sensory Physiology, Volume VII/6B. edited by Autrum, H. et al. NY: Springer-Verlag 231Goldsmith, T. & Bernard, G (1974) The visual system of insects in Rockstein, M. ed. The Physiology of Insecta, 2nd Ed. Vol II. NY: Academic Press page 166-272 232Miller, W. (1979) Chapter 3, Ocular Optical Filtering In Comparative Physiology and Evolution of Vision in Invertebrates: Volume 7 / 6 / 6 A of the series Handbook of Sensory Physiology pp 69-143 233Snyder, A. Menzel, R. & Laughlin, S. (1973) Structure & function of the fused rhabdom J Comp Physiol vol 87, pp 99-135 234Chittka, L. & Menzel, R. (1992) The evolutionary adaptation of flower colours and the insect pollinators’ colour vision J Comp Physiol A vol 171, pp 171-181 102 Processes in Biological Vision

The phylogeny of the butterflies is exceedingly complex with many groups within the various groups subordinate to Lepidoptera. As a convenience, this phologeny is reproduced here; Kingdom: Animalia Phylum: Arthropoda Class: Insecta Order: Lepidoptera (Butterflies and moths) Suborder: Rhopalocera Superfamily (at least three) There are multiple suborders and many families within superfamilies below the above descriptors dating from the Palaeocene era, about 56 million years ago. The current Wikipedia is a useful guide in this area as these descriptors change frequently. The Url www.itis.gov is a much more authoritive, but large, taxonomy. Chittka & Menzel focus on the color discrimination between flowers based on “slope detection” associated with a single spectral channel (within stage 1, see below) rather than differential signal discrimination based on channel differencing in stage 2 and subsequent information extraction in Stage 4. Both techniques are used in the color vision modalities of all species known to have such visual capabilities. The slope detection technique is used specifically when performing differential color discrimination between small color differences. Snyder described potential optical coupling and electrical coupling between sensory channels emanating from the ommatidia of Insecta based on his estimates of the detailed histology of various species, and his extended nomogram from Dartnall. The importance of this coupling is less severe based on the theoretical spectra presented in this work and used in Section 3.6.3.1. However, it may be significant in some species. His paper cited above includes two spectral responses that exhibit two relative peaks. He goes to some length to demonstrate it is not due to electrical cross talk due to imprecise probing that resulted in intercellular recording from two separate sensory receptors.

In general, the block diagram and stage designations used in this work are compatible with the sensory modalities of Insecta as described in Section 1.5.1 and further annotated in Figure 3.6.1-2. The visual modality of Insecta is much simpler than that of Chordata, partly because the entire nervous system of the phyla is much simpler. As a general rule, no stage 3 neural circuits are found in Insecta (except possibly in very large or extinct species) that employ pulse signaling and generating what are labeled “action potentials.” The distances between the output circuits of one stage or ganglia and the input circuits of another are generally less than 2 mm, the nominal criteria for introducing stage 3 signal projection techniques. The Retina 3- 103

Figure 3.6.1-2 Annotated visual modality block diagram applicable to Insecta. The diagram is modified from and compatible with that used throughout Chordata. Retina us used to name a group of fused retinula. The nomenclature for axon names have been added for convenience. The subscript x is replaced by the letters UV, S, M or L for the axons of sensory receptor and by the letters O, P, Q or R for the axons of lamina output neurons to indicate the spectral sensitivity of the signals they carry. These axons may also carry spatial information signals. See text.

3.6.1.1 Diversity among eyes of Insecta

As suggested in the previous paragraphs, the diversity among the eyes of Insecta is an order of magnitude greater than among the eyes of Chordata. The visual modality of Insecta,

C can be explored using the same block diagram as used within Chordata, C can be described completely and concisely using the Electrolytic Theory of the Neuron, C employs the same photodetection/de-excitation mechanism as in the eyes of Chordata, C employs the same four spectral sensitivity bands as in chordates, UV–, S–, M –, & L–, C involves animals small enough that they do not encounter the limitation on UV performance, due to absorption by the lens behind the cornea, of large chordates, C employs stage 2 signal processing circuits that appear analogous to those of Chordata (except for the assigned names), C involves stage 4 and stage 5 circuit topologies that are so simple they can frequently be defined at the individual functional engine level, 104 Processes in Biological Vision

On the other hand the visual modality of Insecta,

C employs multiple structural and functional forms of “eyes” C does not employ stage 3 signal projection signals of the pulse (action potential) type, C reorients the sensory receptor active material relative to the axis of stimulation to allow the detection of polarized light using techniques not found in Chordata, C employs a nearly infinite variety of combinations of spectrally sensitive and polarization sensitive receptor configurations within a single sensory receptor grouping (rhabdom), C occasionally uses sensory receptor groups in rhabdom that are so small, they frequently limit the L–channel spectral sensitivity, A fundamental problem in research among the very large numbers of Insecta within an Order, Family and Genus is that members of these groups frequently exhibit different visual characteristics based on their environmental niche or other criteria. These differences make it very difficult to describe the visual modalilty of Insecta without reference to a taxonomy based on the visual capabilities of various species and their grouping into Families, Suborders, Orders and potentially higher groupings. This work will only attempt to highlight a few of these, sometimes subtle, differences among groups (Section 3.xxx). It is extremely difficult to present a “model” eye of even the butterflies of the Class Insecta as Nilsson et al. attempted to do in 1988.

3.6.1.2 Simple versus compound eyes–apposition versus superposition etc.

The eyes of Insecta consist of the unitary ocular or simple eye, and/or a group of fused ocular described as a compound eye. The simple ocular consists of a single optical aperture, the cornea. It is generally followed by a “lightpipe” described as the crystalline lens. The cornea is a simple lens generally characterized as a thin lens by opticians. Conversely, the crystalline lens is generally described as a thick lens. It may exhibit both an entrance aperture with optical power and an exit aperture with optical power. In between these two apertures is a solid material with a graded index of refraction. In the general case, it appears the optics of the simple eye constitutes a non-inverting terrestrial telescope that projects an image of the external environment into the light sensing element of the eye known as an ommatidium in the world of Insecta. The ommatidium contains a group of visual sensory neurons labeled retinula surrounding a central light pipe called the rhabdom. The sensing portion of the sensory neurons, called the rhabdomere are inserted into the rhabdom in a variety of configurations depending on the Order and Species of the particular specimen. Some of these configurations support the sensing of the polarization of the incident light; some do not. Virtually all of the rhabdome of Insecta (with some putative exceptions) sense utraviolet (UV), short (S–), medium (M–) and long (L–) wavelength light.

The compound eye of Insecta consists of a large number of simple eyes fused together to form a large collection aperture eye (generally approaching a hemisphere in form) supporting anywhere from a few thousand to a hundred thousand ommatidia. In the compound eye, each cornea is constrained to a hexagonal shaped aperture to support the overall package as shown above.

The degree of fusion may vary significantly between the apposition form and the superposition form. In the apposition form, each external facet is directly associated with a single detector assembly. The resulting assembly is described as an ommatidium. In the superposition form, the facets form a geodesic dome well separated from the detector assemblies. The external surface of the superposition eye is necessarily spherical. A group of facets direct light from a single source to a single detector assembly. A discussion of the superposition eye will be ignored until Section 3.6.2.4. 3.6.1.2.1 Potentially more complex simple eyes

The literature includes a number of references to an ocellus containing up to 100 retinula, grouped into several ommatidia behind a single cornea (and presumably a single crystalline cone). Until these potential configurations are more fully documented, they will be ignored in this discussion. 3.6.1.3 Fundamental versus complex ommatidia

Differentiating between the fundamental ommatidia, consisting of retinula that extend the length of the ommatidium, and the complex ommatidia, where the retinula are divided into ranks arranged serially along the length of the ommaticium must currently be done primarily using caricatures. Laughlin (1976) describes a fundamental ommatidium in his figure 2 that will be discussed in detail in Section 3.6.3.3. His text suggests the fundamental ommatidium of the dragonfly, Hemicordulia tau, contains only six retinula that extend the length of the ommatidium. Smola (1976) provides cross sections of the worker bee, Apis mellifica, ommatidium that shows eight The Retina 3- 105 retinula of nominal equal cross section within a fundamental ommatidium at a distance of 130 microns below the crystal cone. Smola & Meffert (1975 presented similar information for the cockroach, Periplaneta americana235, and the blowfly, Calliphora erythrocephala236. Their investigations of the blowfly defined six retinula sensitive to the conventional visual spectrum of humans, and two retinula, R7 & R8 with sensitivities in the UV peaking at 344 nm with no sensitivity in the conventional visual spectrum. [xxx get both citation ] Jarvilehto & Moring (1976) indicated a total of eight retinula of similar size in the cross section of the ommatidium of blowfly with each showing significant polarization sensitivity. Zettler & Weiler (1976) provided Figure 3.6.1-3 describing a fundamental ommatidium organization in the fly, Diptera, along with additional information regarding the projection of the sensory neuron axons to the lamina (and R7 & R8 projecting lvf axons bypassing the lamina). The caption and notation supporting this figure suggest the cartridge is summing signals from adjacent ommatidia in order to create a type R–, broadband luminance signal, emanating from the lamina as part of a color vision capability. It is questionable whether all of the ommatidia are “looking” in the same identical direction.

Stavenga & Arikawa (2006) describe a complex ommatidium containing two distinct groups of retinula, R1 to R4 in the distal ommatidium and R5 to R8 in the proximal ommatidium, in the following figure. They also defined three types of complex ommatidium based on a different criteria; the number and variety of rhabdomere sensitive to UV and Short wavelength light. However, they encountered a paradox, “A most curious property of insect ommatidia has thus emerged, namely that the visual pigment expression patterns are not identical in different ommatidia, even though the anatomy of the ommatidia seems virtually indistinguishable.” Rewording slightly, the putative DNA patterns and their proposed expression does not conform to the measured results in the laboratory! Based on this work, the spectral sensitivity of a rhabdom is not dependent on its anatomical characteristics or its DNA coding related to proteins. They were investigating the butterflies of the Order Lepidoptera and further analysis is undoubtedly under way. Sauman et al. (2005) encountered a similar paradox, “Strikingly, an area at the dorsal-most margin of the monarch eye stands out from the rest of the retina: cross-sections of this area showed that the R1 through R8 photoreceptor cells express only DpUVRh mRNA (Figure 3A).” Figure 3.6.1-3 Schematic diagram of the anatomical organization of the fly, Diptera. “Solid black dots represent Stavenga & Arikawa (2006) cite Sauman et al237. as type R1-R6 receptors of fundamental-type ommatidia “all describing a more graded arrangement of sensory looking in the same direction” and converging on one neurons, from retinula extending the length of the cartridge of the stage 2 lamina.” See text. From Zettler & ommatidium to arrangements that clearly exhibit two Weiler, 1976. distinct ranks. “The monarch photoreceptor cells are arranged in a semi-tiered fashion (Figure 1B), in which the cell bodies of the R1 and R2 cells are widest near the crystalline cone and then taper considerably as they approach the basement membrane. The tiny R9 cell sits just above the basement membrane.” Their figure 1B is a characture rather than a micrograph.

235Mote, M. & Goldsmith, T. (1970) Spectral sensitivities of the color receptors in the compound eye of the cockroach, Periplaneta J Exp Zool vol 173, pp 137-146 236Smola,U. & Meffert. P. (1975) A single-peaked UV-receptor in the eye of Calliphora erythrocephala J. Comp Physiol vol 103, pp 353-357 237Sauman, L. Briscoe, I. Zhu, A. et al. (2005) Connecting the navigational clock to sun compass input in monarch butterfly brain. Neuron vol 46, pp 457– 467. 106 Processes in Biological Vision

Sauman et al. also noted an additional variation among monarch butterlies, Danaus plexippus, “Like many insects (Labhart and Meyer, 1999), monarchs possess photoreceptors in the dorsal rim area (DRA) of the compound eye that are anatomically specialized for polarized-light detection (Reppert et al., 2004).” It appears that the order Lepidoptera (butterflies and moths) includes many insects exhibiting complex ommatidia. Simultaneously, the order Odonata (dragonflies & damselflies) includes many insects exhibiting fundamental ommatidia. However, there are 34 orders subordinate to the class Insecta238. Where the differentiation in form occurs in the taxonomy of Insecta is not clear based on this investigation. However, it is an important differentiation when reviewing the literature of the visual modality of Insecta. The URL www.itis.gov provides an excellent entry point to the taxonomy of insects. http://www.itis.gov/ItisDataTools/jsp/hierarchy.jsp provides a list of all Orders within Animalia. It includes at least 28 Orders under the Class Insecta. Each Order varies from a few dozen to one- half million species. [xxx See Excel file Insecta taxonomy under Insecta ] 3.6.1.4 Reconciling the definition of the “pigments” of Insecta vs other animals

As noted by Goldsmith & Bernard on page 166 of the second edition of “The Physiology of Insects, ”As in the first edition, the problem of functional units continues as a recurrent theme.” This quotation from 1974 can be paraphrased in the present. This section will only address the clarification of the terms pigments and chromophores related to vision.

Several distinctions must be made between the common and scientific descriptions related to “pigments.”

These include;

1. the common descriptions used for humanly perceived colors and the standardized descriptions of color science.

2. The distinction between material exhibiting color used for optical shielding purposes, that stored as proto- chromophores, and the actual chromophores of vision.

As noted below in Section 3.6.2.1, the term pigment is almost universally used to describe a material exhibiting a distinctive color as perceived by the human visual system. It is conventionally a bulk material that is observed in artistry and printing by reflected light. A commercially viable pigment has a well characterized spectral response to reflected light as a function of wavelength.

A chromophore is a material that is sensitive to stimulation by light. It may participate in a conventional chemical process when stimulated by light or it may participate in a quantum-mechanical process that leaves the chromophore unchanged in a chemical sense. Only true chromophores are employed in the quantum-mechanical transduction process of animal vision. They are usually found in monolayers of the liquid-crystalline form coating substrates in direct contact with the dendritic elements of sensory neurons (or the dendrites themselves). As used in vision, chromophores are very efficient absorbers of light and appear black by reflected light (they do not reflect a significant amount of light). When stimulated, they assume an electronically excited (quantum-mechanical) state and appear transparent. In the case of the retina of Chordata, the ophthalmologist observes the material behind the chromophore when it is in the transparent excited state. This may be a highly reflective tapetum or some other biological tissue.

Closely associated with the chromophores, from both the chemical structure and physical distance perspectives, are the proto-chromophores (sometimes labeled visual pigments). These materials are usually stored in bulk form, typically described as granules within a nearby cell associated with the sensory neurons. In the case of chordates, these cells are labeled retinal photoreceptor epithelium (RPE). No explicit name for these types of cells have been found in the literature of Insecta. The proto-chromophores are typically retinenes or retinines derived from vitamin A. As developed by Goldsmith & Bernard, most of the pigments associated with the optics of vision are complex, multi-ringed molecules related to the xanthone family. They are readily oxidized or reduced. As noted in the Merck Index, the perceived colors of members of these families, by reflected light, varies widely depending on their chemical environment. The perceived colors of yellow, orange and red are frequently associated with this family. Goldsmith & Bernard continued using the name “ommochromes,” but this term is now archaic. They also describe one of many ways these molecules can be created. They also describe a second family of pteridines, that are

238http://www.insectidentification.org/orders_insect.asp The Retina 3- 107 predominantly red, yellow or colorless. They describe a variety of properties of these materials observed in-vivo. Several investigators have discussed the physical movement of the pigments associated with the optics. It may be the pigments do not move but are either oxidized or reduced in a given region, thereby changing their appearance in that region dramatically (generally causing them to become transparent). 3.6.1.5 Color vision potential of Insecta

As of 2016, there is negligible doubt about the ability of Insecta to demonstrate color vision via electrophysiological means as well as behavioural means.

The subject of color vision within Insecta appears to be as controversial in the recent literature as it was among the literature of Chordata for many years. The discussions have been more complicated because the human investigator can not sense the ultraviolet spectrum, or the polarization parameter associated with each spectrum that play such important roles in insect vision. However, using the multiple stage description of the visual modality of insects, it appears quite definitive that many if not most insects have the capability of sensing a chromatic environment not unlike other animals. The stage 2 signals emanating from the lamina show all of the characteristics required to form a complete color sensitive visual modality. In some cases, it may be based on a trichromatic color spectrum based on UV–, S– and M – light. In the more general case, it is tetrachromatic, including the L– spectrum, plus a polarization component. As an example Sauman et al. (2005) asserted with regard to the Monarch butterfly, Danaus plexippus, “It is therefore likely that monarchs have tetrachromatic vision based primarily on three opsins and a lateral filtering pigment.” Sauman et al. offered no block diagram or circuit schematic to support their assertion. As noted in the previous section of this work, the protein opsin plays no functional role in vision. The primary functional discriminator is the specific member of the Rhodonine family of chromophores used to coat the microvilli grid of the rhabdomere of a particular sensory neuron. The character of this coating cannot be determined by anatomical or histological experiments.

The differential signaling neurons within the “lamina” of stage 2, known histologically as large monopolar cells (LMC) are analogous to the horizontal cells of chordate vision. As in the horizontal cells, the soma plays no functional role in the visual modality. The electrolytic output signals from LMC are functionally bipolar. Their usage has been extended to not only form differential O–, P– and Q–channel signals but also e-vector axis discrimination among polarization channel signals. The three differential signals are the basis of color vision in humans (Section 17.3.3 and specifically Section 17.3.3.6). An extension of the New Chromaticity Diagram to 300 nm would display the complete spectral capability of insects with full spectrum vision. See also Section 3.6.6. 3.6.1.6 A generic eye of Insecta

Figure 3.6.1-4 shows an elaborated simple eye consisting of most of the features to be found in any eye of Insecta. This eye includes the stage 0 optical elements, the stage 1 sensory neural elements, and potentially a ganglia of synapses supporting connection to the stage 2 signal processing neurons located outside the ommatidia of stage 1. Stage 0 includes the light waveguide formed by the outer envelope of the rhabdom. The rhabdom consists of the receptor portions of each sensory neuron, labeled its rhabdomere. These rhabdomere and their associated sensory neurons are frequently stratified into a distal group and a proximal group. In the illustrated complex ommatidium, the rhabdomere are stratified into two assemblies labeled the distal part of the rhabdom (D) and the proximal part of the rhabdom (P). The arrangement of the complete sensory neurons, housed within the distal and proximal parts of the retinula of stage 1 are shown in cross-sections below the distal (D) and proximal (P) portions of the retinula. Each cross-section includes four numbered sensory neurons as shown. The un-labeled lobes are the projected images of the sensory neurons of the other part. The central shaded portion of these representations represent the rhabdomere (receptor portion) of each sensory neuron. The shape of the shaded area varies widely between circular and highly rectangular regions as discussed with regard to their waveguide properties in Section 3.6.2.3.3. 108 Processes in Biological Vision

Figure 3.6.1-4 A generic simple eye and component of the compound eye of Insecta REDRAW FOR QUALITY. Arrow pointing to the right represent incident light. Arrows pointing to the left represent light reflected at the tapetum (if present). CL; corneal lens. CC; crystalline cone. CZ; clear zone. D; distal part of retinula in the complex ommatidium. P; proximal part of retinula in the complex ommatidium. B; basal part of retinula and ommatidium. T; tapetum. PP; primary pigment area. SP; secondary pigment. Not to scale, retinula is typically smaller diameter than corneal lens. A rotation in the geometry of the waveguide is assumed to occur between the distal and proximal parts of the tiered, or stratified, rhabdom as shown by the circular arrow and in the cross sections. Lower left; arrangement of sensory neurons and their rhabdomere. When the e-vector of the incident light is parallel with the microvilli, the light is maximally effective at stimulating the chromophore coating the microvilli. See text. Compare to figure 3a of Stavenga & Arikawa, 2006.

The precise configuration of the crystalline cone–rhabdom interface remains controversial. Nilsson has provided considerable material on this subject, and included an electron–micrograph of the region for Argynnis paphia. It may differ significantly from that of other members of Lepidoptera.

The rhabdom is shown as a square waveguide with a possible twist of 45 degrees in the area of transition between the distal and proximal parts of the stratified, or tiered, rhabdom. This is different than most depictions, such as that of Stavenga & Arikawa to emphasize a point. A circular cross-section guide is ideal for circularly polarized radiation. However, a rectangular cross-section guide is preferred for linearly polarized radiation (Section 3.6.2.3.3). The grid polarizers of the microvilli (containing conductive fluid rather than conductive wires) clearly select, or convert the randomly polarized stimulus to, linearly polarized light within the ommatidium of most species. Altner & Burkhardt have investigated the twist in the rhabdom of the dorsal eye of the male March fly, Diptera, Nematocera, Bibionidae Bibio marci 239 A twist has been observed in many species of Insecta. However, Altner & Burkhardt acknowledge the continuing controversy and provide several citations. If polarized light is not an important functional element in the proximal portion of the ommatidium, the rotation suggested by the circular arrow between the two parts may not be present. Goldsmith & Bernard (page 219) presented the theoretical modal patterns of light in a circular waveguide. However, no empirical evidence was provided that the rhabdome of all members of Insecta conformed to this assumption. The alternative is the well-known rectangular waveguide. It requires a trained eye to differentiate between the modal patterns of the circular and rectangular waveguides. See Section 3.6.2.3.3. Goldsmith & Bernard did not describe or even speculate on the mechanisms related to polarization within the rhabdom. It is doubtful they were aware of the grid-type polarizer described in this work. They reported on

239Altner, I. & Burkhardt, D. (1981) Fine structure of the ommatidia and the occurrence of rhabdomeric twist in the dorsal eye of male Bibio marci (Diptera, Nematocera, Bibionidae) Cell Tis Res 607-623 The Retina 3- 109 experiments using crustaceans where the microvilli were excited by lateral light relative to the axis of the rhabcom. They employed crustaceans because of the larger dimensions of the microvilli structures within a rhabdom. They described the orientation of the e-vector of polarization relative to the grid of microvilli based on this non- vivo excitation (page 249). Their description is counter to the more recent text of Hecht quoted below. “The simplest linear polarizer in concept is the wire-grid polarizer, which consists of a regular array of fine parallel metallic wires, placed in a plane perpendicular to the incident beam. Electromagnetic waves which have a component of their electric fields aligned parallel to the wires induce the movement of electrons along the length of the wires. Since the electrons are free to move in this direction, the polarizer behaves in a similar manner to the surface of a metal when reflecting light, and the wave is reflected backwards along the incident beam (minus a small amount of energy lost to joule heating of the wire). For waves with electric fields perpendicular to the wires, the electrons cannot move very far across the width of each wire; therefore, little energy is reflected, and the incident wave is able to pass through the grid. Since electric field components parallel to the wires are reflected, the transmitted wave has an electric field purely in the direction perpendicular to the wires, and is thus linearly polarized. Note that the polarization direction is perpendicular to the wires; the notion that waves "slip through" the gaps between the wires is wrong240.” Wehner has provided figures 5 & 6 showing both rectangular and circular rhabdoms in a variety of families of Insecta241. His caption to figure 5 appears to contain a typographical error. The diameter of the microvilli are typically between 40 and 70 nm rather than μm based on his scale bar. He includes an extensive discussion of the twisting of the retinula within a given ommatidium. His discussion of E-vector navigation on page 321 is useful but probably archaic. “At least in the bee, only the UV receptors are involved in E-vector navigation.” His spectral sensitivity of the M –channel of the worker bee in figure 29 provides an excellent match to the theoretical 532 nm spectrum of this work.

Nilsson et al. have provided a major work in the visual modality of butterflies. However, they did not identify the feature of the ommatidia that acts as a polarization analyzer (the term is used to define either of two analyzers, one introducing polarization and a second used with a detector to identify the principle axis of the e-vector of the polarized light present. They also did not consider the potential for rectangular waveguides even though many investigators have provided data suggestive of this possibility. All of their citations in this area were to close collaborators. It may be useful to augment their paper with additional information from the fiber optics community242 (Section 3.6.2.3.3). Their paper is reviewed in Section 3.6.2.3.4.

The stack of rhabdomere associated with an individual sensory neuron are described at lower left. Each sensory neuron supports a large number of individual dendrites that in-turn support a large number of microvilli arranged in a grid-like structure that is coated with the appropriate chromophoric material. Each of these structures is labeled a wafer in the figure. It is to be demonstrated that the grids of all of the wafers are aligned relative to the incident light. This would appear to be the most efficient arrangement. These grids introduce the polarization sensitive feature of insect vision (Land et al., 1991 fig 27). The spacing between the microvilli of a specific wafer is less than the wavelength of the light to which the ommatidium is sensitive. Thus the grid-type polarizer can act as a modulator of the light sensed by the sensory neuron. It can also act as a polarization analyzer for the light passed through the distal part of the retinula. Optimally, the portion of the rhabdom beyond the distal portion would be rotated 45 degrees to optimize the projection of the now rotated polarization vector associated with the remaining light, in species still utilizing polarization in the sensing mechanism of R5-R-8. The number of wafers supporting a given sensory neuron can be compared to the number of discs in the outer segment of the sensory receptors of the chordate retina243. As demonstrated by the re-crystallization experiments reported by Arikawa et al. (1999a), there is no opsin required for the operation of the ommatidium of the insect retinula (Section 3.6.4).

The term cartridge is used variously in the histology literature when speaking of various families of Arthropoda. In some cases, it refers to the above parts of the ommatidium. In other cases, it is used to describe the individual sensory neurons (R1 through R8 or R9). The most appropriate usage has been to describe the ganglia associated with a given ommatidium but located behind the basal (or basement)

240Hecht, E. (1990) Optics, 2nd ed., Addison Wesley ISBN 0-201-11609-X. Chapter 8. 241Wehner, R. (1976) Structure and function of the peripheral visual pathway in Hymenopterans in Zettler, F. & Weiler, R. eds. Neural Principles in Vision. NY: Springer-Verlag page 292 242Marcuse, D. (1982) Light Transmission Optics, 2nd Ed. NY: Van Nostrand Rheinhold 243Land, M. Laughlin, S. et al. (1981) handbook of Sensory Physiology, Volume VII/6B. edited by Autrum, H. et al. NY: Springer-Verlag page 313, fig. 27 & 28 110 Processes in Biological Vision

membrane of the eye. These cartridges constitute individual elements in the lamina of stage 2. In some concepts, the ommatidium also contains some synapses between the axons (generally identified by their associated receptor number of the stage 1 sensory neurons (ex., R3) and the neurites of stage 2 signal processing neurons (generally identified by their sensory neuron source but using the letter L (ex., L-3). These synapses may occur in knots (or ganglia). The individual neurons of stage 2 are identified by a different nomenclature. In other concepts the axons of the sensory neurons extend to beyond the physical boundary of the ommatidium and into the lamina of stage 2.. Each simple eye consists of a complex stage 0 optical system that can vary significantly among families and species. In the generic form shown, the corneal lens (CL) consists of a two-element lens, sometimes described as a corneal lens and an associated corneal process (CP). In a variety of moths, the initial corneal lens exhibits an antireflection coating (AR) created by a “nipple array” of projections from the material of the cornea (Section 3.6.3.4.1) but with an average, or effective, index of refraction of one half of the difference in index between the cornea and air. This lens group is associated with the subsequent crystalline cone (CC). The spacing may be negligible or significant in a given species. The character of CC has not been clearly elucidated in the literature. However, Nilsson et al. have described it as consisting of two optical elements forming a terrestrial (or non-inverting) telescope. In such a configuration, the acceptance angle of the proximal aperture of the waveguide formed by the rhabdomere may be significant. Most histologists have described stage 0 like Ribi, 1987, as consisting of a dioptric apparatus with both a cornea and a crystalline lens (cone). Such descriptions appear in many textbooks and journal articles but will not be adequate in the following discussions.

While the rhabdom is typically shown as cylindrical, electron microscope images typically demonstrate the envelope of the rhabdomere is a parallelepiped, more nominally a square. Land et al. have shown this relationship in their figure 24 in 1991 for the compound eye of Daphnia. This condition is also demonstrated by the modes of axial energy transmission through the rhabdom. Furthermore, it is the only form that would accommodate the polarization introduced by the grid-polarizers formed by the microvilli of the individual rhabdomere (Section 3.6.3.1).

Each of the sensory neurons introduces its sensory receptor component, the rhabdomere into the central optical waveguide created by the rhabdom. The input end of the rhabdom accepts light from the preceding stage 0 optical elements (within the acceptance angle of the waveguide). Many ommatidia of Insecta exhibit a striated or tiered arrangement of the sensory neurons. Neurons labeled R1–R4 in the distal group (D) and neurons R5-R8 in the proximal group (P). The rhabdomere are able to intercept a designated spectral portion of the light projected into the waveguide. This light is transduced into an electrical signal within the dendrites (microvilli) of the sensory receptor neuron. This electrical signal is processed by the two Activa (biological transistors) found in each sensory receptor neuron and appears in the axoplasm and at the pedicle of the axon of the neuron. The signal is passed from the pedicle of the axon via a synapse to one or more orthodromic neurons forming the Lamina and/or distal medulla of stage 2. There is an unresolved question of whether the ninth sensory receptor neuron (R9) is actually light sensitive (and belongs to stage 1, or is actually a signal processing neuron of stage 2. Its location and properties appear quite similar to the so-called eccentric cell of the retinula of the crustacean Limulus. Ribi (1987) notes, “The ninth proximal retinula cell. . . makes no synaptic contacts in the lamina.” Laughlin (Land et al., 1991, page 173) minces his words regarding the eccentric cell of Limulus, “The eccentric cell is, judging by the poorly developed microvilli, and its position in the ommatidium, a specialized photoreceptor. Its particular function is to integrate recepor input and then transient the resulting signal to the brain. . .Simultaneous intracellular recordings from retinual cells and the eccentric cell show that they are linked. . .” The simple eyes, and particularly the compound eyes, are reported to contain a variety of bulk pigment particles (PP) along the area adjacent to the crystalline cone (CC). Both eyes are generally reported to contain a variety of bulk pigment particles (SP) stored in the area surrounding the ommatidium, but at a distance outside of the waveguide wall that makes their influence on the light within the rhabdom questionable. It is more likely these pigment particles are used to form the chromophores deposited on the surface of the microvilli forming the rhabdomere of a particular sensory neuron. Some of the above particles appear to be quite dynamic in their presence and/or appearance. Consistent data in this area is difficult to obtain. The particles are frequently translucent (and sometimes fluorescent when subjected to ultra-violet stimulation). Whether the particles move or merely change their opacity is usually not discussed or demonstrated. Their visual appearance is frequently characterized poorly using terminology that does not conform to the rules of additive and subtractive color used in the printing and other industries. The precise spectra of these particles is needed to understand their precise roles. The basal section (B) is associated with a ninth cell that is usually described as the ninth sensory neuron receptor (R9). As noted above, there is considerable question whether this is in fact a sensory neuron of stage 1 or a signal processing neuron of stage 2 (similar to the eccentric cell of Limulus). R9 is generally reported to not contain or be The Retina 3- 111 associated with any pigment particles, and to not contain any axons (R-9) connecting to any orthodromic lamina via a neurite (L-9). In other representations, the basal section is associated with a “monopolar cell,” not unlike the eccentric cell of Limulus. The monopolar designation is extraneous, the cell exhibits all the functional characteristics of a normal neuron, with 3-6 definable electrical terminals, including two associated with the axon, an electrical power connection and a electrical synapse connection at the pedicle. The tapetum (T) is frequently considered a continuous structure serving more than one ommatidium in the compound eye. It is generally perforated to allow the routing of axons and/or neurites through its surface. Ribi also makes an observation likely to be important with respect to color vision in insects, “investigations suggest that Papilio augues has at least one lamina-cell fibre, L-1, which receives additive inputs from all svf (short visual fibres from each receptor) and (long visual fibres) lvf 1 and lvf 2.” This would suggest P aegues creates a broadband spectral channel similar to the R–channel of this work and found throughout Chordata. Ribi also notes, “Comparable L-neuron types in each cartridge, with inputs from receptor cells displaying different spectra sensitivities are found in other insects, (example, bee & dragonfly).” Ribi goes on to note the presence of L-fibres with broad spectral sensitivities and other L-fibres with apparently narrow spectral sensitivities commensurate with differencing between the UV–, S–, M – & L– to generate relatively narrow spectral sensitivities corresponding to the O–, P– & Q– channels of chordate vision. Ribi discusses the interaction of spectrally different signals interacting antagonistically and notes, “In P. aegeus, narrow peaks are not spread through a spectrum between 500 nm and 550 nm (exactly as predicted in this work, and found among Chordata). Ribi’s discussion did not address the question of signals of different polarization sensitivities being combined to form alternate (and potentially parallel) O–, P–, Q– or R– channel signals.

The draft cited in the longer URL of the Invertebrate Brain Platform. “Optic Lobe,” asserts the signaling within the insect eye conforms to the sequence indicated in the above figure. In some discussions, the lamela is described as the first of two medulla. The lamina, lamina ganglionaris, is described as “second-order visual neurons that receive direct input from the photoreceptors (= retinula cells).”The lobula is described as the second, or proximal, medulla. The reference also indicates the visuotopic field of each eye is preserved in the topology of the stage 1, stage 2 and initial stage 4 circuits “similar to arrangements in the mammalian visual system.” Ribi makes a slightly stronger assertion from a geometrical perspective, “Together the photoreceptor axons project to a single neuropil unit called a cartridge in the first synaptic region layer, the lamina. . .This retina-lamina projection preserves angular information exactly.” Ribi also defines a “pseudocartridge” of axons from a given ommatidium before they merge with other axons at the cartridge of the lamina. He also notes, “No synaptic contacts or invaginations between the nine retinula- cell axons could be recognized at this level.” Ribi presents considerable information about the course of neuron axons, labeled L-fibres, within the lamina. He also indicates that the color sensitivity of these fibres had not been determined sufficiently to describe a pattern to them. The cited reference also notes the Lobula complex (labeled stage 4 in the figure) includes both a lobula and a lobula plate that perform processing in parallel. The lobula system is reported to identify objects by their shapes.

As noted in Section 3.3.1, the anatomical and histological designation of neurons as monopolar, bipolar and multipolar have no meaning in the electrophysiology of these neurons. The terms relate to how the neurons are arranged for optimum physical packaging. The soma does not play a significant role in electrophysiology except to sometime incorporate the amplifiers known as Activa. At other times the Activa are located at the junction of the dendritic and axonal elements regardless of the location of the soma. All neurons exhibit three electrical contacts with their environment for signaling; an input neurite structure (potentially consisting of both dendrites and podites) and a signaling output structure (an axon and its pedicle). There are also three electrical contacts related to the power supply that may be closely aligned with the above signaling contacts.

The reference distinguishes between intrinsic neurons as arborizing within stages 2 & 4 while projection neurons connect the major engines (knots of neurons) of the optic lobe and the central brain (lobula) Some of these projection neurons are centripetal and centrifugal as indicated in the above figure. The continuing discussion of the neurons in that reference is in serious need of a figure like that above to aid interpretation of the text. It is important to note that the organization of the chromophore coated microvilli (extensions of a dendritic structure) are aligned with their axis perpendicular to the axis of the incident light. This feature was documented by Waterman (Land et al., 1991, page 312) after crediting Goldsmith & Bernard244. The result is the different sensory neurons of an ommatidium are sensitive to the polarization of the incident light as well spectrally selective regarding that light. As a result and as discussed further in the next section, the output of the sensory neurons is proportional to the product of the intensity and polarization of the spectrally selected light

244Goldsmith, T. & Bernard, G (1974) The visual system of insects in Rockstein, M. ed. The Physiology of Insecta, 2nd Ed. Vol II. NY: Academic Press 112 Processes in Biological Vision

The cited paper from the Invertebrate Brain Platform provides extensive description of the neurons and neural paths associated with the lamina, medulla and lobula, but without describing the wavelength sensitivity of the signals in these neural paths.

A clear understanding of the ommatidia of a variety of butterflies has emerged during the turn of the 21st Century. Ribi245 has described the morphology of the ommatidia grossly and the neural circuits involved in significantly greater detail. This Ribi paper cites a considerable number of his papers that will not be cited in detail here, but only with respect to their year. As an example, Ribi (1978) focuses on his concept of screening pigments. The papers provide a large number of light and electron micrographs accompanied by considerable text. However, they lack any circuit diagrams of the neural system. The text is frequently open to various interpretations that a set of circuit diagrams could avoid.

[ 3.6.1.6.1 The dimensions of the rhabdom of butterflies EMPTY

The absolute dimensions of the rhabdome of various Orders through Species of Insecta vary widely and significantly. Some of the rhabdom appear to be circular, however, most are shown to be rectangular in basic form. The effective form of the particular rhabdom can be determines using the techniques of Nilsson et al. (1988) discussed below.

3.6.2 The morphology of the compound eye of Insecta

The compound eye of Insecta appears to employ an ommatidium very similar in morphology to the simple eye (including individual crystalline lenses) but combines many thousands of ommatidium in a single fused structure. Each ommatidium exhibits a very narrow field of view (on the order of 1.5 degrees). The complete compound eye provides a total field of view of nearly a hemisphere in most cases. Ribi describes the compound eye of his butterfly as consisting of 19,000 ommatidia in both sexes and the flattened fused cornea as covering an area of 9.6 mm2.

The compound eye of Insecta is used in an almost endless variety of families within the kingdom. Only a few families have been investigated in any detail, but even some of these families are highly diverse. Stavenga & Arikawa provided a review in 2006 (now dated but useful) that includes many conceptual details relative to a number of the species within several families246. As noted in Section xxx, it appears that a few, if not most, of these families use an unusual form of vitamin A defined as Vitamin A3. This form appears to be utilized by insects feeding primarily on carrion where the vitamin is further oxidized following death. The use of Vitamin A3 by Diptera is reported in Section xxx. 3.6.2.1 The compound eye of the butterfly according to Arikawa & colleagues

During the last two decades, Arikawa and colleagues have built on the earlier work of Stavenga and provided extensive information concerning primarily butterflies. At least three papers will be analyzed (at least partially) below; Bandai et al., 1992, Arikawa et al., 1997, and and Chen et al., 2016. Other papers will be introduced when specific information is important to the understanding of the subject matter.

There are cautions that need to be reviewed before analyzing these papers. 1. It is important to distinguish between a pigment (in bulk form and generally stored separately from a rhabdom) from an active chromophore (present in liquid crystalline form deposited on a microvilli, or in the case of chordate eyes on the disks of the outer segment. These forms can exhibit significantly different spectra even though they consist of the same chemical molecule. A pigment denotes a material that is observed visually by reflected light whereas a chromophore is a material that absorbs light passed through it. The color of a chromophore resulting from observing the color that passes through it is the complement of its absorption spectrum (Section 17.3.4.3).

245Ribi, W. (1987) Anatomical identification of spectral receptor types in the retina and lamina of the Australian orchard butterfly, Papilio aegeus aegeus D. Cell Tissue Res vol 247, pp 393-407 246Stavenga, D. & Arikawa, K. (2006) Evolution of color and vision of butterflies Arthropod Structure & Development vol 35, pp 307-318 The Retina 3- 113 2. When in the liquid crystalline form, the chromophores may exhibit anisotropic absorption spectra. Generally, the spectrum used in vision is the narrow band spectrum with high sensitivity when excited by light traveling parallel to the axis of the conjugated atoms within the molecule. 3. Most “color” recording media, such as the human eye, printed material using conventional 3-color subtractive printing techniques, and conventional television equipment will not sense and cannot present information present in the samples under examination. As a result, a pigment (observed by reflected light) related to a UV chromophore may appear to reflect little light over a broad spectrum. When observed using the above equipment by transmitted light (sometimes described as back-lighted), the UV chromophore may appear to be transparent. The granules present in the RPE of chordates are examples (Sections 4.5 & 4.6.2.2.3). In [Figure 4.5.1-1], the chromophores are described by their absorption spectra. [Figure 4.6.2-6] shows actual imagery of the RPE by transmitted light from Wolken, 1966. 4. When the appearance of a pigment or chromophore is described as whitish visually, it is important to realize that the human eye will report narrow band light between 380 and 420 nm as whitish or milky white (what is defined as Lilac in Section 2.1.1.6.1 of this work). Such light is obviously not related to the perception of white occurring when short wavelength light at a net 494 nm (P = 0.00) and long wavelength light at a net 572 nm (Q = 0.00) are mixed (Sections 17.3.3 & 17.3.4). 494 & 572 nm constitute the Hering Axes of human color space. 5. The labels used by the Arikawa investigators (and many others) do not recognize the difference between the names of colors observed by reflected light and those observed by transmitted light. The labels are frequently significantly different from those adopted by the US National Bureau of Standards and the CIE (Section 2.1) While the difference between purple and magenta may appear semantically trivial, purple is the name of a spectral color (generally at 410 nm and observed by transmission through a filter or spectrometer), while magenta is a non-spectral color (consisting of a mixture of red, ~625 nm, and blue, 470 nm) observed by reflection in the printing industry. Magenta is frequently labeled 532c nm, the complement of the peak of the M – channel chromophore at 532 nm.

The problem is highlighted by a statement in Goldsmith & Bernard (page 188), “The red granules contain chiefly reduced xanthommatin; they absorb maximally at 540 nm, . . .” A material absorbing maximally at 540 nm has a complement at 540c nm and appears as magenta when the source of the illumination has a color temperature of 6500 K (sunlight) and the human eye is not chromatically adapted. If the color temperature of the source is less than 6500 K, the source is deficient in blue and the observer will label his perceived color as red instead of magenta. In many cases, the investigator is not sufficiently trained to know when the light source is inadequate or recognize the difference between magenta and red.

6. The chromophores of chordate vision do not fluoresce under normal in-vivo conditions. They bleach. That is they absorb the stimulant energy and reconfigure to a long-life transparent electronic state (Sections 5.3.5 & 5.5.15).

The distinction between purple and magenta is not observed in the papers cited in this section. Neither is the distinction between yellow (570 nm) of the CIE and the yellow of these papers (520 nm). Two features of figure 4 in Arikawa et al. (1999) should be noted. The absorption coefficient of both the “yellow” and “red” screening pigments are quite low (they are nearly transparent), and the “yellow” curve, if it showed greater absorption at short wavelengths, would be called a “minus blue” filter in the photographic field. A minus blue filter is typically perceived as bronze by the human eye, not yellow. 5. “Lucifer yellow CH, lithium salt is a water-soluble dye with excitation/emission peaks of 428/536 nm. It is a favorite tool for studying neuronal morphology, because it contains a carbohydrazide (CH) group that allows it to be covalently linked to surrounding biomolecules during aldehyde fixation” according to Thermo- Fisher Scientific. They note this material is “For Research Use Only. Not for use in diagnostic procedures.” The 428 nm emission line is less intense than the 536 nm line. The emission at 536 nm is more yellowish–green (where yellowish is an adjective modifying the noun green) than yellow using the NBS and CIE nomenclature. A much better yellow “pigment” is strontium chromate, SrCrO4 with a peak reflectance at 572.3 nm . The visual modality of chordata does not use any chromophore absorbing near 570 nm. Yellow is a perceived color based on computation within the central nervous system (CNS), Section 17.3.4.2. 6. The use of “pigment “will continue to be used in both situations in this section to avoid confusion between quotations from the literature and assertions related to that literature in this work. 114 Processes in Biological Vision

Bandai et al247. provide material on the physical arrangements and additional data on the experimental protocol used by Chen et al. and Arikawa et al. whose papers are discussed below. Figure 3.6.2-1 shows the orientation of elements involved in their experiments. The Bandai et al. paper provides a wealth of other polarization and spectral data. They do not demonstrate that their probe has a high probability of only penetrating one sensory neuron during a given insertion. They do note that “Because 95% of our data were obtained from R1-4, it appears that our electrodes usually tracked in the distal half of the retinula, so that there was only a small chance that R5-8 would be impaled, and virtually no possibility that R9 would be.”

Figure 3.6.2-1 Stimulus condition and typical responses for Bandai et al. The intent was for the micro-electrode to penetrate only a single photoreceptor soma in an ommatidium near the center of the left eye. Note the linearity in the generator potentials developed in response to the log of stimulus intensity and the strength of the recorded signal. These are not action potentials. No absolute voltage is given. From Bandai et al., 1992.

Figure 3.6.2-2 builds on the layout of the butterfly ommatidium shown earlier and defines the polarization sensitivity of the receptors in one type of ommatidia. The polarizations of R3 & R4 are at 90 degrees to that of R1 & R2. The polarization of R5 & R7 and R6 & R8 are at 45 degrees to the above pairs and are at 90 degrees to each other. These values are consistent with the grid-type polarization analyzers generated by the microvilli of individual rhabdomere and the 45 degree rotation of the grids of the rhabdomere of R5-8 required to operate optimally behind the rhabdomere of R1-4 (Section 3.6.2.3).

247Bandai, K. Arikawa, K. & Eguchi, E. (1992) Localization of specral receptors in the ommatidium of butterfly compound eye determined by polarization sensitivity J Comp Physiol A vol 171, pp 289-297 The Retina 3- 115

Figure 3.6.2-2 Polarization sensitivity curves predicted from the microvillar orientation of the Papilio xuthus photoreceptors. The labels of figure 12 indicating color sensitivity have been added to this figure. From Bandai et al., 1992. 116 Processes in Biological Vision

As noted above, Chen et al. have studied the compound eye of the butterfly, Graphium sarpedon, quite intensely but without the benefit of any block diagram or schematic of the visual modality or the sensory receptors. Their supplementary material presents their raw findings without significant additional discussion. They provide 19 spectra combined with a polarization diagram for each (in pairs lettered A through V). Many of these pairs appear to relate to the same UV-, S-, M- & L- channels of this work. Figure 3.6.2-3 shows the statistical correlation between the Chen et al. channels compared to the theoretical responses of the Rhodonines believed to be used as primary photoreceptors throughout the animal kingdom. The single channels indicated by letters appear to be well matched to the individual theoretical spectra shown. As noted in Section 3.6.3, the potential 2 photon —> 1 exciton mechanism or the alternate 2 exciton —> 1 free electron mechanism may represent a second order mechanism moving the wavelength of maximum spectral sensitivity into the 610 nm region. Their polarization responses have been discussed above. The D and O channels of Chen et al. appear to represent linearly mixed signals from two photoreceptors of the UV- and S- type or the UV- and M- type respectively.

The spectral sensitivities of the F, N, Q, R & S channels of Chen et al. exhibit more complex spectra than those discussed above. The F, Q & R channels exhibit peak sensitivities near 500 nm that Chen et al. label BG. Spectra with a peak sensitivity of 500 nm have frequently been associated with a putative “rod” photoreceptor in mammals in the past. Chen et al. have struggled with these spectra and note;

“The enigmatic BG photoreceptor class is extremely rare; we only found a few clearly labeled examples, both of which were R3/4; of type I ommatidia. This is puzzling, because R3/4 in type I ommatidia are usually dG (dorsal) or O (ventral) receptors.” And “The BG receptors, which peak around 500 nm and have a long tail extending toward the long wavelength region, also cannot be explained by the identified visual pigments. However, their spectral sensitivity can be reproduced if we assume the existence of another visual pigment peaking at 480 nm, R480.”

Physical chemistry offers an alternate explanation for the peak at 500 nm. This peak appears to represent spectra obtained from the isotropic sensitivities of all photoreceptors when stimulated from other than their preferred direction (See Section 5.5.9.2). Along their preferred axis, the pigments exhibit a different spectral absorption directly related to their resonant conjugated Figure 3.6.2-3 Correlation between spectra of Chen et al. structures. These conjugated structures exhibit and theoretical rhodonine spectra of this work. Top frame, different peak sensitivities due to the specific length of the letters relate to the spectra shown in their supplemental their conjugated structures (Section 5.5.8.2) and material as they align to the lower frame. The lower frame directly calculable using the Helmholtz-Boltzmann shows the nominal individually normalized (first order) Equation. These rhodonine pigments and the associated spectra of the mammalian retina based on vitamin A1. computations are in complete support of the “one Above the spectra are the commonly associated labels for photoreceptor class–one rhodonine” rule. This rule is a the individual spectra. See text. more precise rendition of the “one receptor–one visual pigment” rule quoted in two different forms (pages 5 & 10) in Chen et al. without citation. This clarification of the rule may aid Chen et al. to simplify their discussion of a great many opsins (their figure 5)–many more than needed to accommodate even their multiple potential The Retina 3- 117 photoreceptor spectra. In their discussion, they define an opsin, after citing Arikawa et al248., as “L2 (and L1) opsins are supposedly of the protein part of green-absorbing visual pigment and L3 opsins are of red-absorbing visual pigment.” In this work, various opsins form the disks of mammalian outer segments of the photoreceptors and the microvilli of the sensory receptors of non-mammalian species. These opsins are physically coated with the liquid crystalline form of the actual visual pigments, the rhodonines. There is no direct relationship between the opsins (actual proteins) and the rhodonines which are not proteins but conjugated arenes derived from retinol (vitamin A) through further oxygenation. The rhodonines are not “chemically” attached to proteins. The mammalian rhodonines are produced in the retinal photoreceptor epithelium (RPE) totally separate from the extrusion of the opsins by the sensory receptor neurons. There are three distinct sets of rhodonines based on the three recognized forms of vitamin A as described in Sections 1.2.1.1 and 6.3.4.4.2. It is important to distinguish between a pigment (in bulk form and generally stored separately from a rhabdom) from an active chromophore (present in liquid crystalline form deposited on a microvilli, or in the case of chordate eyes on the disks of the outer segment. These forms can exhibit significantly different spectra even though they consist of the same chemical molecule. A pigment denotes a material that is observed visually by reflected light whereas a chromophore is a material that absorbs light passed through it. The color of a chromophore resulting from observing the color that passes through it is the complement of its absorption spectrum (Section 17.3.4.3). When in the liquid crystalline form, the chromophores may exhibit anisotropic absorption spectra. The use of pigment will continue to be used in both situations in this section to avoid confusion between quotations from the literature and assertions related to that literature in this work.

This clarification may also explain the D and O spectra of Chen et al. wherein this analysis asserts these spectra are linear summations of the spectra from two distinct photoreceptors that each satisfy the “one receptor–one visual pigment” rule. It may also help them simplify their discussion of the expression of various mRNAs within a single sensory neuron, or sensory neuron photoreceptor.

Spectra N and S of Chen et al. cannot be characterized at this time. N exhibits a peak at 400 nm but consists of only three data points. S exhibits a peak near 560 nm with a wide range bar at 540 nm. It is possible that additional test data would confirm the S spectra corresponds to the theoretical M- channel spectra of this work.

The cited paper by Arikawa et al. is important in further describing the ommatidia of Insecta. However, much of its empirical calculations and broad speculations need to be reviewed carefully. They note in their introduction, “the nature and composition of spectal receptors in the ommatidia has yet to be elucidated? Their goal was to move closer to that elucidation. This work does not support the concept of self-screening of a pigment in place of a more precise quantum-mechanical calculation using the Helmholtz-Boltzmann Equation and concept. This may be a question of semantics. If they mean self-screening within an ommatidium by rhabdomere of different peak spectral sensitivity along the axis of the rhabdom, this work would suggest the term cross-chromophore, inter-chromophore or inter-rhabdomere optical screening along the axis of the rhabdom. If they mean, self-screening within the rhabdomere associated with a single sensory neuron receptor (Section 5.3.5.3.1), it is recommended the Helmholtz- Boltzmann conceptual equation combined with the Pauli Exclusion Principle of quantum physics/chemistry is a more direct means of accounting for the phenomenon. As a matter of fact, the first wafer of rhabdomere are excited by the appropriate spectral light and turn transparent. As a result this first wafer does not screen the second wafer. The second wafer then absorbs the incident light before becoming transparent. This progressive increase in transparency is the mechanism behind adaptation as well as the observed “bleaching” of the receptors in vision. The geometry of the disks of Chordata and the microvilli of Insecta (and probably also of Mollusca), cause the absorption process to operate like a series of independent absorption filters spaced at nominally equal intervals. This work also questions the utility of their figure 2 in describing the character of the “pigment area.” The area shown exhibits a relatively low density of pigment material in terms of the bulk of the cytoplasm of the enclosing cell. The paper does report the presence of chromatic material stored in the area surrounding the rhabdom. This material appears to be stored in separate cellular structures from the sensory neurons, similar to the arrangement in the retinal photoreceptor epithelium (RPE) of mammals. These pigments are used to replace the chromophores lost

248Arikawa, K. Scholten, D. Kinoshita, M. & Stavenga, D. (1999b) Tuning of Photoreceptor Spectral Sensitivities by Red and Yellow Pigments in the Butterfly, Papilio xuthus Zool Sci Of Japan vol 16(1): pp 17-24 118 Processes in Biological Vision

over time from the microvilli (possibly through phagocytosis as in mammals). While Arikawa et al. assert the rhabdom acts as a waveguide for light, they did not review the requirements of such a guide. Their text suggests the surrounding pigments, rather than the fluid matrix surrounding the rhabdom forms the wall of the guide. This work takes the opposite view, the stored pigment plays no significant role in projection and absorption of light within the interface with the surround. In fact, figure 3 of their paper shows the stored pigment is not in direct contact with the periphery of the rhabdom but immersed in the cytoplasma of a cell. The difference in index of refraction between the two sides of the interface is the critical factor. The pigment is shown stored within the lemma of other cells interdigitated with cells R1-4 and R5-8. Figure 3.6.2-4 reproduces figure 3d and 3e of the Arikawa et al. (1999b) paper. In this figure, R-ommatidium refers to a long wavelength sensitive structure with peak sensitivity in the 600 nm region. Y-ommatidium refers to a medium wavelength sensitive structure with a peak sensitivity in the ~520 nm region. The 520-532 nm region is considered to absorb in the olive-green, or yellowish-green in this work rather than yellow at 578 nm as defined by the 1976 UCS Chromaticity Diagram (Section 2. 1.1.3). It is not obvious from these electron micrographs whether a process of phagocytosis can be accommodated in the plane of these images. It is also difficult at the scale of these images to determine if the microvilli are extruded by one sensory cell and absorbed at another cell of different function. As noted in the caption, whether the microvilli are curved or straight should not be implied from these two frames alone. Furthermore, the structure of R9 is not shown in these frames. The amount of polarization associated with the curved microvilli may be greatly reduced. As in the case of Mollusca (Section 1.7.2.2), the role of the putative ninth receptor (R9) in Chen et al. and in Arikawa et al. is unresolved. It may play an alternate role similar to that of the eccentric cell in the eyes of the horseshoe crab, Limulus, (Xiphosura polyphemus) . In that species (Appendix D), the analog of R9, the eccentric cell, appears to act as a combined stage 2 (signal processing) and stage 3 (encoding) neuron for visual signals. In Arikawa et al. (page 19 under Results), it is noted “The soma of the basal photoreceptors, R9, has no evident pigmentation.” This statement suggests the cell does not perform a sensory function but the choice of words allows for a variety of interpretations related to the situation.

As shown in [Figure 1.7.2-2], Chen et al. number the individual sensory receptor neurons within an ommatidium differently than does Horridge in [Figure 1.7.2-4]. The terms rhabdom, rhabdomin and rhabdomere appear to be used differently thant earlier investigators cited above. [xxx check this ]

The polarization sensitivities of the enumerated single channels appear quite varied. The difference between the dorsal and ventral portions of the eyes of the bluebonnet butterfly, Graphium. sarpedon may be due to the desire to achieve polarization sensitivity in conjunction with stereopsis as in the Arthropoda (Section 1.7.2.1.3). The higher degree of scatter in the data points of cases D, N & O may also be relevant. In D and O, they may confirm the measurements were obtained from electrophysiology capturing and summing the signals from two spectral types of sensory receptor neurons simultaneously as discussed above. As noted by Chen et al., the polarization shown by the remaining sensory receptor neurons show several Figure 3.6.2-4 Electron micrographs of transverse sections patterns; of Papilio retina. d; R-ommatidium, cut through the region slightly distal to the transitional zone. The pigment clusters C The samples A, B, C, D, L, M, N, O & V of the appear electron-dense (arrowhead) The rhabdomeral supplementary data show maximum spectral sensitivity microvilli of R1-4 are curved, whereas those of R5-9 are for e-vector angles of 0 and 180 degrees. These straight (not shown). E; Y-ommatidium. The pigment samples are all labeled R1-2 with the additional clusters appear electron-lucent (arrowhead). The notation that A, B, C, & D are from the dorsal portion rhabdomeral microvilli of R1-4 as well as R5-9 (not shown) of the eye. Samples M, N, O & V are associated with are straight. 1-4; photoreceptors R1-4. Bars = 1 micron in the ventral portion. d and e. See text. From Arikawa, 1999b. The Retina 3- 119 C Conversely, samples E, F, G, H, P, Q, R & S show maximum spectral sensitivity for 3-vector angles near 90 degrees. These samples are all labeled R3-4. Samples E, F, G & H are labeled dorsal. Samples P, Q, R & S are labeled ventral.

C Samples T, U & V show peaks at 45 degrees and nulls at 135 degrees. They are all labeled R5-8 and ventral.

C Sample K is an anomaly, it exhibits a very low degree of polarization with a maximum near 135 degrees and a minimum near 45 degrees. This sample is labeled R5-8 and dorsal. Recently Chen, Awata, Matsushita, Yang & Arikawa have provided a more advanced cartoon related to the butterfly ommatidium249 than shown for Mollusca above and in Section 1.7.1.2. The cartoon was discussed briefly in Sections 1.2.1 & 1.7.2.1.1. Their paper is extensive but does not provide any block diagram or schematic of the butterfly eye except for a relatively simple cartoon. Their figure 1a is presented as Figure 3.6.2-5. The unlabeled central hatched column represents the active absorption column of ommatidia, the rhabdom. A better annotated figure 1 for Papilio xuthus is provided in Arikawa et al. (1999b). See Section 3.6.3.4 for a better rendition of the stage 0 optics.

They define three distinct types of ommatidia based on their fluorescent properties. Type I does not fluoresce, type II fluoresces strongly and type III fluoresces weakly. Types I (42.3%) and III (48.0%) represent 90% of their samples. A vast majority of their ommatidia did not fluoresce significantly!. They note in their results, “This fluorescence is only observed in ommatidia of the ventral eye region. The dorsal region has no fluorescence, . . .” Since all of these eyes contain the appropriate chromophores to support vision, it can be deduced that fluorescence is not a characteristic of the chromophores, but relate to some other material. Page 3 of their paper provide more details on this subject.

They indicated the presence of a selection of fluorescing pigments; a UV fluorescing pigment near the top of the ommatidium and a reddish pigment below the letters C, C’ in the figure. Both pigments surround the active receptor area defined by the central column, the rhabdom. These pigments may complicate their experimental protocol and results in their discussion. Whether these pigments are excited within the natural environment was not discussed in the paper. Based on closer evaluation of their electron- Figure 3.6.2-5 The ommatidia of Graphium Sarpedon. micrographs, it does not appear these pigments play a Two distinct sets of photoreceptors are shown, R1-R4 and role in active photoreception. These pigments appear R5-R8. The letters C, C’, D & E relate to other inset images to be stored in cells equivalent to the RPE of the in the original figure. Receptor R9 is shown near the basal membrane. See text. From Chen et al., 2016.

249Chen, P-J. Awata, H. Matsushita, A. Yang, E-C. & Arikawa, K. (2016) Extreme Spectral Richness in the Eye of the Common Bluebottle Butterfly, Graphium sarpedon Front Ecol Evol vol 4, article 18 | http://dx.doi.org/10.3389/fevo.2016.00018 120 Processes in Biological Vision

chordate eye and distinctly removed from the matrix surrounding the rhabdom Their extensive investigation identified photoreceptors with 15 distinct spectral sensitivities. However, they only identify eight or nine distinctive locations within the nominal ommatidium. They appear to focus their attention on the soma of the individual photoreceptors rather than the active sensory material of each sensory neuron, the individual rhabdomere, within the central column, the rhabdom. See the next section for a discussion of the ommatidia of other members of Arthropoda. They indicated surprise at their results, based on intracellular electrophysiological evaluation, and their discussion indicated a fundamental group of four photoreceptor types providing a typical tetrachromatic capability (UV, S, M & L). They noted, “Do G. sarpedon use all 15 spectral receptors for seeing colors, i.e., is their vision pentadecachromatic? This has to be checked by behavioral experiments, but it is rather unlikely.” Some of the treatments of their ommatidium are brutal (microwaved for 30 seconds) considering the delicacy of chromophores (bandgaps of one to three electron-volts). Depending on the amount of liquid present and the power level, this could reduce the Rhodonines to retinenes. Some of their treatments are similar to the equally brutal treatments of Seki et al. and Suzuki et al. described in Section 5.5.15.4. Figure 2 of Bandai et al. provides a more revealing caricature of three adjacent ommatidia from another butterfly family, Papilio xuthus, and assert the ommatidia are identical to those in Papilio augeus. It shows four distinct elements leaving each ommatidium through the basement membrane; one from each R9 cell, a group from the distal sensory neurons (R1-4), a separate group from the proximal sensory neurons (R5-8) and a fourth path that cannot be defined from the figure and is not addressed in their text. No citation is given for the figure although a reference to Ribi (1987) is provided.

Chen et al. describe the peak sensitivities of the dorsal type I ommatidia photoreceptors in their figure 5 (shown here as Figure 3.6.2-6) as 354, 455, 541 & 599 nm respectively. These values are in good statistical agreement with the theoretical peak sensitivities of this work (342, 437, 532 & 625 nm, where the 625 nm value may be subject to second order effects not considered in the theory) for terrestrial mammals. Terrestrial mammals employ rhodonines based on vitamin A1. There are suggestions in the literature that Insecta may employ rhodonines based on vitamin A3 during at least part of their lifetime. This may result in differences on the order of a few nanometers between the two sets of values given above (Section 6.2.2.4). Chen et al. indicate the long wavelength peak sensitivity in the butterflies was contentious among investigators. The sample size of four does not support their smoothed curve for the L–channel, particularly in the absence of range bars on a majority of the points in that spectrum. The old paper of Bernard suggested the peak among nine species of butterflies was at 610 nm250. The right skirt of their long wavelength receptor appears to support a peak value of about 610 nm and conform to the theoretical shape developed in Section 5.5.10 based on the Helmholtz–Boltzmann Equation. The non-Gaussian nature of the spectral sensitivities of the Rhodonines predicted by the Equation indicates it is best to use the half-amplitude points on a given spectral response to more precisely describe the spectral sensitivity of a particular photoreceptor. However, the width between the half-amplitude points is species dependent and depends on the amount of chromophore present of a given type due to Pauli’s Exclusion Principle. Alternately, it appears that most species employ similar amounts of each chromophore to assure a reasonably uniform spectral sensitivity for the complete eye when the signals from the individual photoreceptors are summed exponentially (Section 15.xxx or 17.xxx). If it is desired to use an mathematically derived peak in the spectral response for a signaling channel, the average value between the two half-amplitude values can be used. The absorption spectrum labeled BG in Chen et al. has a peak absorption near 500 nm. This is normally the isotropic absorption of all photoreceptors when they are stimulated by light perpendicular to their normal axis of excitation. This spectrum has frequently been associated with a “broadband” receptor described (inappropriately) as a “rod” in mammalian eyes. The BG channel does not exhibit the broad spectrum required of a “rod.”

250Bernard,G. (1979) Red-absorbing visual pigment of butterflies. Science 203, pp 1125–1127 .doi:10.1126/science.203.4385.1125 The Retina 3- 121

They only speculated briefly in their discussion concerning the reason for the other spectral types. They introduced considerable mRNA information. The mRNA data did not correlate well with their spectral sensitivities. They provide no block diagram or schematic accounting for their “surprisingly” large number of different spectral responses. Their supporting material attempts to rationalize some of their data. Their supplementary material is accessed from the right column of their first on-line web page. This data will be discussed after the following sub- section. 3.6.2.1.1 The high frequency limitations of butterfly visual sensory neurons

Bandai et al. noted on page291, that the shortest stimulus pulse they could use without reducing the amplitude of their measured generator potentials compared to longer pulses was 30 ms. This is an important measurement describing the high frequency Figure 3.6.2-6 Spectral sensitivities of photoreceptors of bandwidth limit of the Activa circuit found within the type 1 ommatidium of Graphium sarpedon retina. Values sensory neurons of the butterflies and probably a vast are means plus their associated statisitical ranges. Their number of small members of Insecta. A time constant four specific chromatic channels are described along with a of about 20 ms can be inferred from their observation. BG channel that may be the intrinsic absorption of the As shown in the universal P/D equation of sensory sensory material when excited by light orthogonal to the biology (Section 3.6.3.2), this time constant (and the conventional description. The number of samples for the associated delay time) is a function of the stimulus BG and sR spectra were quite small. See text. From Chen level. This time constant should not be associated et al., 2016. with the time constant of the transduction process which is much faster (in the microsecond or shorter range) 3.6.2.1.2 Is R9 an eccentric cell?

A question arises as to the spectral sensitivity and the purpose of the cell labeled R9 in much of the work investigating Insecta. Some histological studies do not recognize the cell at this location as a sensory neuron. Many such studies note there are no nearby/associated granules of proto-chromophores.

Similar investigations relating to the crustacean, Limulus, has provided more detailed information showing that the cell encountered in the ommatidia at this location is not a stage 1 sensory neuron at all but is a stage 2/3 signal processing/signal projecting neuron. The nomenclature stage 2/3 is used because of the apparent primitive character of the neural system of Limulus vision. Because of the relatively large size of Limulus, it requires the axons of some neurons to be greater than 2 mm long. Because of this requirement, it appears this single eccentric cell is used like many stage 3A stage neurons to both perform simple signal processing (summation and differencing) as well as generating action potential pulse streams encoding the result of these activities.

Figure 1 of Bandai et al. shows R9 as a single sensory neuron “where the bilobed R9 photoreceptor contributes microvilli to the rhabdom.” However, they then note, “R9 is pigment free.” It is not clear if their test equipment was capable of recording the complete transmitted or reflected spectrum of a UV sensitive material. Humans are not capable of observing such a spectrum visually. This observational limitation is found throughout the papers analyzed in this section. It is also virtually impossible for humans to observe the chromophores potentially present in thin (typically one molecule thick) liquid crystalline layers because of the nearly instantaneous adaptation of such individual layers to the transparent condition upon even minimal photon excitation. It is noted that Ribi, 1987, shows only a single lobed R9 located behind R1 in figure 1.but a residual or remnant of a second lobe (also in the accompanying unstained transmission electron micrograph of figuer 2c. These questions can not be satisfactorily resolved until more detailed information is gathered concerning R9 of the insect ommatidia until additional information is gathered from this and various other species. The light used to stimulate the cells in these investigations must be applied along the long axis of the rhabdom hosting the R9 cell. 3.6.2.2 Special features of the polarization mechanism 122 Processes in Biological Vision

The next paragraph is specific to the butterflies of the family Papilio. As noted earlier, there appears to be considerably different polarization sensitivity among butterflies in general. The principles described would apply to other species, but their grid polarizer arrangements might be different. The microvilli, being water-based liquid filled, act as electrical conductors. In their grid arrangement, with a pitch of approximately 0.1 microns, roughly a fifth of the wavelength of the incident light, they act as a grid type polarizer commonly found in the morphology of many animals (butterflies and hummingbirds as examples). In the ommatidia, this grid polarizer is used to detect polarization in the stimulating light. The polarizers of the distal portion of the rhabdom introduce a unique situation. Figure 3.6.2-7 reproduces figure 3(e) of Arikawa et al, (1999). Note that the light passing through the rhabdom (into the paper) encounters either horizontal or vertical polarizing grids. As a result, all of the light reaching later sections of the rhabdom has its e-vector parallel to either the horizontal or vertical axis. This is true of all of the light, even that not absorbed by the rhabdomere shown. As a result of these two orientations, the light not absorbed by the first rank of receptors will be either circularly polarized or polarized at a nominal 45 degrees from the light polarized by the grid polarizers of the first rank since these components add vectorially. If the resultant light is circularly polarized, fifty percent of the light will be lost when interrogated by the grid polarizers of the second rank. On the other hand, if the light is polarized by 45 degrees relative to the grid polarizers of the first rank, they would be optimally absorbed by the second rank of sensory receptors if their grids were oriented at 45 degrees to those of the first rank. This situation explains the observations of Ribi (1987, figure, 1b) that neurons R5-8 are rotated in their spatial position by 45 degrees relative to R1-4. It also questions or clarifies the predictions of Bandai et al (1992, page 295). The polarization sensitivity of sensory neurons R5-R8 will be rotated by 45 degrees relative to R1-4 but this will not be indicative of the polarization sensitivity of these neurons to the light incident at the rhabdom because this rotation is internal to the rhabdom.

Note how the rhabdomere of this figure defines a nominally square cross-section optical waveguide, with walls of the guide parallel to the grid of either the horizontal or vertical grids associated with different rhabdomere.

Some electron microscope images show significant in-plan curvature of the microvilli of a given rhabdomere wafer. No analysis has been carried out to understand or explain this phenomenon. [Xxx see figure 3.6.3-4d ]

3.6.2.3 Detailed specific features of the optics of the ommatidia

[xxx edit re index of refraction ] There is considerable information in the histology literature that is important to understanding the operation of the ommatidia of Insecta, with emphasis on the butterflies. However, specific crucial information has not been located, such as the index of refraction (preferably as a function of wavelength) for the fluid within the rhabdom and the index of the fluid surrounding the rhabdom. Thus, the assumption that the rhabdom acts like an optical waveguide cannot be Figure 3.6.2-7 Electron micrograph of the grid polarizers substantiated at this time. As a result, many of the within the rhabdom of Papilio. The grid polarizers formed conclusions in this section must be considered by the R1 and R2 sensory receptors are arranged vertically tentative while awaiting these index values. XXX edit and those of R2 and R4 are arranged horizontally. These grids act as polarization analyzers for any light passing The difference in index of refraction between the through them that is not absorbed by these sensory contents of the rhabdom and its immediate surround receptors. As a result, all of the light leaving this region of creates a very effective barrier to the light traveling the rhabdom, must be polarized either horizontally or within the rhabdom. This is particularly true where the vertically. The phase angle between the e-vectors due to waveguide is long and thin. In this case the light these polarizers is not known. Scale bar = one micron. See within the guide incurs total internal reflection text. From Arikawa et al., 1999. whenever it approaches the “wall” of the guide. Any material (pigment or otherwise) more than ¼ wavelength (about 0.1 micron) away from the wall of the waveguide will have negligible impact on the light within the guide. As noted in figure 3b of Ribi (1987), most of the pigment granules highlighted are more than 0.1 microns (typically >2 microns) from the nominal wall of the waveguide. With these spacings, the waveguide model of The Retina 3- 123 Arikawa et al. (1999b) appears oversimplified251. The “pigment area” of their figure 2 should be described as the “surrounding area” with the appropriate index of refraction. The pigment contained therein, and its index of refraction, should be shown as separated from the interface with the rhabdom interface by a significant distance. Nilsson et al. have provided a 25 page analysis of the optical system of the butterfly252. It includes a valuable set of light and electron micrographs from various perspectives. They initially note, “The fundamentals of waveguide optics and Fourier optics may be unfamiliar to many biologists. . . .” They also note, “The agreement between theory and experiment is now so good that we can safely take the fly compound eye as being of the best understood optical systems in the animal kingdom.” Land, a member of the Nilsson team, has also been active in investigating the chordate eye. However, the “best understood” assertion can be questioned. The human eye is also very well understood. The paper is very sophisticated and precise from the perspective of an optician. Additional precision in the indices of refraction can be hoped for in the future, but this is a difficult measurement for biological material (which is frequently subject to shrinkage). Section 3.6.3.2 provides a summary of the available indices for two species. The values given are preliminary and not totally consistent. The major conclusions of Nilsson et al. is that the stage 0 optics of the simple and compound eyes of Insecta can vary between members of Arthropoda in a systemic manner and many species use different modifications to the basic optical formulation. The so-called crystalline cone should actually be shown as consisting of two optical elements (lenses), most often resulting in an afocal (non-inverting or terrestrial) telescope.

251Arikawa, K. Scholten, D. Kinoshita, M. & Stavenga, D. (1999b) Tuning of Photoreceptor Spectral Sensitivities by Red and Yellow Pigments in the Butterfly Papilio xuthus Zoo Sci (Japan), vol 16(1), pp 17-24 252Nilsson, D-E. Land, M. & Howard, J. (1988) Optics of the butterfly eye J Comp Phys vol 162, pp 341-366 124 Processes in Biological Vision

Figure 3.6.2-8 reproduces their figure 1. It is accompanied by the assertion, “a simple lens-waveguide model of the ommatidium, where the cornea provides most or all of the optical power, must now be abandoned.” As shown, the cornea becomes a thick lens with optical power at each surface and the corneal cone is an optically independent element also of the thick lens configuration. Their figure 2 shows the clearly defined separation between the corneal process (CP) and the crystalline cone. Other investigators assign a different shape to the corneal process. Stavenga & Arikawa introduce a clearly defined separation between the crystalline cone and the rhabdom, along with several caricatures (not to scale). The separation is labeled the clear zone (CZ). They assert the CZ can be quite wide in some superposition eyes. but do not consider the acceptance angle of the individual rhabdom as a waveguide.

They deduced the index of refraction of the rhabdom to be 1.36. Section 2.2.2.3, presents a reported value of 1.41 for the outer segment in Chordata. The surrounding environment was reported to have an index value of 1.36. The difference between 1.36 and 1.41 is sufficient to support a robust optical wall for a waveguide. See Section 3.6.3.5.2. [xxx copied from SCORE notes. edit into proper text form ] The coloration along the ommatidia external to the rhabdom is usually described using casual language. To be more useful, the actual spectrum of the coloration is needed, particularly since the spectrum may have a significant UV spectral component not visible to human eyes or most photographic techniques.

The pigment particles near the proximal end of the crystalline lens are well characterized from an opticians perspective, they are designed to prevent unwanted light from entering the waveguide formed by the rhabdom and reducing the interfering with the absorption of the light from the desired field of view of the stage 0 optical system. In that role, the pigment can be described as a “pupil stop.”

The term epi-illumination is frequently used in the study of the rhabdom of insects. It refers to a microscope employing frontal illumination as opposed to illumination transiting the specimen. A metallurgical microscope operates by epi- illumination. Electron microscopy provides a different perspective on the neural circuitry within the rhabdom that historical images obtained using light microscopy. The Figure 3.6.2-8 The demonstrated stage 0 optical system of e-m imagery of xxx shows the presence of axons the simple and compound eye of Insecta ADD OTHER neurites and synapses at the stage 1/stage 2 interface in SYMBOLS. The cornea ( C) is shown as a “thick” lens. such profusion as to suggest a neuropil, or neural knot, with a newly defined corneal process (CP) exhibiting optical at these locations. power. The corneal cone (CC) is also shown as a thick lens with potentially variable properties among species. The Those studying the compound eye of insects frequently primary pigment cells (PPC) and the accessory pigment cells describe a pseudopupil. “In the compound eye of (APC) play a much more dynamic role than previously invertebrates such as insects and crustaceans, the described. The role of the pigment shown within the pseudopupil appears as a dark spot which moves across retinula (RC) take on a different role when surrounding the rhabdom (Rh). From Nilsson et al., 1988. The Retina 3- 125 the eye as the animal is rotated253.” 3.6.2.3.1 The tapetum & secondary pigment found in many butterfly and moth species

Stavenga & Arkikawa have described a tapetum behind the R9 neuron at the end of the rhabdom in a wide variety of species (figures 1 & 3a). They described the character of the tapetum as highly reflective over a wide wavelength spectrum created by an air-filled tracheole (citing Land & Nilsson254). The tracheole should be described as gas- filled. It is likely that the gas in these voids does not have the composition of “air.” It is more likely CO2, N2 or O2, gases that are readily available individually within animal tissue. Such gas-filled tracheole create “total internal reflection at their surface because of the large difference in index of refraction between the tissue and the gas. The same technique is used in the head of dolphins and other members of Odontoceti, the toothed whales, to form total internal reflecting surfaces at auditory frequencies. Their figure 1 also shows a large amount of secondary pigment extending up along the sides of the crystalline cone to the intersection with the cornea or “cornea process.” This pigment appears to significantly limit the performance of a superposition compound eye described conceptually in figure 5. Bernard & Miller have provided an electron micrograph providing considerable detail relating to the form of tapetum, Figure 3.6.2-9 in the buckeye butterfly, Precis lavinia. Each of the tapetum shown in the longitudinal cross section of the ommatidia consists of about forty distinct slabs perpendicular to the direction of the incident light. The spacing of the slabs, although regular is not equal. The average spacing is about 0.25 microns (250 nm). In a typical Bragg dielectric mirror, layers of high and low index material are interleaved. If the spacing of these paired layers is one quarter wavelength, constructive phase interference is obtained for the reflected light (a very high quality mirror). By varying the spacing of the paired layers logarithmically, the spectral band of the reflection can be broadened (as in the log-periodic antenna of radio)255. Note the variable spacing of the slabs near the top of the electron micrograph. The spacing variation need not be very great to achieve significant spectral broadening. It is possible the alternate slabs are gas filled (providing a very large difference in index of refraction between members of the paired layers of the Bragg dielectric mirror.

The range of wavelengths that are reflected is called the photonic stopband. Within this range of wavelengths, light is "forbidden" to propagate through the structure. Increasing the difference in index of refraction between the materials of the Bragg pairs increases both the reflectivity and the bandwidth. The reflectivity of Bragg dielectric mirrors are also sensitive to the transmission mode of the incident energy. Typically, the TE mode alone is highly reflected by this stack, while the TM modes are passed through. Thus, an ommatidium employing a Bragg dielectric mirror can act as a polarizer under light adapted conditions. Under dark adapted conditions, it will be less effective. It is important to explore the polarization sensitivity of an ommatidium to various stimulus intensity levels as well as to various spectral wavelengths.

253Land, M. Gibson, G. Horwood, J. & Zeil, J. (1999) Fundamental differences in the optical structure of the eyes of nocturnal and diurnal mosquitoes. J Comp Phys A vol 185 (1), pp 91–103. doi:10.1007/s003590050369. 254Land, M. & Nilsson, D. (2002) The origin of vision In Willmer P & Norman D. ed. Animal Eyes NY: Oxford Univ Press 255Bernard, G. & Miller, W. (1970) What does antenna engineering have to do with insect eyes IEEE Student J vol 8, pp 2-8 126 Processes in Biological Vision

The reflection of light by the tapeum back through the ommatidium and into the exterior environment constitutes the “eye shine” or “eye glow” described by many environmentalists when a bright light is used to illuminate the head of an animal at night. It should be noted that the eye becomes light adapted for the individual spectral channels of the eye stimulated by the light source. The adaptation is typically accomplished within less than a second in most animals (Section xxx). In light adaptation, the chromophores coating the rhabdomere become essentially transparent (less than 1% absorption over the length of the rhabdomere stack). They are described as “fully bleached” in the vernacular but this term is misleading. A less than one percent absorption is fully effective at high light levels. This bleaching is an effective part of the adaptation mechanism providiing the eye with a wide dynamic intensity range. This adaptation causes the light reflected by the tapetum to appear much brighter than the external coat of the animal. The spectrum of the reflected light is related primarily to the spectrum of the incident light and the spectrum of the dielectric mirror. Stavenga explored the properties of eye shine from several species of butterfly256, but did not address the variability of the reflectance related to the polarization properties of transmission mode within their rhabdome and the potential of a Bragg mirror as the tapetum. This additional parameter might place a different perspective on the the Stavenga text that noted “Eye regionalization suggests that different eye areas have special functions (Bernard and Remington, 1991).” Special functions might refer to special capabilities whether these were used operationally or not. Stavenga also noted, “In a comparative study of a Figure 3.6.2-9 A tapetum based on reflection from a stack number of heliconian species that all lacked a distinct of dielectric plates in Precis lavinia at X 3875. Two arrows dorsal area, we found that the ratio of the differently at upper right indicate two layers of othrogonally orineted coloured facets can change markedly across the eye microvilli of the rhabdom ( r). The parallel bars represent suggesting that heterogeneity and regionalization exist the tapetum (t) of each rhabdom. P; pigment of the basal universally in butterfly eyes.” He provided several pigment cells. See text. Scale bar = 1 micron. From citations. The employment of different tapetum with Bernard & Miller, 1970. different Bragg mirror properties may change the interpretation of Stavenga’s observations regarding screening pigments.

Goldsmith & Bernard have noted the presence of both specular and diffuse reflector types of tapeta. They have also discussed the relevance of such reflector types to the light polarization mechanism (page 254). Many butterflies and virtually all moth species exhibit what has been described as a nipple array covering the surface of the cornea (Figure 6 in Stavenga & Arikawa). By inspection, this array of protrusions at an array spacing considerably less than the wavelength of interest in vision, an optician would define this array as an intermediate index of refraction surface constituting an antireflection coating on the cornea. The tapetum and antireflection coating of the cornea should be considered at the detail level of the optical analysis of Nilsson et. al., and included tn th overall analyses of the optics in Insecta vision. 3.6.2.3.2 Index of refraction for ommatidia of blowfly and honey bee

256Stavenga, D. (2002) Reflections on colourful ommatidia of butterfly eyes J Exp Biol vol 205, pp 1077-1085 The Retina 3- 127 The best data on the index of refraction for Insecta is that from Goldsmith & Bernard for the blowfly, Calliphora, honey bee, Apis and the firefly, Photuris257. Figure 3.6.2-10 reproduces their figure 8. Note the values are given to three decimal values after the decimal point. They do discuss possible errors in these values and note the value of 1.311 is questionable because the region contains cytoplasm and should exhibit a value greater than that of water at 1.333. Note the scale at the bottom of the left frame. The question of shrinkage between the in-vivo and in-vitro values after physical manipulation of the eyes was not discussed. They also provide excellent background on the optical system of these species as recognized in 1974. Their discussion should be compared to that of Nilsson et al. of 1988. Nilsson et al. (page 360) suggest the index for the rhabdom of Xois arctoa should be near 1.36 if the index of the surrounding matrix is taken to be 1.34. The lower frame shows profiles of constant index of refraction are shown as well as ray paths for paraxial rays (1 and 2) and parallel, oblique rays (3 and 4). The contours clearly define a graded index optical system. The outer contour also indicates optical power for the end of the cone adjacent to the rhabdom.

257Goldsmith, T. & Bernard, G (1974) The visual system of insects in Rockstein, M. ed. The Physiology of Insecta, 2nd Ed. Vol II. NY: Academic Press pg 206 128 Processes in Biological Vision

Figure 3.6.2-10 Longitudinal sections of dioptric portions of ommatidia with indices of refraction as determined by interference microscopy with light of wavelength 546 nm.. Left; the blowfly, Calliphora (Seitz (1968). Right; the honey bee, Apis (Varela & Wiitanen, 1970). Bottom; cornea and exocone of the firefly, Photuris (Seitz, 1969). See text. From Goldsmith & Bernard, 1974.

3.6.2.3.3 The rhabdom as a waveguide The Retina 3- 129 Several sensory modalities of animals employ dielectric waveguides, particularly optical waveguide technology in vision and acoustic waveguide technology in hearing. The analyses up through the 1980's of the rhabdom operating as a dielectric waveguide was early and superficial. The only discussions found in the litereature of Insecta involved rhabdom of circular cross section. During the 1970's, the subject of dielectric waveguides was under very intense study in a parallel field, fiber optics. This section will broaden the framework for analyses of the visual waveguides of Lepidoptera based on this work. It will describe both cylindrical and rectangular dielectric waveguides and the relevant features of each. Goldsmith & Bernard presented the theoretical modal patterns of light in a circular waveguide258. However, no empirical evidence was provided that the rhabdom of all Insecta conformed to this assumption. The alternative is the well-known rectangular waveguide. As noted by comparing the next two subsections and the actual photographic images of rhabdom acquired by Nilsson, it requires a trained eye to differentiate between the theoretical modal patterns of the circular and rectangular waveguides and the modal patterns observed in the laboratory. The theoretical modal presentations of Nilsson are only presented in black and white while the presentations of Goell provide more informative gray scale images. As a general rule, the rhabdom of Insecta, like the outer segments of Chordata, do not scale with the size of the animal. In general, either component must have a minimum dimension of about 2.6 microns in order to support a robust L–channel (red) sensitivity. Smaller diameter rhabdome will provide less robust L–channel performance. As discussed in detail in Nilsson, it is important that the modal pattern at the entrance to the waveguide accept the energy within the Airy disc of the preceding portion of the stage 0 optical system if maximum energy transfer into the rhabdom is to be achieved. However, it is generally desired that the modal pattern of the rhabdom sum both the first and second order modal patterns for the longest wavelength required by the animal and its niche. This summary modal pattern should be broader than the Airy disk of the preceding optical train. Nilsson et al. have provided an electron micrograph of the crystalline cone–rhabdom interface for Argynnis paphia. The shape of the cone differs significantly from that of the cone of their figures 6 & 7, and the above figure for the index of refraction at various points. The distance between the cone and rhabdom may also differ significantly.

The previous sub-section has provided best estimates of the dielectric indices of refraction supporting waveguide operation. The values are not of the desired precision but are useful for pedagogical purposes.

[xxx copied from section 3.6.1.6 ] Wehner has provided figures 5, 6 & 8 showing both rectangular and circular rhabdome in a variety of families of Insecta259. His caption to figure 5 appears to contain a typographical error. The diameter of the microvilli are typically between 40 and 70 nm rather than μm based on his scale bar. The rectangular rhabdom have an aspect ratio of at least 2:1 and are not easily represented by an inscribed or circumscribed circle. He includes an extensive discussion of the twisting of the retinula within a given ommatidium. Images from other investigators provide similar geometries for many other members of Lepidoptera.

Marcus has provided a comprehensive volume on the properties of both circular and slab dielectric waveguides. It is the nominal Bible of the fiber optics community260. The book provides additional valuable definitions related to waveguides in general. When the data for two one-dimensional slab waveguides are combined orthogonally, the results relate to a single rectangular waveguide. Between the circular and rectangular waveguides, virtually all rhabdom found among Insecta can be described. Only the rectangular dielectric waveguide can describe rhabdom with an aspect ratio on the order of 2:1. By reducing the aspect ratio, the modal patterns of the general rectangular dielectric waveguide reduce to those of the square waveguide and appear virtually identical to the modal pattern of circular waveguides. The rectangular dielectric waveguide is clearly the more general description of the rhabdom of Insecta. 3.6.2.3.4 The circular waveguide modes of Heteronyrnpha merope

Nilsson et al. provided photographic records of the actual standing wave patterns within the rhabdom of their butterfly, Heteronyrnpha merope. Figure 3.6.2-11 shows their idealized (calculated) equivalents of the amplitude functions and the modal patterns They also labeled the types of standing waves encountered and proceeded to determine the dimensions of the rhabdom required to support these modes. The figure and its caption contain a large amount of critical information. In this species, many of the mode patterns are polarized linearly as indicated by the

258Goldsmith, T. & Bernard, G (1974) The visual system of insects in Rockstein, M. ed. The Physiology of Insecta, 2nd Ed. Vol II. NY: Academic Press page 219 259Wehner, R. (1976) Structure and function of the peripheral visual pathway in Hymenopterans in Zettler, F. & Weiler, R. eds. Neural Principles in Vision. NY: Springer-Verlag page 292 260Marcuse, D. (1982) Light Transmission Optics, 2nd Ed. NY: Van Nostrand Rheinhold 130 Processes in Biological Vision

second column. Using the cut-off factors shown, the size of the related waveguide dimension can be calculated with some precision. The component modes beginning with a TE or TM are linear modes. Those beginning with HE or EH are circular modes. The amplitude functions describe the electronic phase of the various portions of the intensity patterns shown. Within the scope of their investigations, they note in their caption that where pairs of circular waveguide modes are shown, they always occurred in pairs that generated the associated linear modes shown.

Figure 3.6.2-11 “Terminology and characteristics of the waveguide modes with cut-off values (Vc) below 5.5. The LP (linearly polarized) terminology denotes sets of modes that combine to give a linearly polarized result, The constituent modes (TE, TM, HE and EH) form groups with the same or nearly the same cut-off. These modes may show complex patterns of polarization but they are always excited in pairs that produce a linearly polarized LP mode. (The subscripts describe the state of polarization and number of lobes in the pattern.) The intensity patterns (derived as the square of the amplitude) are shown as they appear at the waveguide aperture. Changing angle, position or wavelength of excitation, causes some modes (like the 2nd) to appear in more than one form (Snitzer and Osterberg 1961). Our use of 1st, 2nd, 3rd etc. denotes those families of modes that share the same or similar cut-off values.” The amplitude function and appearance (intensity) are calculated (idealized). Figure and caption from Nilsson et al., 1988.

Nilsson et al. discuss the mathematics of circular waveguides in considerable detail in their section 3, including development of the critical cutoff frequency as a function of diameter and the dimensionless cutoff parameter, VC. Similar equations apply to the cutoff frequency (wavelength) rectangular waveguides. To support their analyses, they changed to the rhabdom of a different species of small nymphalid butterfly, Xois arctoa. Their discussion describes various cutoff wavelengths using color names from the vernacular rather than those defined scientifically by the US National Bureau of Standards (now NIST) or the CIE (Section 3.6.2.1). They showed in the laboratory nd that the cutoff wavelength of the rhabdom of Xois arcoa is 590 nm for the 2 mode (LP11) at VC = 2.4. They physically measured the diameter of 10 rhabdom of three animals of this species and obtained a diameter of 1.86 ± 0.12 microns (s.d.). With these values they were able to solve their equation (6) for refractive index of the rhabdom of 1.362 based on their assumption that the surrounding tissue had an index of 1.34. The Retina 3- 131 Note the technical problem in this last computation, a problem encountered frequently in the biophysics literature. A value precise to four digit accuracy is calculated from values only good to 2 and 3 digit accuracy. They proceed to rationalize their logic ending with the statement, “A rhabdom refractive index of 1.36 is thus likely to be close to the true value.” Thus, they revert to a claim of three digit accuracy based on only two digit accuracy in their measurements of the diameter and VC. A long wavelength limit of 590 nm for the cutoff wavelength of the rhabdom provides an explanation why no L–channel spectrum (typically peaking in the 600-625 nm region) is recorded from the eyes of many species of Insecta. The rhabdom would need to have a diameter of 2.828 times the longest wavelength to propagate such energy, i.e., a diameter of 1.77 microns to propagate energy with a wavelength of 625 nm (0.625 microns). Nilsson et al. do note the significant range of diameters in the rhabdome of butterflies (section 3e). They assert that most butterflies have rhabdom in the 2 micron diameter range and that Melanitis leda is unusual in having a rhabdom of 4.5 microns diameter. The rhabdom of Heteronympha merope is given as 2.1 microns. These butterflies would have no difficulty of propagating the L–channel spectrum through their rhabdome. The diameter of the outer segment of the human photoreceptors is usually taken as 2 micron diameter or marginally larger (Appendix L). In a review, Stavenga discussed a wide range of functional variations among insects261. [xxx combine with other introductory material ] A similar set of equations apply to a rectangular waveguide formed by a high index inside material surrounded by a low index material, as found in fiber optics. Nilsson et al. note some of the energy propagated along such a dielectric waveguide is present in the space outside of the boundary between the two materials. However, the density of this energy falls off exponentially within a very short distance of the interface relative to the wavelength of the energy involved (Section 4.4.3 of Processes in Biological Hearing and citation 123 therein). The vast majority of electron micrographs show the pigments, of any type, outside of this distance. 3.6.2.3.5 The rectangular dielectric waveguide modes

Dielectric waveguides play a major role in the modern quantum optics of thin films and fiber optics. The knowledge gained in these fields is directly applicable to the waveguides of biology. Goell has provided the foundation for and required analyses to understand the modal patterns of a rectangular dielectric waveguide262. A more recent and expansive analyses involving even more complex arrangements of dielectric materials is by Bierwirth et al.263.

Goell proposed to achieve an analysis of the rectangular dielectric waveguide by matching the known character of the fields outside a dielectric interface with the similar fields inside the dielectric interface. He recognized immediately that the fields would involve both conventional and modified Bessel Functions rather than simple trigonometric functions. He used a very sophisticated analysis. He also noted;

C “unlike metallic waveguides, the field patterns of dielectric waveguides are sensitive to refractive index differences, wavelength and aspect ratios.” C “Since the rectangular dielectric waveguide modes are neither pure TE nor pure TM, a different scheme must be used. The scheme adopted is based on the fact that in the limit, for large aspect ratio, short wavelength, and small refractive index difference, the transverse electric field is primarily parallel to one of the transverse axes. Modes are y x designated as E mn if in the limit their electric field is parallel to the y axis and as E mn if in the limit their electric field is parallel to the x axis. The m and n subscript are used to designate the number of maxima in the x and y directions, respectively.” Goell’s major conclusions include; C “The results of the computations show that the circular harmonic method for analyzing rectangular dielectric waveguides gives excellent results for waveguides of moderate aspect ratio.” Figure 3.6.2-12 shows several computed modal patterns for rectangular waveguides of modest aspect ratio. The

261Stavenga, D. (1992) Eye regionalization and spectral tuning of retinal pigments in insects TINS vol 15(6), pp213-218 262Goell, J. (1969) A Circular-Harmonic Computer Analysis of Rectangular Dielectric Waveguides Bell System Technical Journal vol 48(7), pp 2133–2160 263Bierwirth, K. Schulz, N. & Arndt, F. (1986) Finite-difference analysis of rectangular dielectric waveguide structures IEEE Trans Microwave Theory and Tech vol 34(11), pp 1104-1114 132 Processes in Biological Vision

2 1/2 equation for the script B, = (2b/λ0)C(nr - 1) .

Figure 3.6.2-12 Modal patterns for rectangular dielectric waveguides of modest aspect ratios. The aspect ratio, a/b = 2, the index of refraction difference was 0.01 and the script B is a parameter provided in the text. From Goell, 1969.

3.6.2.4 The superposition (scotopic) eye of Insecta

Exner (1891) introduced the concept of the eye of Insecta changing optical configuration as a function of the external illumination intensity. He based the proposition on his observation of the change in the degree of pigmentation between the crystalline cones of the compound eye with time. His proposal was that the optics of the eye changed from an all refractive (dioptric) design to a design involving both refractive and reflective (catadioptric) optics. His proposition was based on a number of assumptions; C the receptive field of the rhabdom at its entrance considerably exceeded the cone of light delivered by the cornea and crystalline lens of a single ommatidium to its rhabdom, C in the absence of blocking pigment, light entering a cornea some distance from a specific rhabdom could be reflected by the wall of the crystalline lens in order to arrive at the reference rhabdom, C as a result, the light collection efficiency of a given rhabdom could be increased significantly under low light conditions. The technology of the 1890's did not provide adequate answers regarding the feasibility of this approach. However, it was noted the increase in sensitivity would be accompanied by a significant loss in acuity of the overall eye. Between 1890 and 1970, the drudgery of performing an adequate optical ray-tracing was quite high. With the advent of the desktop digital computer, the drudgery was greatly reduced but only a few people knew how to perform even simple “thin lens” ray tracing, much less the more sophisticated ray-tracing associated with “thick The Retina 3- 133 lens” designs of biology. Goldsmith & Bernard (1976) reviewed the pros and cons of the superposition lens concept in considerable detail in 1976, citing the inconclusive analyses of Horridge (1969-1972) and the opposing arguments of Hausen (1973). They introduce the challenge of “semantics and the superposition eye” on page 225 and address the question of adequate investigative protocols on page 261. The question of whether an inverted or non-inverted (erect) image is presented to the rhabdom by the cornea and crystalline lens is also debated. They also note the inadequacy of the precision of the indices of refraction up through the 1970's. At least three digit accuracy is required to the right of the decimal point, with four digits preferred. As noted in the preceding figure, even three digit precision is not commonly available in the biological literature for Insecta. There may be significant differences between species. Nilsson et al. reviewed the potential superposition eye of Insecta in 1988 based primarily on the Australian nymphalid, Heteronympha merope, butterfly. They suggest both optical designs may have evolved from the same proto-design. They also presents a more modern analysis of the performance of both types of eyes based on both geometrical ray tracing and refractive ray-tracing applied to thick lens systems. As noted earlier, their conclusion was the most important eyes employed a non-inverting terrestrial telescope design that was afocal. It did not produce a real image at the focal plane (entrance to the rhabdoma). Nilsson et al. summarize their position in two parts, 1. “Originally, we considered focal and afocal systems to be discrete and even opposite solutions to apposition eye design, and no intermediates appeared to make sense. However, when we considered wave and geometrical optics together we came to realize that under some circumstances alternative interpretations of the same optical system could legitimately co-exist. Some of these problems are considered in the Discussion.” 2. “In retrospect, it seems not surprising to find evidence for afocal apposition optics in butterflies because it has been known since Exner (1891) that most other lepidopterans possess an afocal system in their refracting superposition eyes. In this other major type of compound eye - the superposition eye - an erect superimposed image from many facets is formed on the retina, and this is most commonly realized by an afocal system in each facet. Our findings in butterflies thus provide a link between apposition and superposition eyes that otherwise seemed unrelated.”

The findings of Nilsson et al. depend on their ray-tracings that were not described or shown. They also did not discuss the gradient index form of the crystalline lens of their butterflies. Their section 2 does provide the dimensions and indices they used in their analyses. They did note, “The limited resolution of light microscopic techniques is, however, inadequate to provide detailed information about the narrow cone stalk. Electron microscopy (Fig. 3) demonstrates a more complex structure in the cone stalk, and it is clear that the stalk region cannot be optically homogeneous. A detailed analysis of the optical structure of the cone stalk seems to be beyond the reach of available techniques, but we can still conclude that, whatever is in there, it behaves as a lens-cylinder.”

They conclude that the crystalline lens is a complex optical system of its own, “The other, and more important, conclusion is that a simple lens-waveguide model of the ommatidium, where the cornea provides most or all of the optical power, must now be abandoned. A ro1e has to be found for the powerful lens situated just distal to the rhabdom tip.” They then discuss modeling the “thick lens” of the crystalline cone as a series of “thin lenses.” This position alone shows they were not experienced or competent in thick lens optical ray-tracing. They do indicate the limited utility of their approach, in a highlighted paragraph on page 348, and indicate their limited skill in optical ray-tracing. No current optician would dare propose such a crude modeling approach. The design illustrated in their figure 6 suffers from significant coma, a condition not introduced or discussed in their text. The closing paragraph of section 2c (page 351) is instructive.

[xxx The remarks of Nilsson et al. re waveguides in sections 3a and 3b need to be addressed in a different section of this work. ] [xxx The visibility of the standing wave patterns within the rhabdom can be observed exteernally for various families of butterflies and moths. Note this in section xxx. Excellent numbers for the size of the rhabdom based on which modes are suppressed as a function of wavelength. ] The discussion in the Nilsson et al. paper is extensive and informative. It deserves further study by those performing laboratory experiments. The discussion ends with, “A natural question to ask is whether a similar evolutionary sequence has led to the superposition eyes in other insect orders such as the Neuroptera, Trichoptera and Coleoptera. As with the Lepidoptera, a resolution of these questions depends on finding isolated instances of apposition eyes in groups where the superposition design is dominant.” Their figure 18 reverts to caricatures to show the similarities and inter-convertibility of apposition and superposition eyes, but in an interesting way. 134 Processes in Biological Vision

In 2002, Land & Nilsson reexplored the merits and feasibility of biological optical systems of the apposition and superposition types at the level of an introductory text. While presenting a large number of caricatures, they did not provide optical designs resulting from modern ray-tracing that confirmed the validity of many of their caricatures. They did provide a clear cross section of a superposition type compound eye, Figure 3.6.2-13. They also presented empirical evidence of the quality of the image formed at the individual rhabdom (pages 162 & 169). Their discussion of adaptation in such an eye was quite superficial and did not address the actual mechanism of adaptation associated with the “bleaching” of the chromophores. A more modern analysis employing optical ray-tracing software capable of handling thick lenses consisting of gradient index of refraction materials would be most welcome in this area. It does appear the optical character of the crystalline cone differs considerably between the apposition and superpositions eyes. 3.6.2.5 Morphology of stage 4, information extraction, and stage 5 cognition

Strausfeld has attempted to describe the entire visual modality of two species of Dipera, Musca domestica and Calliphora erythrocephala in block diagram and 264 camera lucida form based on histology . The material Figure 3.6.2-13 Superposition eye of the nocturnal dung is extensive but may differ in terminology from this beetle, Onitis westermanni. CC; crystalline cone, CZ; clear work at the detail level. zone, rh; rhabdom layer. Vertical background striation is an artifact of the copy process. From Land & Nilsson, 2002, Strausfeld describes the number of neurons present in attributed to S. Caveney. the fly, 3.4 x 105 in total, with 76% of these related to vision, Within the visual modality, “68% invade the medulla, 12% invade the lamina, 18% invade the lobula and 2% are derived from the lobula plate.” He goes on to describe the visual modality in considerable detail. However, since he did not recognize the stratified neurites consisted of the dendritic (non-inverting input) tree and the poditic (inverting input) tree, the material requires reinterpretation. Many of his figures are suggestive of signal differencing that is generally associated with the horizontal and amercine neurons of chordate retina. His description of the amercine neurons makes it clear that they have a pedicle even though the axon may be of minimal length. He does assert the visuotopic mapping of the external environment is maintained at least as far as the medulla (and potentially the early neurons of the lobula. He subdivides the global lobula into two portions, the lobula and the lobula plate. He goes on to map many outputs from stage 5 leading to the efferent command neurons of stage 6.

Strausfeld introduces two sections related to the electrophysiology of the fly visual modality but does not provide any circuit diagrams, interconnection diagrams, waveforms or detailed descriptions. His discussion is quite detailed but not necessarily as consistent as it would be if supported by such diagrams and/or waveforms.

Figure 3.6.2-14 from Sauman et al. (2005) describes the plan view of the visual modality of the monarch butterfly during their investigations of the butterfly’s navigation capabilities. They employed a Cry-staining procedure; “Cryptochrome (CRY) is colocalized with PER and TIM and is a blue-light photoreceptor involved in photic entrainment.”

264Strausfeld, N. (1976) Mosaic orgainzations, layers, and visual pathways in the insect brain. In Zettler, F. & Weiler, R. eds. Neural Principles in Vision. NY: Springer-Verlag Sections 2.6 & 2.7 The Retina 3- 135

Figure 3.6.2-14 Visual modality of monarch butterfly visual modality. Figure is a composite showing two distinct mappings shown in red. Left of the cut line; Dorsal Rim Axonal Projections and their Relationship to CRY-Positive Fibers. Fiber is best identified as an lvf fiber between a retinula and the medulla. Right of the cut line; Schematic representation of CRY-positive neurons and their axons. RE, retina; LA, lamina; ME, medulla; LO, lobula; PL, pars lateralis; PI, pars centralis; SOG suboesophageal ganglion. See text. From Sauman et al., 2005.

3.6.3 The electrophysiology of the compound eye of Insecta

Ribi has focused on the use of electron microscopy to define the neurological circuitry associated with the ommatidia of several insect species. In Ribi, 1987, the same numbering system is used for the retinula of an ommatidia as in the papers of Section 3.6.2. By combining the text from Ribi and others with that of the Invertebrate Brain Platform, a reasonably clear understanding of the circuitry of the insect eye can be obtained.

The signals derived from the sensory receptor neurons (R1 through R8 or R9) do not individually indicate the polarization of the spectrally selected light. However, by comparing the amplitude of the signals from two orthogonally arranged receptors sensitive to the same spectral wavelengths, an indication of the polarization angle of the source light can be estimated. Bandai et al. have described the polarization sensitivity of the sensory neurons within an ommatidium. They describe the polarization sensitivity of neurons R1 and R2 of the distal capsule as perpendicular to that of R3 and R4 of the same capsule. In the proximal capsule, R6 and R8 are perpendicular to R5 and R7 and at 45 degrees to the polarization sensitivity of the sensory neurons of the distal capsule. However, they were less sure of the spectral performance of these sensory neurons in the butterfly. They note, “5. We conclude that R1 and R2 are either UV, violet or blue receptors whereas R3 and R4 are green receptors. Some R6 and R8 are red receptors.” They offered no comment about spectral performance of the R5 and R7 receptors. They provide good data in their Table 1 but identify a violet receptor spectrum peaking at 402 nm. Their Standard Error relative to the Mean was quite large for R5 with a peak sensitivity at 402 nm. This work predicts their narrow spectrum in figure 6 is actually a difference spectrum between UV– sensitive and S– sensitive receptors. Their peak spectral sensitivities are systematically shifted to longer wavelengths in the UV– and S– spectra and to shorter wavelengths in the M – and L– spectra relative to the predicted peaks of this work. This situation could be due to the small sample sizes they used. Ribi (1987) has described the interconnections of many of the sensory neurons and orthodromic neurons. However, he notes, “Although we have conclusive electrophysiological recordings the structure of the retinula-cell endings and second-order neurons is poorly understood.” This work notes that when Ribi discusses the pigments associated with the various sensory neurons, he is speaking of gross pigment and not the monolayer of liquid-crystalline chromophoric material coating the individual microvilli. Ribi is not definitive as to whether he is describing the pigments as perceived by reflected or transmitted light. His figure 1 is complicated. In summary, the spectral performance of the insect sensory neurons are reported as follows; 136 Processes in Biological Vision

Author Ribi Bandai Arikawa Chen Year 1987 1992 1999 2016 Species Papilio Papilio Papilio Graphium Dorsal Ventral Sample - - 15-20 ~15 nm 20 20 spacing Receptor R1 UV UV, v, S UV UV R2 UV UV, v, S UV R3 S M M M R4 S M M M R5 M [red pig.] - - L/M L R6 M [red pig.] ?L L/M L R7 M [red pig.] - - L/M L R8 M [red pig.] ?L L/M L R9 - - [no pig.]

UV (342) 380 364 354(8) S (437) 450 460 455(20) M (532) 540 522 520 541(24) L (625) 610 601 600 599(4)

1. The “red pigment” in brackets was not further defined. It should probably be described as magenta pigment (being the complement of the M–channel absorption spectrum of yellowish-green). 2. Several of the papers noted that the R9 receptor was not accompanied by any pigment clusters.

Chen has defined three distinct specializations of ommatidia within the compound eye of individual butterflies of Graphium. In general the sensitivity of the sensory neurons is consistent among the specializations based on their Table 1 but R5-8 of the dorsal ommatidia were reported as diverse as indicted. In some cases the divergence from the nominal peak absorption was large (640 nm versus a mean of 599 nm in one case). They noted in this regard,

“We extensively manipulated the relative proportions of R541, R582 and R599 in our attempt to reproduce the spectral sensitivities” of several of their “receptors in the ventral type II ommatidium, and also tried incorporating various pigment filtering effects. However, we were unable to obtain satisfactory fits to our data.”

The numbers in parenthesis at lower right are the numbers of receptors used to determine the average peak sensitivity. The wavelengths in parentheses at lower left are theoretical values for the non-Gaussian chromophores rounded to three digit accuracy. The value of 625 nm is a first order prediction that may actually occur at shorter wavelengths due to second order mechanisms. In all of the experiments in the above Table, the monochromator sample spacing was too coarse to justify three digit precision. In the Chen paper, a set of 22 “narrow band” filters were used without specifying the actual spectral profiles of the filters. The spectral sensitivity of R1 through R8 of Ribi (1987) are based on Horridge (1983). The color of the pigment associated with R5 through R8 by Ribi is appropriate for these pigments if observed by reflected light. The lower case v associated with the values of Bandai (1992) is used to indicate a reported value not supported by theoretical considerations. Many of the pigment labels used by the authors in the table are casual and do not differentiate between their color by transmitted light (UV, violet/blue, green or red) from their color by reflected light (colorless, cyan, yellow or magenta). The colors observed by transmitted light are associated with the descriptor, additive color, whereas the colors observed by reflected light are associated with the descriptor, subtractive color. The energy threshold for exciting a sensory neuron in Insecta has not been found in the literature. In humans, and probably a wide variety of species within Chordata, the energy required is approximately 2.0 electron volts (46 kCal/mole) based on Sliney (Section 12.5.2.4). This value suggests excitation by photons at wavelengths in the red may require a 2 photon —> 1 effective exciton mechanism (Section 12.5.2.4), or an alternate 2 exciton —> 1 free electron mechanism, in order to excite the sensory neuron (thereby generating a free electron within the dendroplasm). Either form of this second order mechanism may cause a shift in the peak L–channel spectral sensitivity to the region of 610 nm. The Retina 3- 137 3.6.3.1 The neural circuitry of stage 1 of the eye of Insecta

Figure 3.6.3-1 is the proposed neural circuit of the sensory neuron of Insecta adapted from the similar circuit of the eye of Chordata. A complete development of this circuit topology is provided in Section xxx. The major difference is in the implementation of the microvilli of the sensory neuron. In the eye of Chordata, the microvilli (microtubules in that technical field) are arranged in a circular chalice (or goblet, Section 4.3.2) that contains the disk stack of the outer segment of each photoreceptor neuron. In Insecta, the microvilli extend from the dendrite to form a grid that is perpendicular to the long axis of the rhabdom (top frame) This grid (labeled wafer 1) is followed by wafer 2 and subsequent wafers for the length of the rhabdomere. Each wafer consists of a group of microvilli connected to one or a series of dendrites all associated with a single sensory neuron. Each wafer is coated with a layer of one of several Rhodonine chromophores (probably on both sides). The grid of each wafer is a functional structure by itself; the microvilli of the grid are spaced at about 0.2 microns and together they form the polarizer associated with that rhabdomere. When the e-vector of the incident light is aligned with the direction of the microvilli of the grid, it is maximally effective at stimulating the chromophores coating the microvilli. The middle frame shows the electronic components within the sensory neuron that are formed by various internal lemma (not shown) and specifically modified sections of external lemma (shown as a jagged line). The lower frame shows the same electronic components configured to be more recognizable to an electrical engineer. The circuit is generally known as an unbalanced differential pair of Activa (transistors), or alternately a cascode circuit. The base terminal of the left Activa is shown open (not connected to any other element) while the base of the right Activa is shown connected to a battery (labeled (3) for electrical bias. The delay element represents the slow ionic conduction of electrical charge along the length of the axoplasm within the axolemma leading to the pedicle of the axon.

The left Activa forms the adaptation amplifier and the right Activa forms the distribution amplifier since it drives the axon which may distribute a voltage signal through synapses with a variety of orthodromic neurites. The “base” of the left Activa is stimulated quantum-mechanically by the excitons created in the chromophoric liquid crystal due to absorption of a photon. For each exciton created, the absorption coefficient of the liquid crystal is reduced (making it more transparent) until the exciton is transferred to the left Activa. The entire sensory neuron is a logarithmic device that can accommodate a wide range of stimulus conditions..Under daylight conditions, the chromophores of most eyes are reduced to about one percent of their dark adapted condition (they are significantly bleached).

Goldsmith & Bernard (1974, page 175) reported the unusual charge distribution associated with the microvilli based on electron-microscopic examination; “the walls of the microvilli are more electron opaque that the interiors.” The walls (exterior lemma) form the first (left) Activa. The opaque regions represent the site of the electrostenolytic process converting glutamic acid to GABA and CO2 while generating a negative electrical potential in the dendroplasm relative to the external milieu (Section 8.6). The electrostenolytic mechanism introduces a component of the impedance labeled (1) in the figure. On the same page, Goldsmith & Bernard provide additional information about the dimensional character of the microvilli grid. These features vary widely among Figure 3.6.3-1 Proposed morphology and circuit diagram of species as indicated by the taxonomy in their Table I the sensory neurons of Insecta ADD. (page176). The potential significance of these structures to the polarization sensitivity of these insect eyes is noted (page 177 & Section V,C). The labels IPM (inter-photoreceptor matrix), INM (inter-neuron matrix) and OLM (outer limiting membrane) are left 138 Processes in Biological Vision

in this representation because it is important to provide an oxygen free IPM to prevent premature chromophore oxidation. On the other hand, the INM must accommodate chemicals rich in oxygen moieties/legands. The labels OS (outer segment) and IS (inner segment) have a less defined meaning in the rhabdom of Insecta than in the chordate eye. 3.6.3.2 The Photo excitation/De-excitation equation of the eye of Insecta

It is proposed that the photo excitation/de-excitation (P/D) mechanism of Insecta is the same as for the rest of the animal kingdom. Photons are absorbed, generating excitons and the excitons stimulate the specialized lemma of the microvilli while simultaneously being de-excited. The stimulation of the microvilli lemma allows an electron to enter the dendroplasm shared with the rest of the microvilli of that dendrite as a “free electron.” This overall mechanism is given the name transduction. Note, it does not involve any atoms, ions or molecules moving through the lemma of the neuron. The (P/D) mechanism associated with the transduction of photons into an electrical current within the sensory neuron is complex. However, the quantum-mechanical differential equations describing this mechanism have been developed and solved (See Section 7.2). The response is significantly different for stimulation by an impulse function versus a much longer duration square pulse. Except for the flash associated with lightning, it is unusual in the natural environment to encounter an impulse or square pulse where the leading edge of the pulse occurs in less than a few milliseconds. The light adaptation function is dependent on the amplitude of the stimulus. It can be only a few microseconds.

Through the 1970's, it was difficult to obtain a commercial optical shutter mechanism with a fast enough rise time to avoid impacting the recorded rise time of the test set combined with the sensory receptor of the specimen. An electro-optical shutter was generally required. Pulsed light emitting diodes (LED’s) had not entered the commercial marketplace. Currently, it is important to demonstrate the shape of an impulse or a square pulse is adequate for use in P/D mechanism evaluations.

[xxx The complete equation for the P/D mechanism in response to an impulse function is shown here in the stand- alone version of this section. The definition of all of the parameters appears in Section 7.2

Eq. 7.2.4-1

under the condition that σ •F•τ not equal 1.00

Note the impulse function “i” includes three distinct terms, a scale factor, a delay term and an amplitude term. Several auxiliary terms are used to quantify the complete function as a function of temperature and stimulus intensity. The P/D response to a square pulse is even more complex and will not be displayed here. xxx] It should be noted that the amplitude term includes two separate exponential terms where the multiplier to each “t” is defined as the reciprocal of a time constant. However, the two time constants can not be summed or differenced to give a net time constant applicable to the settling phase of a P/D response. The Retina 3- 139 The amplitude term, with the time constant in the first exponential being a function of F accounts for the shape of the complete monopolar description of the P/D response. The delay term introduces an absolute delay in the time scale before the amplitude term departs from the horizontal base line.

Each term in the total response, i(F,t,j,σ,τ) is a function of the stimulus intensity in photons/sec/unit area, F. 3.6.3.2.1 The stage 1 P/D response to an impulse function

Figure 3.6.3-2 reproduces the nominal response of the P/D mechanism to excitation by an impulse from Section 7.2 based on the human eye of Chordata. The P/D Equation involves two separate time constants, an attack time constant controls the rise time of the leading edge of responses through an important parameter, the product of the time constant and the quantum flux absorbed by the chromophores coating the disks of the outer segment of the sensory neuron receptor in Chordata or the microvilli of the rhabdomere of the sensory neuron receptor in Insecta (and Mollusca). The second (decay) time constant controls the relaxation of the response back toward a steady state level during long stimulation periods. Once stimulation has ceased after a long period of stimulation, where a steady signal level has been reached, the decay time constant is the only parameter controlling the decay of the response back to baseline. Hodgkin attempted unsuccessfully to develop and solve the P/D Equation during the 1950's, and then tried to represent this mechanism using the Poisson Equation with poor results. The P/D equation exhibits a unique condition (when σCFCτ = 1.000) where the two time constants give way to a single time constant. The response under this condition takes the form of the Poisson Equation and has been named the Hodgkin Solution in his honor.

Figure 3.6.3-2 Theoretical impulse responses based on the P/D Equation. The latency of the mechanism is shown by the delayed departure of the waveforms from the baseline, as a function of the peak flux density, F, in photons/micron2- sec. For other temperatures, the time scale can be multiplied by the appropriate value of KT. The value of sigma, σ, is appropriate for perpendicular illumination or a stack of individual disks. The Hodgkin Solution (σCFCτ = 1.000) occurs at F =12.

The value of sigma, σ, for the eye(s) of Insecta will be different because of the different configuration and orientation of the rhabdomere relative to the incident light compared to the disks of Chordata. This configuration and orientation may even vary within Insecta because of the desire to achieve different sensitivities to polarized light. 140 Processes in Biological Vision

Note the change in the apparent rise time constant with different values of the photon flux density in quantum units, F. When quoting an apparent rise time constant the investigator must associate it with a specific flux density or illumination intensity to be meaningful. The Retina 3- 141

3.6.3.2.2 The stage 1 P/D response to square pulse stimulation ADD

There are a wide variety of responses by insects to square pulse light simuli. For reasons not entirely clear to this theoretician, the investigators tended to use very long square pulses, frequently 500 ms. The effective time constants of the relevant eyes tend to be much shorter than 500 ms. As a result, the features of the recorded responses tend to be poorly displayed. Laughlin (Land et al 1981, chapter 2) provided a variety of responses with long stimuli duration for a variety of Arthropoda (dragonflies, house flies & Limulus). Laughlin (1976 in Zettler & Weiler) provided a set of waveforms for both the retinula and “large monopolar cell,” LMC, of the dragonfly, Hemicordulia tau. While not all recorded at the same stimulus intensity, they show a variety of conditions that can be addressed based on knowledge of the theoretical P/D mechanism that he did not have available, and the subsequent mechanisms within the retinula and the lamina of various eyes of Insecta. Electrical signal saturation at the output axoplasm of the sensory neuron is particularly evident in his figures and in Figure 7.2.6-4 of this work. It shows the measured impulse response waveforms of Baylor (1979) overlayed with the theoretical equation for various stimuli intensity (without significant discussion of the variation in delay with stimulus intensity). The specimen was a toad. Laughlin also ignored or was unaware of the variable delay in the response functions as a result of different stimuli intensities (Section 7.2). This delay was illustrated in the previous figure showing the response of the endothermic animal eye (at 37 C) to an impulse function. These delays can be significantly longer for the eyes of exothermic animals. Figure 3.6.3-3 shows the P/D response of a sensory neuron to square pulse excitation of constant intensity for its duration.

Figure 3.6.3-3 Theoretical P/D response to a square pulse stimulus. For illustration only, use equation to establish actual values. Stimulus 200 ms long beginning at time zero. Responses are each of 200 ms duration following a delay inversely proportional to the stimulus intensity. Decay time constants after cessation of stimulus are all 12.5 ms. F is the stimulus intensity. Note the potential saturation level associated with the maximum axon current of the first Activa of the sensory neuron. Parameters are those of the previous figure. At F = 10, the response is below the Hodgkin Condition and of slightly different form than that at F = 17.5. See text. 142 Processes in Biological Vision

It should be noted that the P/D response varies with the intensity of the excitation but remains a continuous function. The overshoot should not be described as a phasic portion with the rest considered tonal. The terminology is inappropriate. The overshoot “settles” to a steady state value determined by the intensity of the stimulation. The overshoot is approximately 45% for F = 140; 30% for F = 70; 15% for F = 35; 10% for F = 17.5; and 0% for F = 10. The initial rise for each waveform is proportional to the product of the stimulus intensity and the first time constant of the P/D Equation. These slopes are not related to any simple time constant in the P/D equation, and are not a simple exponentials. The apparent time constants shown between 50 and 250 ms are not real. The shape of the settling, after the initial peak involves a combination of the two time constants within the P/D equation. When the stimulation ceases, each response returns to the baseline according to the real second time constant of the P/D equation only. 3.6.3.2.3 Inversion of the P/D response by stage 2 neurons

Land et al. (1991) presented an extended study of the noise associated with signals measured in the retinula of various members of Insecta. Their page 247 used patch clamp techniques to provid excellent electrophysiological data confirming the P/D mechanism is utilized in the dragonfly at several specific locations in the signaling chain. They used 500 ms stimulation pulses that result in more complex responses than illustrated in the above figure. They show the waveforms measured at the axon of their large “monopolar” cell (LMC) are clearly inverted relative to the waveforms in the axoplasm of the preceding retinula axon. This inversion suggests the LMC acts like a horizontal cell of stage 2 in the chordate retina, as detailed in Figure 3.6.3-4. The insert at lower right shows the electrical schematic of the Activa. The schematic is the same as that for the horizontal cell of the chordate retina used elsewhere in this work. This figure is a simplification of one from Boscheck shown below.

The label large monopolar cell of histology is highly inappropriate when describing the electrophysiological (functional) performance of this neuron type. Functionally, it is a horizontal neuron with a bipolar electrical signal at its pedicle. The Retina 3- 143

If all of the svf signals applied to this stage 2 neuron synapsed with only the dendritic (non-inverting) tree, the output signal would constitute a summation of spectrally (and possibly polarization) distinct signals. The output signal would represent a broad spectral signal not associated with any sensory neuron of stage 1. It would represent a R–type signal of this work. If the svf signals applied to this stage 2 neuron synapsed with both the dendritic tree and the poditic (inverting) tree, the resulting stage 2 output signal would correspond to O–, P– or Q– type of signals representing components of the chrominance channels of vision. Alternately, for input signals representing signals of different polarization from stage 1, they could represent a new group of polarization signals not defined among the eyes of Chordata. The above figure was modified from Wehner based on the availability of considerably more electrophysiological data. The Wehner figure, attributed to the worker bee, may be a significant simplification of an earlier figure by Boschek (1971) based entirely on histological research265. The Boschek figure also appears in Goldsmith & Bernard (1974, page 196) where it is attributed to the common fly, Musca domestica.

3.6.3.2.4 Generator potentials versus action Figure 3.6.3-4 Synaptic connections in the cartridge of a potentials in Insecta worker bee based on data. . . . Black outline unshaded; the LMC (type L1) and its ramifications. Black shaded and The stage 1 sensory neurons of Insecta produce what hatched; short visual fibers, (svf from retinula cells). Black are defined as generator potentials via the P/D outline & shaded; centrifugal fibers. A, B & C; mechanism. These waveforms are entirely tonic stratification of laminar neuropil. Active (analog) in character. They are not action potentials or electrophysiological elements; Activa shown at the junction any other kind of phasic waveform. between the dendritic tree on the left, the poditic tree on the right hand, and axon of the Activa extending below the As a general rule, Insecta does not employ action Activa. A membrane (internal lemma) is shown rising potentials, specifically phasic signals generated as vertically from the Activa to separate the dendroplasm from monopolar pulses by stage 3A analog to pulse the podoplasm of the neuron. The termination of this lemma converting neurons. There may be an exception in the is undefined, and unimportant, at this time. Right; case of very large (and possibly extinct) members of representation of the electrical schematic of the Activa. See Insecta. Pulse type neural signals (action potentials) text. Modified from Wehner, 1976. are typically required when neural signals must be propagated for more than two millimeters before reaching a synapse (or Node of Ranvier). In Chordata, less than 5% of the signals carried within the neural system involve phasic (pulse mode) signals. 95% of all signals within Chordata are tonic (analog mode) signals (Section 10.1.1.2). The eccentric cells of Limulus, and potentially the neuron labeled R9 in ommatidia, can be modified through evolution to generate stage 3 pulse output signals. This modification typically involves wrapping the long axons in myelin. As an alternative, the axon may be closely packed within a group of shorter neurons as found in some members of Mollusca, such as the locomotion neurons of squid. The locomotion neurons are not properly classified as stage 3 neurons. The long visual fiber (lvf) neurons may carry signals, resembling action potentials, that may be generated by eccentric neurons (or neurons labeled R9). Many of these lvf appear to originate in the stage 1 ommatidia and

265Boschek, C. (1971) On the Fine Structure of the Peripheral Retina and Lamina ganglionaris of the Fly, Musca domestica Z Zellforsch vol 118, pp 369-409 144 Processes in Biological Vision

bypass the stage 2 lamina on the way to the more distant stage 3 medulla.

3.6.3.3 Empirical signals associated with the ommatidium and stage 2 lamina

Figure 3.6.3-5 provides a set of analog waveforms related to the elements of both the ommatidium and lamina of the dragonfly, Hemicordulia tau, from Laughlin (1976). As noted most clearly in the 1976 caption to the figure, “the flash intensities are not equal between records.” The vertical calibration bars are all 10 mV. Note the much taller bars at the lowest stimulation levels. The flash duration was always 500 ms and its intensity increases from top to bottom in each column.

Figure 3.6.3-5 Diagrammatic representation of two retinula neurons and one LMC. The axons from the ommatidium were described as short visual fibers. Responses were obtained at different locations, both intracellularly and extracellularly (lamina depolarization column). Interruptions in the flash traces of the lamina depolarization and two associated with the retinula axon were not explained in the original paper. See text. From Laughlin, 1976.

Laughlin did not describe the spectral or polarization characteristics of his light source, except to say it was a point source located at a distance. He referred the reader to his earlier papers. As noted in earlier discussions, the soma of the large monopolar cell plays no active role within the neural system. It is responsible for the homeostasis of the neuron and not signaling. The single Activa (active semiconductor device) within the LMC is found at the junction of the neurites and the axon. Based on the inversion illustrated between the LMC and retinula axon waveforms, the LMC probe was inserted into the axolemma of the neuron. This inversion between the waveforms indicates clearly that the recordings from the retinula axon synapsed with the poditic The Retina 3- 145 terminal of the LMC. [xxx ADD comments about saturation in the retinula axon waveform and in the LMC waveform at maximum stimulation. Many of the waveforms shown can be related to the theoretical waveforms shown above resulting from the square pulse stimulation of a sensory neuron. Those waveforms in row A show negligible overshoot associated with the leading edge of the pulse response and conform th F = 10 in the earlier figure. Waveforms B1 and B3 can be associated with F = 35 to F = 140 of the earlier figure. C1 can be related to F = 140 or higher and suggests some differences in the characteristics of the test set (some test set overshoot and some loss in low frequency bandwidth in the test set). B2 and B3 show clear examples of saturation in the axonal circuit of the Activa within the neuron similar to the truncated form for F = 140 shown in the earlier figure. The truncation is typically associated with a non-exponential decay after cessation of the stimulus, as the Activa within the neuron regains its normal operating condition. C3 and its associated stimulus trace are a bit confusing. The waveform suggests the presence of a two- step square pulse. The waveform illustrates a settling to an intermediate plateau before the stimulus is reduced. The waveform then settles toward a different plateau before the cessation of all stimulation. The waveform then decays exponentially as expected. The waveforms of column 4 can be associated with inverted waveforms from stage 2 neurons where the driving svf signal was applied to the poditic (inverting) tree as discussed earlier. A4 shows some overshoot similar to and inverted A3. B4 and C4 show similarities to F = 140 or higher but may also show excessive peaking in the test set response. The waveform at lower right appears to show excessive peaking and inadequate low frequency response related to either the neuron or the neuron-test set combination. A trained and experienced oscillograph operator frequently observes what appear to be waveforms obtained without a properly compensated probe. In biological investigations, this is a frequent problem where a “home made” probe is prepared from glass tubing and no frequency compensation is provided as part of the probe assembly.

Wehner (1976, page 280) has provided extensive discussion of the histology of the stage 2 lamina (with some material related to the stage 4 medulla) of bees, Apis. He notes significant differences between the retinula within the ommatidia of bees and the members of Lepidoptera. He notes the axons leaving a given ommatidium are frequently twisted in the opposite direction to the twisting of the retinula within an ommatidium. He also provides more background on the ninth neuron frequently described as either R9 or an eccentric cell (page 289) but draws no clear conclusion.

Figure 2 of Zettler & Weiler266 (1976) shows the P/D responses for a sensory neuron, “a marked receptor of type R1- R6,” over an intensity range of 104. They follow the pattern predicted by the P/D Equation. The output signal varies in amplitude by less than a factor of three. Unfortunately, they do not plot the absolute delay associated with each intensity level and they do not specify whether the neuron was dark adapted between stimuli or the order of stimulus (in increasing or decreasing intensity). There appears to be some overshoot associated with their test instrumentation. Much of the discussion provided by Zettler & Weiler appears speculative based on current knowledge if the electrophysiology of vision. See also Section 3.6.1.3 for additional data from Zettler & Weiler regarding stage 2 signal processing. Their figure 6 does quantify the receptive fields of three individual L-type neurons from a lamina. For three different wavelengths, the UV–, S– and M – receptive fields remain identical. This observation leads to the conclusion that the receptive fields of the adjacent ommatidia are very similar and do not vary significantly with wavelength. The observation also contributes some data relative to whether the crystalline cone is a terrestrial telescope or not, and whether the receptive field of adjacent rhabdom are equal and independent of spectral wavelength. Two of the three receptive fields are 4.0 degrees wide at 50% and the third receptive field equals 5.4 degrees regardless of wavelength. Conceptually, such performance is much more compatible with a terrestrial telescope limiting the receptive field than with a fixed size rhabdom waveguide where its receptive field is inherently sensitive to wavelength. 3.6.3.3.1 A cartridge of stage 2 of the domestic fly

Boschek provided significant information about stages 1 and 2 of the house fly acquired with high magnification electron microscopy. His descriptions of the photoreceptors as well as the synapses involved in these stages is extraordinary. He defines ten configurations of synapses based strictly on histology. Unfortunately, Boschek appears to have fallen into a Bayesian trap. He assumed that every region of high electrical opacity along the lemma of a neuron was associated with a synapse. In fact, such an area can be associated with either a synapse, a Node of Ranvier (among chordates) or an electrostenolytic area constituting the electrical power source for a neuron. Many dimensions are provided. Figure 3.6.3-6 shows a partial schematic of a single capsule of Musca domestica with a wide variety of his histologically defined synapses. Two separate LMC, L1and L2 are shown to the same level of detail as the LMC in the figure of Section 3.6.3.2.3. LMC L3 and L4 are shown less completely. The numbers refer

266Zettler, F. & Weiler, R. (1976) Neural Principles in Vision. NY: Springer-Verlag, Section 2.4 page 259 146 Processes in Biological Vision

to his classification of synapse types. The Retina 3- 147

Figure 3.6.3-6 A semischematic summary drawing of the structure and synapses interconnecting stage 1 and stage 2 circuits of Musca domestica. The numerics and arrows define synapses according to Boschek. This work provides an alternate description of many of the putative synapses. L1-4; somata of LMC. EC; epithelial glial cells. R1-6; short visual fibers (SVF) are axons from retinula cells axon terminals. α,β; two of six paired centrifugal fibers. R7, R8, Long visual fibers (LVF) from retinula cells. U; unidentified fiber fragments associated with type 8 synapses. “Broken lines have been used to indicate areas of inadequate evidence.” Annotation added. See text. Modified from Boschek, 1971. 148 Processes in Biological Vision

“Synapse 10" appears to be an electrostenolytic power source drawing glutamic acid from the adjacent epithelial glial cell. “Synapse 9" is inadequately defined using Boschek’s words but appears to also be a site of electrostenolytic activity. “Synapses 1" and “synapses 5" appear to be the primary synapses between the SVF axons and the LMC neurons. “Synapses 2" and “synapses 3" appear to be connecting the axons of the two labeled SVF with the dendrites of the centrifugal fibers, α & β. The centrifugal fibers appear to be the analogy of “bipolar neurons” in the retinas of chordates. They sum signals from varous retinula within one ommatidium to create a broadband signal (channel R) associated with the putative but archaic “rods” of scotopic vision. “Synapses 4" appear to be connecting other LMC to the centrifugal fibers, α & β. “Synapses 8" appear to be an inadequately defined connection to the fragments labeled “U.” R7 and R8 appear to be LVF passing through the lamina without synapsing. Boschek indicates there are six pairs of retinula from a single ommatidium interconnecting with the centrifugal fibers. There are clearly multiple pairs of retinula synapsing with individual LMC cells. These numbers are more than sufficient to support a full color capability in the fly with additional circuitry available to support e-vector angle determination in at least one spectral region. More electrophysiological data is needed to identify the spectral performance of the individual retinula participating in each actual pairing to determine if O–, P– & Q–channel signals are available at the axons of the LMC of each cartridge. The pairing may actually vary with the location of the ommatidium within the overall compound eye.

[xxx add more from Boschek or others on the list of files acquired on 27 April ]

3.6.3.4 Stage 3– Signal projection in Insecta

Signal projection from one stage to the next is less important in Insecta than in Chordata. The distances between various signal processing engines is much smaller and the brain itself is frequently dispersed (rather than be defined as a unitary brain as in chordata). Thus, the need for signal projection over long distances, greater than 2 mm, is rare in inects. The use of monopulse signals (usually described as Action potentials), as oppose to analog generator potentials, are not found in insects.

On the other hand the speed of signal projection within analog neural paths has been found to exceed any reasonable estimate of the speed of molecular or ionic diffusion along axons or other conduits. The fact that signaling occurs at a speed exceeding reasonable molecular or ionic diffusion rates is strong support for the Electrolytic Theory of the Neuron where signaling by electronic charge rather than by ionic charge transport is the proposed mechanism (Section xxx). 3.6.4 Confirmation of the Rhodonines as chromophores of Insecta

During the 1980's a considerable number of papers from virtually a single laboratory made many assertions concerning the chromophores, visual pigments, filtering pigments and their combination using what were believed to be early intracellular recording techniques. There was scant proof that their spectra originated from only a single sensory receptor cell. The author’s also appear to have relied upon their earlier training as no block diagrams, schematics or descriptions of the transduction mechanism were provided. In one notable case267, the nominal absorption spectrum was modeled using the expression 1– 10 0.43xexpression where 10 0.43 = e. Thus, the term 10 0.43xexpression should be replaced by the term e expression as used throughout quantum mechanics. Treating the numeric 0.43 as part of the “total extinction” of the pigment puts their values in conflict with those of most physics investigations. Fortunately, they treated the value 0.43 as a constant of unspecified source and defined another “extinction coefficient per unit length, κ.” The conclusions drawn from these early studies will not be discussed here. [xxx edit to note their emmissive spectrum matched their sensitivity profile in the UV. Indirect evidence that they isolated and recrystallized the liquid crystalline chromophore, Rhodinine(11). ] Arikawa et al. (1999a) claim they successfully isolated and defined the ultraviolet chromophore of vision in

267Smakeman, J. & Stavenga, D. (1986) Spectral sensitivity of blow,fly photoreceptors: Dependence on waveguide effects and pigment concentration Vision Res vol 26(7), pp. 1019-1025, eq. (1) The Retina 3- 149 Papilio268 However, their identification was not totally complete from a chemical or absorption spectra perspective. They performed the re-crystallization of the liquid crystalline material by extracting it from the retina using the formaldehyde method (section 2.4) and precipitating it on a microscope slide substrate and then observed its emission spectra through a light microscope-attached photodiode array (USP-410, Unisoku), equipped with an image intensifier (V1366U, Hamamatsu photonics). The glass of the microscope slide and the optics of the microscope/image intensifier probably limited the measurements (Section 3.6.4.1) The “modified formaldehyde method” used by Arikawa et al. (1999a) is traceable via their citations back to Suzuki et al. as discussed in Section 5.5.15.4 where a question is raised with regard to using formaldehyde. Formaldehyde not only shares a terminal group with the retinal form of the chromophores of vision but is also known as a strong reducing agent due to its electron-deficient carbon. This electron-deficient agent acts as an electrophilic agent when exposed to a resonant molecule such as any of the retinines. It may destroy or modify the resonant form of the retinine (with an i) required for a chromophore exhibiting a non-isotropic sensitivity to light accompanied by a shift in its spectral sensitivity. Arikawa et al. identified the chromophore as a retinene, 3-hydroxyretinol, but immediately qualified their remarks by suggesting it might be the form 3-hydroxyretinal (paragraphs 2.4 & 3.2). A resonant form of the two retinenes, could lead to confusion if one assumes the resonant retinene must be one or the other of these forms. The use of formaldehyde in extraction may have introduced an unexpected complication into their molecular identifications. Their method involved a presumptive identification as they did not demonstrate the character of the atom or group attached to Carbon 5 of these (potentially conjugated diol, and therefore resonant) arenes. This work demonstrates the actual chromophores of vision in the UV are conjugated diols with an oxygen atom at positions 11 and 15 (or 1 and 5 depending on the convention employed) of the aliphatic chain. As a result, the molecule is more appropriately named Rhodonine(11), a retinine where the underlined “i” indicates the diol form of this family (Section 5.5, and specifically Section 5.5.8).

Section 3.2 of Arikawa et al. does not provide a strong conclusion. The final paragraph of that section suggests the chromophores of the eye were in fact resonant Rhodonines and not retinenes (with an e). They note, “The fluorescing pigment appeared to be rather labile. Prolonged UV-illumination with the microscope’s mercury lamp caused a rapid fading of the fluorescing stars (ommatidia sensitive to UV light), within half a minute. The fluorescence pattern fully regenerated after a dark adaptation time of several hours, however.” These temporal intervals are suggestive of a diol based chromophore.

The prefix 3-hydroxy- indicates this retinine family is based on vitamin A3 as described in Section 5.3.3. Isolation of this family from the adult form of Palilio is suggestive of two points; first it is likely the earlier caterpillar and pupa stages of their life cycle also used vitamin A3 in their visual chemistry and unless the family is catadromous like some fish (Section 1.2), it would use that form throughout its life cycle. Second, the fact that both selected butterflies and selected moths employ vitamin A3 would suggest that the Order Lepidoptera also employs vitamin A3, like the Order Diptera (flies), as the principle form of the vitamin used (at least) in their visual modalities. 3.6.4.1 The measured spectra of the UV chromophore, Rhodonine(11) of Papilio

Figure 3.6.4-1 reproduces figure 1a from the Arikawa et al. (1999a) paper. It appears to display the same measured values as used in [Figure 3.6.5-2] below. The graph shows a well formed set of measurements with a peak at ~365 nm and left and right half amplitude points at ~322 and ~410 nm respectively. These values can be compared with the values for the human eye in Table 5.5.10-1 and illustrated in [Figure 3.6.3-3]. These values suggest the short wavelength of the left half amplitude point and the peak amplitude have been impacted by the use of soda-glass optics in the microscope, the light source and the microscope slide used as a substrate for precipitating the chromophore. While the system can be calibrated to eliminate the absorption by the envelope of the light source and the microscope/intensifier, it is not clear the calibration accounted for the microscope slide. In either case, the signal to noise in their measurements is degraded using glass optical components. Arikawa et al. only described the optical equipment used for the emission measurements and not the absorption measurements of the chromophore. Use of quartz optics, or other adequately transmitting optics, are preferred and possibly necessary in these measurements269.

268Arikawa , K. Mizuno, S. Scholten, D. Kinoshita, M. Seki, T. Kitamoto, J. & Stavenga, D. (1999) An ultraviolet absorbing pigment causes a narrow-band violet receptor and a single-peaked green receptor in the eye of the butterfly Papilio Vision Res vol 39, pp 1-8 269Wyszecki, G. & Stiles, W. (19198) Color Science: Concepts and Methods, Quantitiative Data and Formulae. NY: John Wiley & Sons pp 15-24 & 708 150 Processes in Biological Vision

3.6.5 Light and dark adaptation among Insecta

This area will not be explored in detail at this time. The literature related to dark adaptation among Insecta appears to follow the archaic assumptions showing dark adaptation following a two-step exponential curve that is not compatible with the actual mechanisms of the visual modality (Goldsmith & Bernard, 1974, page 233). Goldsmith & Bernard did not provide any data points in their figure describing the dark adaptation function but they did indicate the process was complete in less than 30 minutes. The actual mathematical Figure 3.6.4-1 Absorption spectrum of the UV channel framework of Section 17.6.1 follows a more receptor of Papilio ADD. The experimental data (circles) complicated path that appears similar to the archaic are compared with visual pigment spectra (bold curves) assumption under specific stimulus conditions (figures predicted by a template (Stavenga et al., 1993). Both in Section 17.6.1). The actual mechanism is spectra are limited by the transmission of the optics used to represented by a graph of an “exposine,” a product of a prepare them. See text. From Arikawa, et al. 1999 with low frequency sine wave and an exponential. added annotation. Depending on the intensity of the last stimulus level, and the position and diameter of the test area, the function becomes a single exponential and then progresses into the exposine form as illustrated.

Figure 17.6.1-4 of this work can be compared to figure 14 of Goldsmith & Bernard.

The remarks of Nilsson et al. in Section 3b about behavioral observations related to dark adaptation can be reinterpreted based on the above citations. When they speak of a dark adapted eye, they are referring to an eye that has not been stimulated for a period on the order of 30 minutes. Under this condition the opacity of the rhabdom is very high and “very little or no light comes back.” This is a theoretical statement. As soon as any stimulation is applied, the rhabdom becomes light adapted within a few milliseconds and its opacity is greatly reduced. As the opacity is reduced, applied orthodromic light is reflected back through the rhabdom and eye shine is observed. This is the applicable empirical statement. Whereas, the mode patterns are the same in the light and dark adapted eyes, it is only in the light adapted eye that the opacity is low enough for investigators to observe light that has passed through the rhabdom in both directions. 3.6.6 Summary performance of the visual modality of Insecta

Insecta vision: form, function and neural circuitry can be described in considerable detail based on the above material in Section 3.6.

The figure in Section 3.6 presents the relevant block diagram for organizing the technical information known concerning the visual modality of Insecta and defining the major stages and neural engines employed within that modality of Insecta. [xxx modify to show polarization channels ] Figure 3.6.6-1 displays a re-labeled schematic of stages 0, 1 and 2 proposed to represent the schematic diagram for Insecta adopted from a previous version developed for Chordata. The acceptance patterns shown between stage 0 and stage 1 represent the acceptance pattern of the rhabdom considered as a waveguide for light. The lamina represents the lamina/medulla of the above figures. The axons emanating from the photoreceptors are labeled RX where X can be UV, S, M or L to represent the spectral sensitivity resulting from the rhodonine chromophore employed in the rhabdomere of each sensory receptor. Similarly, the axons emanating from the lamina are labeled LX where X can be O, P, Q or R depending on whether the circuit in the lamina is a differencing circuit or a summing circuit. The Retina 3- 151

Figure 3.6.6-1 Signal Processing within stages 0, 1 & 2 of Insecta. The figure is modified from a similar schematic for Chordata. For Insecta, the acceptance patterns for all sensory neuron receptors are the same and controlled by the aperture of the rhabdom, and the optical properties of the cornea and crystalline lens. The labeling of the axons emanating from the stage 1 sensory neuron receptors and the stage 2 lamina are those developed in the text. See text.

Details of the chemistry and quantum-molecular performance of the chromophores of animal vision are developed in Section 5.5 of the text, “Processes in Biological Vision,” available on line270. The chromophores, given the name of Rhodonines, are the active part of the conceptual material rhodopsin. The Rhodonines are chemically derived from retinol (vitamin A) but are not chemically attached to the protein named Opsin. When in use, the Rhodonines are deposited on the microvilli of the rhabdomere portion of the sensory neural receptors in the form of a liquid crystal. The detailed structure of the microvilli has not been located in the literature of Insecta. They may consist of a neural portion and an Opsin portion. As shown in the figure, each sensory neuron receptor contains two electronic amplifiers, defined as Activa, in series plus associated circuit elements (not shown). The first amplifier is called the adaptation amplifier. It dominates the adaptation mechanism controlling the performance of the entire modality based on its level of stimulation. Since these adaptation amplifiers are independent of the amplifier in other sensory neurons within a ommatidium, adaptation is a spectrally selective process. In the compound eye of Insecta, adaptation is not only spectrally selective but independent for each ommatidium. Thus, adaptation level varies independently for the field of view associated with each nominal 1.5 degree field of view of the compound eye. Figure 3.6.6-2 describes the close match between the theoretical spectra of the photoreceptors of Insecta (based on the similar spectra of Chordata developed in this work (Section 5.5.10.1.2) and the measured spectra from Bandai et. al. (1992). The theoretical first order peak sensitivities are 342, 437, 532 & 625 nm. for the UV–, S–, M– & L– channels respectively The precise peak in the theoretical L–channel response is still a question. First order theory says it is at 625 nm. but this may move closer to an effective 610 nm. due to details of the molecular arrangement of

270Fulton, J. (2004) Processes in Biological Vision. http://neuronresearch.net/vision/pdf/5Photochem.pdf#page=47 152 Processes in Biological Vision

Rhodonine(5) or the 2 photon —>1 exciton mechanism introduced in Section 3.6.3. The measured responses show what appear to be some extraneous inputs, inadequate sampling interval to achieve sufficient precision and some potential problems with calibration. The figure has involved multiple scaling activities by editors.. It is recommended that any future investigators refer to the original Bandai et al. data and calculate the theoretical spectra using the equations in Table 5.5.1 of this work271. Note the important parameters in the fundamental Helmholtz- Boltzmann Equation are the half amplitude values for the short and long wavelength skirts of each chromophore. The values n that Table were derived to match the spectra of the human eye. Future measurement activities should employ narrower sample spacing in the wavelength interval of maximum interest and narrower band width filters in the monochromators used. Such precision would provide measured half-amplitude values for the species under evaluation and for purposes of recomputing the Helmholtz-Boltzmann Equation for that species.

Figure 3.6.6-2 Comparison between theoretical and measured spectral responses of Insecta based primarily on the data for the butterfly, Papilio The comparisons stress the necessity of making measurements at intervals of 5 nm or less to achieve adequate precision. The lower left frame suggests a potential calibration problem in the protocol for measuring such spectra. The measured spectra far from the theoretical waveforms that are not equal to zero (shown as dashed & in color) suggest some signal pickup from adjacent sensory, or other, neurons. See text. Data from Bandai et al., 1992.

The microvilli of the sensory receptors form very effective grid-type polarizers that act as analyzers of the polarization of the incident light. There may be considerable variation in the arrangement of these grid-type polarizers. In the case of Papilio as a minimum, the polarizers associated with sensory neurons R1-R4 rotate the e- vector of the light reaching R5-R8 by 45 degrees, thereby explaining the 45 degree rotation of the latter neurons relative to the former. The same polarization mechanism is shared with other members of Insecta and Mollusca. To confirm the above specifications, investigators will need to update their protocols and test equipment. The

271Fulton, J. (2004) Processes in Biological Vision, http://neuronresearch.net/vision/pdf/5Photochem.pdf#page=74 The Retina 3- 153 imaging equipment used to investigate the insect eye should have a spectral response of 300-700 nm (minimum of 325-650 nm). Electronic imaging devices of this spectral bandwidth are commercially available but probably in black and white (broad spectrum) only. However, by using tailored illumination to collect light from only one spectral band at a time to create a set of “separation” images and then print composite images using false color to indicate the UV channels (and potentially the polarization of individual spectral channels), useful composite images can be obtained. Such separation images (or separation negatives) or commonly used in the printing industry. To record the image light from specimens, it is necessary that all of the optics involved support the required spectrum. Quartz optics are available to support the imaging devices and light sources involved. The monochromators and recording photocell equipment used to record spectral data must also accommodate the above spectral requirement. Such equipment is readily available, however separate monochromators and photocell equipment are frequently used to accommodate the complete spectrum. In these cased the calibration of the equipments should all be calibrated to an accuracy of 5 nm or better. It is proposed that the appearance of the bulk pigments by reflected light is significantly different than the appearance of monolayers (liquid crystalline) of chromophore deposited on the microvilli of the sensory neuron receptors. These two appearances are typically complementary in the language of the printing industry and the community dealing in definitions related to light. In those communities, “magenta” is quite different than “reddish.” It can be argued that many of the references to a reddish pigment in histology actually refers to a “magenta” pigment that includes reddish and bluish components; the actual absorption of the related chromophore peaks in the greenish area of the visible spectrum. Use of the label “pigment” should not be used to describe the morphology or electrophysiology of the sensory neurons unless care is taken to specify whether the appearance relates to reflected or transmitted light. Arikawa and Stavenga encountered this problem in 1997 (page 2502, lower right) where they note, “In transmitted light, the ommatidium appear either yellow or (more or less saturated) red.” As noted in Section 3.6.3.1, their yellow is actually a yellowish-green based on the spectral peak at 536 nm of their photoluminescent indicator, Lucifer-yellow. Yellowish-green has a complementary color at 536c nm. This work supports and this paragraph provides the foundation for the last sentence in their abstract, “The different pigmentations are presumably intimately related to the various spectral types found previously in electrophysiological studies.”

Investigators need to carefully evaluate the Excitation/De-excitation Equation of Section 3.6.3.2 to recognize the critical necessity to define the intensity of the stimulant in their test protocol. Defining this stimulant intensity is complicated by the spectral sensitivity of the rhabdom as a whole and individual groups of spectrally sensitive rhabdomere within a single rhabdom. It is also complicated by the spectrally sensitive adaptation mechanisms within each wensory neuron associated with a specific signaling channel. 154 Processes in Biological Vision

TABLE OF CONTENTS 4/30/17 3 Description of the Retina...... 1 3.1 Introduction ...... 1 3.1.1 Background ...... 2 3.1.1.1 Order and tetra-chromaticity in the photoreceptor arrays of the retinas ...... 2 3.1.1.2 Comparison of retinas of different phyla ...... 4 3.1.2 A framework for discussion of the chordate retina...... 5 3.1.2.1 The plan view perspective of the retina...... 5 3.1.2.1.1 A panoramic view of the photoreceptors of the retina ...... 5 3.1.2.1.2 Global recording of other layers of the retina...... 5 3.1.2.1.3 Local recordings of the plan view of the retina ...... 6 3.1.2.2 The profile view perspective of the retina...... 6 3.1.2.3 The signaling architecture of the retina...... 6 3.1.2.3.1 Expansion of spatial processing within the Top Level Schematic . . . 6 3.1.2.3.2 Subdivision of retinal layers or interdigitation ...... 7 3.1.2.3.3 Initial tabulation of signal processing roles within the retina ...... 8 3.1.3 Additional concerns in experiment design ...... 10 3.1.3.1 Lack of a detailed model...... 10 3.1.3.2 Lack of consistent control of the motion of the eyes...... 10 3.1.3.3 Lack of precise control of stimuli ...... 10 3.1.3.4 Importance of controlling the stimuli orientation ...... 11 3.1.4 Matters specific to the organizational structure of the chordate retina...... 11 3.1.4.1 Matters of scale...... 11 3.1.4.2 Fovea versus other terms...... 11 3.1.4.3 Amercine cells...... 12 3.1.4.4 Matters of architecture...... 12 3.1.5 Matters of photoreceptor cell classification ...... 12 3.1.5.1 Background--Rods and cones ...... 12 3.1.5.1.1 The clinical attempt of Shultze to identify rods in 1860-70 ...... 13 3.1.5.1.2 Attempts at redefining the retina during the 1940-70s ...... 14 3.1.5.1.3 Attempts at redefinition during the 1980-90s ...... 17 3.1.5.1.4 Attempts at locating rods during 2000-11 & culminating in 2016 . . 19 3.1.5.2 “Rods” and “cones” are not functional descriptors...... 20 3.1.5.3 On the subject of “red rods” and “green rods” ...... 21 3.2 Morphology of the chordate retina ...... 21 3.2.1 Anatomical Level...... 24 3.2.1.1 The brain/blood barrier ...... 25 3.2.1.1.1 Membranes separating the laminates...... 25 3.2.1.2 Layers of the Retina and some statistics ...... 26 3.2.1.2.1 The neural laminate ...... 27 3.2.1.2.2 The photoreceptor laminate ...... 28 3.2.1.2.3 The RPE laminate...... 28 3.2.1.2.4 Laminate dimensions...... 29 3.2.1.3 Other anatomical features ...... 31 3.2.1.3.1 The visual streak versus an elongated fovea ...... 31 3.2.1.3.2 The optical disk (or blind spot) ...... 31 3.2.1.3.3 The macula or macula lutea...... 31 3.2.1.3.4 The signal paths on the neural laminate surface...... 33 3.2.1.3.5 The tapetum...... 34 3.2.1.4 The optic nerve ...... 35 3.2.2 Gross histology of the retina ...... 35 3.2.2.1 The sensing laminate...... 37 3.2.2.1.1 The RPE sub-laminate...... 37 3.2.2.1.2 Orientation of photoreceptors in the outer segment sub-laminate . . 38 3.2.2.1.3 Spatial parameters of the mosaics of the outer segment sub-laminate ...... 38 The Retina 3- 155 3.2.2.2 Geometrical patterns in retinal arrays ...... 41 3.2.2.3 Statistical parameters of the complete mosaic of the outer segment sub-laminate RE-OUTLINE...... 44 3.2.2.3.1 Statistical parameters of the complete mosaic(s)...... 45 3.2.2.3.3 Statistics of the foveola only ...... 51 3.2.2.3.4 Major axes of the foveola mosaic...... 51 3.2.2.3.5 Limiting resolution of the foveola of the retinal mosaic...... 51 3.2.2.4 Statistical parameters of the chromatic mosaics of the outer segments...... 53 3.2.2.4.1 Chromatic mosaics based on trichromatic ...... 54 3.2.2.4.2 Chromatic mosaics based on tetrachromatic assumption...... 55 3.2.2.4.3 Statistical parameters of the individual spectral channel mosaics . . 55 3.2.2.5 The neural laminate RE-OUTLINE...... 57 3.2.2.5.1 Signal paths through the laminate ...... 58 3.2.2.5.2 Sign conserving amplifiers found in the luminance channels ...... 61 3.2.2.5.3 Amplifiers for both sign conserving and sign reversing paths..... 61 3.2.2.5.4 Cell configurations within the laminate...... 63 3.2.2.5.5 Axon sizes within the Optic Fiber Layer ...... 63 3.2.2.5.4 Cell sizes within the laminate...... 64 3.2.2.5.4 Hydraulic elements within the laminate...... 64 3.2.2.6 Subdivisions of the neural laminate ...... 64 3.2.2.6.1 Spatial parameters of the mosaics of the ganglion cells in cat..... 65 3.2.3 Fine histology of the photoreceptor layer of the retina ...... 66 3.2.3.1 The archaic representations of Curcio et al. showing inner segment ellipsoids . 67 3.2.3.2 The more recent work of Roorda & Williams showing the retinal face ...... 68 3.2.3.3 Putative arrangement of photoreceptors in the human eye ...... 70 3.2.3.4 Candidate photoreceptor groupinng based on this work ...... 71 3.2.3.5 Putative arrangement of glia cells acting as light pipes ...... 73 3.2.3.6 The “text rewriting” work of the Roorda team with optimized AOSLO...... 74 3.2.4 Electronic architectural level ...... 79 3.3 Metabolism of the chordate retina...... 79 3.3.1 Static considerations related to a cell ...... 79 3.3.1.1 The Neuron...... 79 3.3.1.2 The RPE cell...... 80 3.3.2 The vascular supply to the retina ...... 80 3.3.2.1 Blood flow to the INM and most neural laminates ...... 81 3.3.2.2 Blood flow to the IPM, RPE and photoreceptor cells ...... 82 3.3.2.3 Block Diagram of the Metabolic Components ...... 82 3.3.3 Dynamic considerations...... 83 3.3.3.1 Bulk characteristics...... 83 3.3.3.1.1 Studies in heat generation within the retina...... 84 3.3.3.1.2 Studies in Oxygen consumption ...... 84 3.3.3.2 Detailed characteristics...... 85 3.3.3.3 Steady state characteristics ...... 85 3.3.3.4 Transient characteristics ...... 86 3.3.3.4.1 Slow transients...... 86 3.4 Functions of the Chordate Retina...... 86 3.4.1 Functional levels...... 87 3.4.1.1 The morphological level ...... 87 3.4.1.2 The physiological or signal function level ...... 87 3.4.1.2.1 The spectral signal level...... 89 3.4.1.2.2 The signal channel level...... 89 3.4.1.2.3 The signal projection level...... 91 3.4.2 The center-surround phenomenon (temporary home)...... 92 3.4.2.1 Types of experiments...... 92 3.4.2.2 Span of stimuli versus span of neurons...... 93 3.4.2.3 Interpretation of experiments...... 94 3.5 Electrophysiology, morphology & function of the eyes of Mollusca ...... 95 3.5.1 The compound eye of Mollusca ...... 95 3.5.1.1 Multispectral mollusc retina ...... 96 3.5.1.2 Details of the rhabdom ...... 97 3.5.1.2.xxx The retina of Pecten maximus ...... 98 3.6 Electrophysiology, morphology & function of visual modality of Insecta ...... 99 3.6.1 Background ...... 101 3.6.1.1 Diversity among eyes of Insecta ...... 103 156 Processes in Biological Vision

3.6.1.2 Simple versus compound eyes–apposition versus superposition etc...... 104 3.6.1.2.1 Potentially more complex simple eyes...... 104 3.6.1.3 Fundamental versus complex ommatidia ...... 104 3.6.1.4 Reconciling the definition of the “pigments” of Insecta vs other animals.... 106 3.6.1.5 Color vision potential of Insecta...... 107 3.6.1.6 A generic eye of Insecta ...... 107 3.6.1.6.1 The dimensions of the rhabdom of butterflies EMPTY ...... 112 3.6.2 The morphology of the compound eye of Insecta ...... 112 3.6.2.1 The compound eye of the butterfly according to Arikawa & colleagues ..... 112 3.6.2.1.1 The high frequency limitations of butterfly visual sensory neurons ...... 121 3.6.2.1.2 Is R9 an eccentric cell? ...... 121 3.6.2.2 Special features of the polarization mechanism...... 121 3.6.2.3 Detailed specific features of the optics of the ommatidia ...... 122 3.6.2.3.1 The tapetum & secondary pigment found in many butterfly and moth species...... 125 3.6.2.3.2 Index of refraction for ommatidia of blowfly and honey bee .... 126 3.6.2.3.3 The rhabdom as a waveguide ...... 128 3.6.2.3.4 The circular waveguide modes of Heteronyrnpha merope ...... 129 3.6.2.3.5 The rectangular dielectric waveguide modes ...... 131 3.6.2.4 The superposition (scotopic) eye of Insecta ...... 132 3.6.2.5 Morphology of stage 4, information extraction, and stage 5 cognition ..... 134 3.6.3 The electrophysiology of the compound eye of Insecta ...... 135 3.6.3.1 The neural circuitry of stage 1 of the eye of Insecta ...... 137 3.6.3.2 The Photo excitation/De-excitation equation of the eye of Insecta...... 138 3.6.3.2.1 The stage 1 P/D response to an impulse function ...... 139 3.6.3.2.2 The stage 1 P/D response to square pulse stimulation ADD ..... 141 3.6.3.2.3 Inversion of the P/D response by stage 2 neurons ...... 142 3.6.3.2.4 Generator potentials versus action potentials in Insecta ...... 143 3.6.3.3 Empirical signals associated with the ommatidium and stage 2 lamina ..... 144 3.6.3.3.1 A cartridge of stage 2 of the domestic fly ...... 145 3.6.3.4 Stage 3– Signal projection in Insecta ...... 148 3.6.4 Confirmation of the Rhodonines as chromophores of Insecta ...... 148 3.6.4.1 The measured spectra of the UV chromophore, Rhodonine(11) of Papilio . . . 149 3.6.5 Light and dark adaptation among Insecta ...... 150 3.6.6 Summary performance of the visual modality of Insecta ...... 150 The Retina 3- 157

Figure 3.2.1-1 CR Cross section through a human retina, ...... 23 Figure 3.2.1-2 The fovea of monkey, Macaca, irus ...... 24 Figure 3.2.1-3 Embryogenesis of the retina showing cellular origin of the various layers ...... 26 Figure 3.2.1-4 CR Electron micrograph of the photoreceptor and nuclear laminates of the bullfrog...... 28 Figure 3.2.1-5 CR A fundus photograph matched with a meridional light micrograph of the macular region .... 29 Figure 3.2.1-6 Cross-sections of a macaque retina taken in 460 nm (blue) and 525 nm ...... 32 Figure 3.2.2-1 A caricature of the central one-third of the human fovea ...... 36 Figure 3.2.2-2 Ultrahigh-resolution spectral OCT image of living human macula...... 37 Figure 3.2.2-3 Tangential section through inner segment layer of Macaca nemestrina near the fovea ...... 39 Figure 3.2.2-4 The human photoreceptor mosaic in the fovea centralis...... 40 Figure 3.2.2-5 CR Micrographs of foveal “cone” inner segments at fixation point of monkey ...... 41 Figure 3.2.2-6 The proposed fundamental array used in the spherical human retina ...... 42 Figure 3.2.2-7 Fermat spiral patterns...... 43 Figure 3.2.2-8 Dubra’s analysis of the regularity of the peripheral photoreceptor mosaic...... 44 Figure 3.2.2-9 The reinterpreted photoreceptor and neural densities of Osterberg ...... 47 Figure 3.2.2-10 Ratio of rods, cones and total photoreceptor cells to ganglion cells in a primate retina ...... 48 Figure 3.2.2-11 CR Horizontal section through a region of a fixed human retina...... 49 Figure 3.2.2-12 Change in photoreceptor spacing with eccentricity ...... 50 Figure 3.2.2-13 A measurement of contrast sensitivity versus spatial resolution in the human eye ...... 52 Figure 3.2.2-14 Caricature of the signal paths found in the retina of the Chordate ...... 60 Figure 3.2.2-15 CR Capillary bed in the neural laminate behind the macular region ...... 64 Figure 3.2.2-16 Caricatures of bipolar neurons within the retina of rat...... 65 Figure 3.2.3-1 CR [Color] Pseudo-color images of the human retina ...... 70 Figure 3.2.3-2 Potential mosaic of photoreceptors based on a trichromatic approach ...... 72 Figure 3.2.3-3 Potential photoreceptor array based on a tetrachromatic human retina ...... 73 Figure 3.2.3-4 Eye motion trace computed from an image sequence showing vertical (y) component of eye motion ...... 76 Figure 3.3.1-1 Operation of the typical living cell with the input and output signals added ...... 79 Figure 3.3.1-3 Vascular circulation within the retina ...... 81 Figure 3.3.1-4 The equivalent circuit of the vascular system of the eye...... 82 Figure 3.3.2-1 Perceived brightness in response to a high contrast image transition ...... 85 Figure 3.4.1-1 Overall Schematic Diagram of the Processes in Animal Vision ...... 90 Figure 3.4.1-2 Block diagram of principal signal paths of the eye ...... 90 Figure 3.5.1-1 Diagram of the retina of Octopus ...... 96 Figure 3.5.1-2 Early prototypical photoreceptor of Mollusca...... 97 Figure 3.5.1-3 Structure of the dual retina of Pecten maximus ...... 99 Figure 3.6.1-1 Anatomy of the head of an insect...... 100 Figure 3.6.1-2 Annotated visual modality block diagram applicable to Insecta ...... 103 Figure 3.6.1-3 Schematic diagram of the anatomical organization of the fly, Diptera ...... 105 Figure 3.6.1-4 A generic simple eye and component of the compound eye of Insecta REDRAW ...... 108 Figure 3.6.2-1 Stimulus condition and typical responses for Bandai et al ...... 114 Figure 3.6.2-2 Polarization sensitivity curves predicted from the microvillar orientation of the Papilio xuthus . . 114 Figure 3.6.2-3 Correlation between spectra of Chen et al. and theoretical rhodonine spectra of this work ...... 116 Figure 3.6.2-4 Electron micrographs of transverse sections of Papilio retina ...... 118 Figure 3.6.2-5 The ommatidia of Graphium Sarpedon ...... 119 Figure 3.6.2-6 Spectral sensitivities of photoreceptors of type 1 ommatidium of Graphium sarpedon retina . . . 120 Figure 3.6.2-7 Electron micrograph of the grid polarizers within the rhabdom of Papilio ...... 122 Figure 3.6.2-8 The demonstrated stage 0 optical system of the simple and compound eye of Insecta ADD .... 124 Figure 3.6.2-9 A tapetum based on reflection from a stack of dielectric plates in Precis lavinia ...... 125 Figure 3.6.2-10 Longitudinal sections of dioptric portions of ommatidia with indices...... 128 Figure 3.6.2-12 Modal patterns for rectangular dielectric waveguides ...... 132 Figure 3.6.2-13 Superposition eye of the nocturnal dung beetle, Onitis westermanni ...... 134 Figure 3.6.2-14 Visual modality of monarch butterfly visual modality ...... 135 Figure 3.6.3-1 Proposed morphology and circuit diagram of the sensory neurons of Insecta ADD ...... 137 Figure 3.6.3-2 Theoretical impulse responses based on the P/D Equation ...... 139 Figure 3.6.3-3 Theoretical P/D response to a square pulse stimulus ...... 141 Figure 3.6.3-4 Synaptic connections in the cartridge of a worker bee based on data ...... 143 Figure 3.6.3-5 Diagrammatic representation of two retinula neurons and one LMC ...... 144 Figure 3.6.3-6 A semischematic summary drawing of the structure and synapses...... 147 Figure 3.6.4-1 Absorption spectrum of the UV channel receptor of Papilio ADD...... 149 Figure 3.6.6-1 Signal Processing within stages 0, 1 & 2 of Insecta ...... 151 Figure 3.6.6-2 Comparison between theoretical and measured spectral responses of Insecta ...... 152 158 Processes in Biological Vision The Retina 3- 159

(Active) SUBJECT INDEX (using advanced indexing option) 2 photon ...... 116, 136, 152 3D...... 84, 118 95% ...... 114, 143 action potential...... 61, 91, 104, 121 Activa...... 27, 59-62, 80, 86, 88, 97, 110, 111, 121, 137, 141-145, 151 adaptation...... 18, 20, 21, 33, 34, 50, 55, 57, 66, 77, 82, 83, 86, 101, 117, 121, 126, 134, 137, 138, 149-151, 153 adaptation amplifier...... 66, 82, 137, 151 afterimage...... 64 amplification ...... 88 arborization ...... 2, 3, 6, 92, 93 attention...... 31, 37, 54, 93, 120 autocorrelation...... 45 axoplasm ...... 110, 137, 141, 142 Bayesian...... 74, 145 Bayesian trap ...... 145 bilayer...... 79 bioelectrochemistry ...... 84 bistratified ...... 59, 62 bleach ...... 113 bleaching ...... 18, 70, 86, 117, 126, 134 blood-brain barrier ...... 25 BOLD...... 5, 149 Bragg dielectric mirror...... 125 broadband...... 15, 20, 66, 105, 111, 120, 148 b-wave ...... 16 calibration...... 39, 144, 149, 152, 153 cerebellum ...... 89 CIE...... 71, 78, 113, 130 CIE 1976 ...... 78 coma...... 133 compensation...... 13, 36, 37, 56, 69, 74, 145 compound eye ...... 1, 95, 100, 104-106, 108, 110-112, 114, 116, 123-125, 132-136, 148, 151 computation ...... 113, 131 computational...... 31, 62 confirmation...... 20, 33, 148 consonance...... 70 continuum ...... 17 cross section...... 23, 32, 36, 38, 68, 78, 92, 97, 105, 125, 129, 134 cross-section...... 5, 22, 25, 31, 35, 38, 107, 108, 122 dark adaptation...... 83, 149, 150 data base...... 63 database ...... 5, 6 DG ...... 116 diode...... 79 diol...... 149 disparity...... 51 DNA...... 105 drusen...... 36 Duplex Theory...... 13, 21 dynamic range ...... 14, 72, 85, 88 eccentric cell ...... 110, 111, 118, 121, 145 electrostenolytic process ...... 84, 137 entrainment...... 134 ERG...... 16, 63, 99 evoked potentials ...... 92 evolution...... 1-4, 13, 84, 95, 99, 101, 112, 143 exothermic animals...... 141 expanded ...... 15, 65, 74 exposine ...... 150 external feedback...... 59, 65 FD-OCT...... 36 160 Processes in Biological Vision

feedback...... 28, 59, 65 Fourier transform...... 45, 51 GABA ...... 3, 137 Gaussian...... 35, 120, 136 genetics...... 57 glia...... 18, 73 glutamate ...... 3, 65, 83, 84 glycolysis...... 84 half-amplitude ...... 120, 152 Hodgkin Condition ...... 141 Hodgkin solution ...... 139 homogeneous...... 64, 133 hyperacuity ...... 56 inverting...... 9, 61, 62, 104, 110, 123, 133, 134, 143, 145 lactate...... 84 latency ...... 61, 139 lateral geniculate ...... 4, 62 Lepidoptera ...... 102, 105, 106, 108, 129, 133, 145, 149 light adaptation...... 18, 126, 138 Limulus ...... 110, 111, 118, 121, 141, 143 liquid-crystalline ...... 106, 135 locomotion...... 143 marker ...... 53 microvillar ...... 114 microvilli ...... 90, 97, 107-112, 114, 117, 118, 121, 122, 125, 129, 135, 137-139, 151-153 midbrain...... 91 monopulse ...... 148 morphogenesis ...... 25, 26, 42, 43 MRI ...... 87 myelin...... 143 N2...... 125 narrow band...... 113, 136 navigation...... 109, 134 neural coding ...... 92 neurite...... 111 neurites...... 65, 89, 100, 110, 111, 124, 134, 137, 144 neuropil ...... 111, 124, 143 neurotransmitter ...... 3 Node of Ranvier...... 143, 145 noise...... 18, 27, 28, 142, 149 non-inverting ...... 61, 62, 104, 110, 123, 133, 134, 143 Nyquist ...... 42, 51-53, 55 OCT...... 13, 26, 36, 37, 74, 79 P/D equation...... 121, 139, 142, 145 parametric...... 85 parvocellular...... 12, 35, 59, 61, 63, 78, 89, 91, 92 perceptual space...... 78 phylogenic tree ...... 1 plasticity...... 42 poda ...... 60, 62 podites ...... 111 poditic ...... 61, 62, 65, 92, 93, 134, 143-145 point of regard ...... 31 POS ...... 51 Pretectum...... 44, 51, 59, 61, 62, 89, 91 protocol ...... 17, 57, 77, 114, 119, 152, 153 pulvinar ...... 75 Pulvinar pathway...... 75 quantum-mechanical ...... 106, 117, 138 resonance...... 67, 87 reticulated...... 64 The Retina 3- 161 retinine...... 1, 149 retinitis pigmentosa ...... 25 rhabdom ...... 29, 96, 97, 99, 101, 104, 105, 107-110, 112, 117-125, 127-134, 137, 138, 145, 150, 151, 153 rhabdome ...... 104, 108, 112, 126, 129, 131 rhabdomere ...... 96-98, 104, 105, 107-110, 114, 117, 118, 120, 122, 126, 137, 139, 150, 151, 153 rod intrusion...... 13 simple neurons...... 79 stage 0 ...... 107, 110, 119, 123, 124, 129, 150 stage 1 ...... 75, 88, 102, 107, 110, 111, 121, 124, 137, 139, 141, 143, 147, 150, 151 stage 1A...... 88 stage 1B...... 88 stage 2 ...... 57, 72, 73, 75, 77, 88, 102, 103, 105, 107, 110, 111, 118, 121, 124, 142-145, 147, 151 stage 3 ...... 89, 100, 102, 104, 118, 143, 144 stage 3A...... 89, 121, 143 stage 3B...... 89 stage 4 ...... 102, 103, 111, 134, 145 stage 5 ...... 75, 103, 134 stage 6 ...... 134 stereopsis ...... 118 Stiles-Crawford ...... 78 stratified...... 107, 108, 134 stress...... 7, 55, 152 synapse...... 15, 17, 58, 59, 75, 77, 79, 110, 111, 143, 145, 146, 148 threshold...... 9, 12, 14, 50, 60, 61, 77, 78, 136 tomography ...... 26, 36, 74 topography ...... 11-13, 29, 46, 56, 67, 74, 78, 79, 87 topology ...... 4, 13, 45, 79, 88, 111, 137 transduction ...... 1, 63, 80, 88, 106, 121, 138, 148 translation...... 42, 63, 88 trans-...... 54 tremor...... 4, 10, 12, 13, 51-53, 56, 68, 69, 74-78, 97 type 1...... 8, 120 type 2...... 8 type I ...... 58, 116, 119, 120 type II...... 58, 119, 136 type III...... 119 Verhoeff’s ...... 26, 36, 37, 80, 82 visual cortex...... 10, 66 vitamin A1...... 116, 120 vitamin A3...... 112, 120, 149 Voroni diagram ...... 44 waveguide ...... 18, 53, 55, 78, 107, 108, 110, 118, 122-124, 128-131, 133, 145, 148, 150 white matter...... 33 Wikipedia...... 44, 102 xxx...... 1, 2, 4, 24, 32, 36, 38, 46, 57, 67, 73, 78, 98, 104, 112, 120, 122, 124, 126, 133, 137, 148 [xxx ...... 76, 78, 97, 105, 106, 118, 122, 124, 129, 131, 133, 138, 145, 148, 150