The NEURONS and NEURAL SYSTEM: a 21st CENTURY PARADIGM

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. A few citations have yet to be defined and are indicated by “xxx.”

James T. Fulton Neural Concepts 1 (949) 759-0630 [email protected]

December 25, 2011 Copyright 2011 James T. Fulton

1 2 Neurons & the Nervous System

[ phase out SSC’s in favor of OR’s or GR’s. [ edit carbonyl (C=O) versus carboxyl (COOH) usage. screwed up in places ] [ sulfide in america; sulphide in britain ] [ establish a clear demarcation between using H-best and C-best ] [ be sure Section 8.2 and 8.3 touch on anatomical computation to support chapter 0. 8 Stage 1 & 2, Signal Generating & Processing Neurons1

“Science is made up with facts as a house is made from stones. But a collection of facts is no more a science than a pile of stones is a house.” —Poincare' , Hypotheses in Physics (1952) “In order to understand any part of nature, one must have both experimental data and a theory for interpreting the data and predicting new data.” – Shepherd, Outline of a Theory of Olfaction, 2005

8.1 Introduction

The sensory neurons serve disparate purposes but exhibit a remarkable uniformity at the morphological, cytological and electrophysiological level. The visual and auditory modalities are focused on sensing of the distant external environment. The olfactory and gustatory modalities are focused on the near and even nearer external environments. The remainder of the external sensory modalities typically (with the exception of radiant heat) involve physical contact with the source of the stimulation.

Adopting Poincare’s above thesis, the goal of this chapter is to show how our collection of facts describe an architecturally identifiable structure of complexity and beauty.

The signal processing (stage 2) neurons immediately behind the sensory neurons vary more widely than do the sensory neurons, primarily in whether they are present at all. The retina and the olfactory modalities employ significant signal processing immediately adjacent to their sensory neurons. The auditory and gustatory modalities on the contrary, avoid stage2 signal processing adjacent to the sensory neurons and employ stage 3 signal projection channels between the sensory neurons and more distant signal processing elements that may be considered part of the divergent stage 2 signal processing or or of the convergent stage 4 signal manipulation elements of the modality architecture.

When discussing the sensory neurons in a consistent lexical context, there is a problem with the use of the terms cilia and microvilli. Elsaesser & Paysan wrote an elaborate paper addressing this duality without defining their terms and coming to any conclusion about the correct terminology2. They did provide over one hundred citations. The term cilium is from the Latin for eyelash and generally is considered a rigid structure. Unfortunately, villium also comes from the Latin and colloquially refers to curly hair. These terms are often used interchangeably in the literature. In this work, the goal is to use cilium and cilia when referring to structures emanating from the dendritic structure of a sensory neuron that are “rigid” or must maintain a column like character. The rigid structures are typically filled with rigid liquid crystalline proteins participating in a piezoelectric transduction process. The photoreceptors maintain a column-like structure for optimal optical

1December 25, 2011

2Elsaesser, R. & Paysan, J. (2007) The sense of smell, its signaling pathways, and the dichotomy of cilia and microvilli in olfactory sensory cells BMC Neurosci vol 8(Suppl 3):S1 Signal Generation & Processing 8- 3 efficiency. Villium and villi will be used when referring to structures emanating from the dendritic structure of a sensory neuron that are not rigid and need not maintain a column-like structure. These structures are typically filled with a fluid like material and reticuli that are flexible in character. At the cytological level, the protein filled cilia transfer their signal to a first amplification stage within the soma of the neuron whereas, the microvilli of sensory neurons have been modified to create an active electrolytic device between their outer lemma and their inner reticulum. In this work, the visual, olfactory and gustatory sensory neurons exhibit microvilli; the auditory and many of the somatosensory sensory neurons exhibit cilia that are sensitive to dynamic loadings and motions. All of the sensory modalities appear to employ the same type of signaling and present a nearly identical interface to the saliency map within the CNS. It will be shown that the signaling architecture, methodologies and techniques within the different sensory modalities are essentially identical. While the visual and auditory modalities are very well understood and interpreted (especially employing the Electrolytic Theory of the Neuron) the modalities employing chemo– thermo– and mechanico–receptors are less well understood. The literature of these modalities at the electrophysiological level is relatively thin. As a result, this work will hypothesize in these areas based on the similarity to the more studied modalities. Only additional laboratory work, based on these hypotheses will allow confirmation or refutation of these hypotheses.

One area supporting the theory and the comparability of the sensory modalities is the adaptation characteristic 2.1 Introduction

The signal generating neurons have the basic task of converting some source of energy into an electrical signal that can be processed and interpreted by the remainder of the neural system prior to taking some responsive action (when appropriate). An initial list of the sensory neurons generally includes those associated with seeing, hearing, touch, taste and smell. Less frequently, the sensory neurons associated with orientation are added to this list. Each of these broad designations can usually be subdivided into several additional descriptors. As an example, the photoreceptors of sight (the visual system) are further divided into spectrally sensitive types. The neurons associated with touch are frequently subdivided into those sensing relative motion and those sensing temperature. These subdivisions of a given modality are sometimes described as qualities within a modality. Shepherd has provided a table of sensory modalities, that he credits to Ganong (1985), that shows some of these groupings3. His listing speaks of the form of incident energy when he generally means the source of the incident energy. The listing by Shepherd only includes a mixture of generic names for the types of sensory cells.

Feducci, editing Torrey’s 5th Edition, provides a brief introduction to the evolution of the sensory neurons4. Shepherd also provided an interpretation (pg 208) of a figure by Bodian (1967) that will be expanded upon here. The caricatures for the modalities other than vision do not allow for signal processing within the neural system supporting that modality. However, it is readily shown that most individual sensory neurons are not connected to the brain by a unique and exclusive neural circuit. Stage 2 signal processing is an effective method of economizing on neural circuitry and is found in nearly every sensing modality. This work will postulate that the excitation energy is quantum-mechanical in form for most, if not all, of the sensory modalities. This prediction is made based on the impulse response reported for a range of these modalities. The result of the postulate is confirmation of the “law of specific nerve energies” attributed to J. Muller in the 1830's. However, the modern interpretation of that law is quite different. The next section will calculate the minimum energy required to excite any sensory

3Shepherd, G. (1988) Neurobiology, 2nd ed. NY: Oxford University Press pg 207

4Feducci, xxx Torrey’s 5th Ed. pg 449 4 Neurons & the Nervous System neuron and create a free electron within the signaling system. Later sections will show that this minimum energy is a critical design parameter within the overall neural system. It defines the technology available to support different sensing modalities. As a guide, these technologies involve the photoelectric, piezoelectric and electrostenolytic effects. These effects are all quantum- mechanical in nature. Both the piezoelectric and electrostenolytic effects appear to be used in multiple implementations that show striking morphological differences. The conversion of the input stimulus to a free electron is known as transduction. It does not rely upon the release or transport of any protein within the neural system. All signaling is performed by the transport of fundamental electrical charges (or the propagation of energy resulting from the motion of such fundamental charges). The last caveat will be discussed in Section xxx where the mode of propagation of action potentials will be addressed. While much of the literature uses the term conductance change to describe a mechanism that either generates or supports the transmission of a neural signal, this work does not. Simple changes in the conductance of an electrical element, other than those associated with the response function of diodes and the Activa, do not occur within the neural system. The changes in conductance associated with diodes (a fixed nonlinear current-voltage characteristic) and Activa (a transconductance that varies with input potential) were described in detail in Chapters 1 & 2. The records in the biological literature inevitably are voltage waveforms responding to changes in the transconductance of one or more Activa and do not represent a simple conductance change associated with a resistive element at all.

This chapter will establish a framework for understanding and discussing the functional performance of these sensory neurons. It will also link the functional performance to the morphology of the neurons. Because the available information is so extensive, the features of the visual system will frequently be used as an exemplar of the general situation.

Most of the sensory neurons rely upon the photoelectric, piezoelectric or thermoelectric effect to generate their electrical signals (sometimes aided by auxiliary molecular materials). The smell and taste sensory neurons remain the least understood at present. They probably rely upon the secretion of an enzyme that reacts with the external molecules carried in the air or saliva within the local morphological structure of the neuron to generate an electrical stimulus.

The neurosecretory role of most (if not all) sensory neurons is not generally appreciated. Without this secretory capability, most neurons would not be functional. This secretory role will be stressed in Section 2.xxx and in Section 4.xxx.

This work does not support the putative chemical-gate structure of neural operation. Nor does it support the concept of a chemically based synapse between neurons. As a result, the term neurotransmitter is not used in this work except to show how that concept compares to the more rational electrolytic proposal. For those trained in the chemical approach, it is important to note that no transfer of heavy metal ions through the lemma of a neuron has never been observed by relatively direct means. The reported observations have all relied on electronic circuitry involving the flow of electrons to support the putative flow of heavy ions. This work will rely upon a companion work5 to provide an extensive review of the literature in these areas. The companion work also develops a complete physiological explanation for the operation of the synapses. This operation is also electrolytic and does not involve any chemical transport across the synaptic gap. An entirely different operating rational is provided for the materials previously labeled neurotransmitters. They are in fact neuro-effectors and neuro-inhibitors. However, their action relates to the electrical power supplies of the neurons and not the transfer of signals across the synapse. If the reader finds it difficult to embrace the electrolytic character of the neural system, it may help to recall that the underlying mechanisms only became known to man during the late 1950's and

5Fulton, J. (2004) PBV and BV Signal Generation & Processing 8- 5 1960's. Theories developed before that time (and reiterated up to the present6) relied upon an inadequate technological base. While those early workers would look upon the ideas presented here as magic (just like they would the liquid-crystal-based display of a hand-held calculator or cell phone), they are well known to the current college graduate in chemistry, physics and electronics. of the sensory neurons. It is clear that all sensory neurons respond while exhibiting several different time constants. They all exhibit the same short term excitation/de-excitation (E/D) characteristic, they all exhibit a band limiting mechanism corresponding to a time constant of a few tenths of a second, and they all exhibit a very long term effect measured in hours to days. Each type of sensory neuron appears to use a slightly different means of overcoming the long term effect. These mechanisms will be discussed in the individual sensory neuron sections.

2.1 Introduction from old hearing or vision xxx

The signal generating neurons have the basic task of converting some source of energy into an electrical signal that can be processed and interpreted by the remainder of the neural system prior to taking some responsive action (when appropriate). An initial list of the sensory neurons generally includes those associated with seeing, hearing, touch, taste and smell. Less frequently, the sensory neurons associated with orientation are added to this list. Each of these broad designations can usually be subdivided into several additional descriptors. As an example, the photoreceptors of sight (the visual system) are further divided into spectrally sensitive types. The neurons associated with touch are frequently subdivided into those sensing relative motion and those sensing temperature. These subdivisions of a given modality are sometimes described as qualities within a modality. Shepherd has provided a table of sensory modalities, that he credits to Ganong (1985), that shows some of these groupings7. His listing speaks of the form of incident energy when he generally means the source of the incident energy. The listing by Shepherd only includes a mixture of generic names for the types of sensory cells.

Feducci, editing Torrey’s 5th Edition, provides a brief introduction to the evolution of the sensory neurons8.

Shepherd also provided an interpretation (pg 208) of a figure by Bodian (1967) that will be expanded upon here. The caricatures for the modalities other than vision do not allow for signal processing within the neural system supporting that modality. However, it is readily shown that most individual sensory neurons are not connected to the brain by a unique and exclusive neural circuit. Stage 2 signal processing is an effective method of economizing on neural circuitry and is found in nearly every sensing modality.

This work will postulate that the excitation energy is quantum-mechanical in form for most, if not all, of the sensory modalities. This prediction is made based on the impulse response reported for a range of these modalities. The result of the postulate is confirmation of the “law of specific nerve energies” attributed to J. Muller in the 1830's. However, the modern interpretation of that law is quite different. The next section will calculate the minimum energy required to excite any sensory neuron and create a free electron within the signaling system. Later sections will show that this minimum energy is a critical design parameter within the overall neural system. It defines the technology available to support different sensing modalities. As a guide, these technologies involve the photoelectric, piezoelectric and electrostenolytic effects. These effects are all quantum- mechanical in nature. Both the piezoelectric and electrostenolytic effects appear to be used in multiple implementations that show striking morphological differences. The conversion of the input stimulus to a free electron is known as transduction. It does not rely upon the release or transport of any protein within the neural system. All signaling is performed by

6Meddis, , R. (1999) The auditory periphery as a signal processor In Day, T. Hohmann, V. & Kollmeier, B. eds. Psychophysics, Physiology and Models of Hearing. Singapore: World Scientific pp 131+

7Shepherd, G. (1988) Neurobiology, 2nd ed. NY: Oxford University Press pg 207

8Feducci, xxx Torrey’s 5th Ed. pg 449 6 Neurons & the Nervous System the transport of fundamental electrical charges (or the propagation of energy resulting from the motion of such fundamental charges). The last caveat will be discussed in Section xxx where the mode of propagation of action potentials will be addressed. While much of the literature uses the term conductance change to describe a mechanism that either generates or supports the transmission of a neural signal, this work does not. Simple changes in the conductance of an electrical element, other than those associated with the response function of diodes and the Activa, do not occur within the neural system. The changes in conductance associated with diodes (a fixed nonlinear current-voltage characteristic) and Activa (a transconductance that varies with input potential) were described in detail in Chapters 1 & 2. The records in the biological literature inevitably are voltage waveforms responding to changes in the transconductance of one or more Activa and do not represent a simple conductance change associated with a resistive element at all. This chapter will establish a framework for understanding and discussing the functional performance of these sensory neurons. It will also link the functional performance to the morphology of the neurons. Because the available information is so extensive, the features of the visual system will frequently be used as an exemplar of the general situation. Most of the sensory neurons rely upon the photoelectric, piezoelectric or thermoelectric effect to generate their electrical signals (sometimes aided by auxiliary molecular materials). The smell and taste sensory neurons remain the least understood at present. They probably rely upon the secretion of an enzyme that reacts with the external molecules carried in the air or saliva within the local morphological structure of the neuron to generate an electrical stimulus.

The neurosecretory role of most (if not all) sensory neurons is not generally appreciated. Without this secretory capability, most neurons would not be functional. This secretory role will be stressed in Section 2.xxx and in Section 4.xxx.

This work does not support the putative chemical-gate structure of neural operation. Nor does it support the concept of a chemically based synapse between neurons. As a result, the term neurotransmitter is not used in this work except to show how that concept compares to the more rational electrolytic proposal. For those trained in the chemical approach, it is important to note that no transfer of heavy metal ions through the lemma of a neuron has never been observed by relatively direct means. The reported observations have all relied on electronic circuitry involving the flow of electrons to support the putative flow of heavy ions. This work will rely upon a companion work9 to provide an extensive review of the literature in these areas.

The companion work also develops a complete physiological explanation for the operation of the synapses. This operation is also electrolytic and does not involve any chemical transport across the synaptic gap. An entirely different operating rational is provided for the materials previously labeled neurotransmitters. They are in fact neuro-effectors and neuro-inhibitors. However, their action relates to the electrical power supplies of the neurons and not the transfer of signals across the synapse.

If the reader finds it difficult to embrace the electrolytic character of the neural system, it may help to recall that the underlying mechanisms only became known to man during the late 1950's and 1960's. Theories developed before that time (and reiterated up to the present10) relied upon an inadequate technological base. While those early workers would look upon the ideas presented here as magic (just like they would the liquid-crystal-based display of a hand-held calculator or cell phone), they are well known to the current college graduate in chemistry, physics and electronics.

9Fulton, J. (2004) PBV and BV

10Meddis, , R. (1999) The auditory periphery as a signal processor In Day, T. Hohmann, V. & Kollmeier, B. eds. Psychophysics, Physiology and Models of Hearing. Singapore: World Scientific pp 131+ Signal Generation & Processing 8- 7 8.1.1 Important background

Before beginning, it is critically important to define the term receptor. Cavallito has documented the origins of this term in chemoreceptor research and notes its foundation in pharmacology11. He notes the great difficulty over the years in arriving at a definition of the term receptor within the pharmacological context. In pharmacology, the concept of a receptor logically revolves about the activity of an exogenic substance introduced into the blood stream. It is assumed the target of that substance contains a specific receiving site on its surface. That site has been defined as a receptor. The concept does not relate to external stimulation of a sensory neuron. Among the sensory modalities (limited to external stimulation for the moment), a distinct mechanism, transduction, is designed to create an electrical signal in response to an external stimulant. This mechanism takes place at a specific input area of the sensory neurons related to the cilia or villi of the input (typically labeled dendritic) portion of the sensory neuron. Because of its sensitivity to extraneous stimulation, this input area is frequently isolated from the blood stream. In the sensory modalities, a receptor is the site of transduction of an external stimulant (a first messenger) into an internal electrical signal (an internal messenger) within the neural system.. The site of sensory neuron receptors is limited to the external interface of the cilia or villi. The site is typically isolated from the effects of pharmacological substances in the blood stream.

It is also important to place the concept of the Structure-Activity Relationship (SAR) in context with the broader field of scientific research. The SAR concept originated long ago when it was virtually impossible to understand the physiological operation of the biological systems within a species. In other fields, it is considered the “black box” approach to science. Introduce an input into the black box and see what the result is. The S in SAR originally stood for stimulant (which was typically describe by a trivial name). As chemistry has developed, the stimulants have been described more completely by their chemical structure, an more recently by their electronic structure as well. Similarly, with advances, the activity resulting from these chemical structures have been divided into both the principle activity and various side effects. While the SAR approach to pharmacology still has its place, a great deal more is known about the internal operation of the biological system. Instrumentation has taken great leaps forward in laboratory investigations and treating the system as a black box is no longer appropriate. Section 8.4.1.4 will describe the current place of SAR in chemoreceptor research. Section 8.6.7 will explicitly show the initial steps in subdividing the SAR concept into a series of intermediate relationships applicable to the olfactory modality. The same subdivision is inherent in the discussions of other modalities.

This work is specifically designed to replace the SAR concept with a concatenated description of each individual step in the operation of the neural system. An associated goal is to show the detailed characteristics and transduction performance of the sensory neuron receptors of each sensory modality. 8.1.1.1 Genetics of the sensory neurons

As will become clear in this chapter, the discussions between the advocates of a labeled-line theory of sensory signal identification and those advocating an across-nerve approach involving multiple neurons is largely irrelevant. All individual sensory signaling paths (invariable of stage 3 type when delivering signals to stage 4) can be traced back through stage 2 to a single sensory neuron or set of sensory neurons. On the other hand, the stage 4 signal manipulation function is charged with interpreting the pattern of signals it receives from groups of stage 3 neurons. Both the inputs to stage 4 and the outputs of stage 4 consist of signals exhibiting a coherence in either time or space relative to the environment that can be described as an across nerve format. Terminology is important; there is no possibility of a cross-neuron hypothesis since it is a single signaling channel. The necessary terminology is the cross-nerve hypothesis (associated with a group of neurons within one nerve). Using the term fiber in place of nerve is poor practice. Erickson has addressed this

11Cavallito, C. (1973) Some trends in the development of structure-activity relationships (SAR) and theory of receptors In Cavallito, C. ed. International Encyclopedia of Pharmacology and Therapeutics, Section 5, Vol 1, Structure-Activity Relationships. NY: Pergamon Press 8 Neurons & the Nervous System terminology problem in an historical but less than precise manner12. 8.1.1.2 Genetics of the sensory neurons

Genetics plays a major/minor??? role in understanding the stimulant sensing complex (SSC’s) associated with the sensory neurons of the neural system. Early work led to the conclusion that the DNA code was nothing but a transcription for all of the proteins in the animal body, with much associated junk code. It is now becoming clear that much of that junk code is extremely important for defining and/or controlling many other processes and materials in the body. This realization has opened new avenues of research in the 21st Century. However, the resultant “deluge” of information has not found its way into an organized structure (a comprehensive theory) where it can be used effectively. Also gaining acceptance is the realization that a single gene may influence many portions of the physiological system, and not control only the genesis of a specific receptor molecule/ligand. In genetic studies, the SSC’s are frequently assumed to be limited to proteins. As a result, they have been described as odor-binding-proteins (OBP) in chemoreception. However, recent work has suggested they may also be lipids created by mitochondria based on instructions in the DNA code. Genetics has remained an observational science until recently. Modern genetics, based on detailed knowledge of the genetic code is at an early stage. Although progress has occurred at an amazing rate, understanding the code still involves great amounts of “junk code” and “pseudogenes”. Attempts to employ “genetic medicine” remain in the experimental state. On the other hand, great success has been achieved using genetically modified (knockout) mice.

The connection between genetics and biology remains largely inferential. Investigators have not had conceptual (intellectual rather than animal) models adequate to the task. As a result, investigators frequently find “what they are looking for.” In the case of vision, they have routinely found three genes representing the three chromophores of vision, based on the conventional wisdom, when it can be shown (Section 8.1.2.x) that Mammalia, including Homo Sapien, exhibits four chromophores. Investigators have routinely not found the gene associated with the ultraviolet chromophore or the putative “rod” chromophore.

In the case of chemorecption, the problem has been more awkward. No model describing a finite set of chemophores has arisen. Therefore, the investigators have assigned genes on virtually every chromosome to the chemoreception process based largely on the fact they code for what are described as G-protein coupled receptors (GPCR). GPCRs consist of three subunits; the α-subunit, which contains the guanine-nucleotide-binding site, and the β- and γ-subunits which function as a heterodimer. Heterodimer in the field of biology is broader term than in organic chemistry. The two subunits may vary in number, location and definition of the individual amino acids. In most cases, the precise molecular structure of the subunits of the heterodimer have not been elucidated. Hence, what precisely they code for is not known at this time.

Strotmann has provided a paper describing “a unique subfamily of olfactory receptors” that illustrated the state of the art in 200113. It asserts each gene of the genetic code specifies a unique olfactory receptor that is a protein. However, they did not proceed to identify any specific receptor and demonstrate that it was a protein. Strotmann identified “five OR37 gene subtypes in the mouse, comprising four functional genes and one pseudogene.” He also noted regarding other genes, “The transcription orientation of four genes is identical, only the first gene in the cluster is positioned in opposite orientation.” He did not address the function of the pseudogene or the functionality of a gene positioned in opposite orientation, or provide any reference. It appears Strotmann did not consider the possibility that the olfactory receptors need not be genetically defined. There is a major literature suggesting the olfactory receptors may not be

12Erickson, R. (2008) A study of the science of taste: On the origins and influence of the core ideas Behav Brain Sci (2008) vol 31, pp 59–105 Paragraph R12

13Strotmann, J. (2001) A unique subfamily of olfactory receptors In Marchlewska-Koj, A. Lepri, J. & Muller- Schwarze, D. eds. (2001) Chemical Signals in Vertebrates 9. NY: Kluwer Academic pp 69-83 Signal Generation & Processing 8- 9 encoded by DNA (Section 8.4). Terminology is a problem for an outsider attempting to understand the genetics literature. Figure 8.1.1-1 provides an overview of that terminology. [ XXX add text ]

Figure 8.1.1-1 from Lancet et al. shows the location of an odor receptor cluster relative to chromosome 1714.

Figure 8.1.1-2 provides more detail relative to an OR cluster from Johnson15.

Figure 8.1.1-1 A scheme depicitng the cluster of olfactory receptor (OR) genes on human chromosome 17, at chromsomal band p13.3. See text. From Lancet et al., 1993.

14Lancet, D. (1993) xxx In Margolis, F. ed. The Molecular Basis of Smell and Taste Transduction. Ciba Foundation Symposium 179 NY: John Wiley & Sons pg 135

15Johnson, B. (1998) xxx xxx 10 Neurons & the Nervous System

Figure 8.1.1-2 ADD Transcription and translation in genetics. From Johnson, 1998.

Figure 8.1.1-3 expands on the previous figure.

Margolis et al. have provided extensive material on the olfactory marker protein(s), Olf-116. Their wording is careful. “We propose tha Olf-1 is a novel olfactory-specific trans-acting factor responsible for directing the expression of genes containing the Olf-1 motif in olfactory neurons. Thus it may play a role in regulating the expression of genes associated with neuronal turnover and olfactory transduction.” Olf-1 may not result in the creation of a protein acting as a stimulant sensing complex (SSC).

For those seeking a broader background in Figure 8.1.1-3 Alternate splicing ADD. From genetics, the lectures of Wolfe on the internet Johnson, 1998.

16Margolis, F. Kudrycki, K. et al. (1993) From genotype to olfactory neuron phenotype: the role of the Olf-1- binding site In Margolis, F. ed. The Molecular Basis of Smell and Taste Transduction. Ciba Foundation Symposium 179 NY: John Wiley & Sons pp 3-26 Signal Generation & Processing 8- 11 in 2005 are recommended17. [xxx not a strong source ] 8.1.1.2.1 Genetics of vision EMPTY

8.1.1.2.2 Genetics of hearing EMPTY

8.1.1.2.3 Genetics of chemoreception

Mombaerts has provided an extensive review of the literature relating the genes believed to relate to the ligands and molecules of the odorant receptors (OR)18. He noted in 2004, based on the conventional understanding of the genome, “Nearly all receptor genes have now been identified as the result of genome sequencing, but few receptor-ligand interactions have been characterized.” While discussing G-protein coupled receptors (GPCR), he concludes, “The Holy Grail of olfaction is within sight, but there is a long way to go—more than half of mouse and human non-chemosensory GPCRs have known ligands, but the vast majority of chemosensory GPCRs remain orphans.” Matching the GPCR’s and their heterodimer peptides analytically is difficult in the extreme. Mombaerts suggests an alternative (page 273) is to isolate an OR that responds to a given stimulant, clone that OR, and demonstrate the cloned OR confers the requisite sensitivity to a sensory cell.

Zozulya et al. have provided a first cut of the entire repertoire of human olfactory receptors19, described as stimulant sensing complexes (SSC) or odor binding proteins (OBP) in this work. The repertoire was the result of a data mining operation. Their strategy was to look for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences.

Zozulya et al. noted,

“Each receptor recognizes multiple odorants, and each odorant binds to multiple receptors to generate specific activation patterns for each of a vast number of distinct smells.

The genes encoding ORs are devoid of introns within their coding regions. Mammalian OR genes are typically organized in clusters of ten or more members and located on many chromosomes. The repertoire of human OR (hOR) genes contains a large fraction of pseudogenes, suggesting that olfaction became less important in the course of primate evolution. Recent studies indicate that some 70% of all hOR genes may be pseudogenes, compared with fewer than 5% in rodents or lower primates.”

The following quotation provides a flavor of their search for human olfactory receptors (hOR):

As a result of the search described above, 347 putative full-length OR genes have been identified in the human genome. This number includes all the previously known, annotated hOR sequences extracted from the public databases. It is feasible that a small number of full-length hORs escaped detection because of frameshifts or other sequencing or assembly errors in the corresponding HTGS entries. We are continuing routine searches using updated versions of genomic sequences to identify such cases. Because of the very high occurrence of OR pseudogenes in humans and the presence of ORs in highly variable parts of human genome, it is also possible that some polymorphic members of this gene family exist in the

17Wolfe, J. (2005) http://www.ucl.ac.uk/~ucbhjow/b241/lectures.html

18Mombaerts, P. (2004a) Genes and ligands for odorant, vomeronasal and taste receptors Nature Rev: Neurosci vol 5, pp 263-278

19Zozulya, S. Echeverri, F. & Nguyen, T. (2001) The human olfactory receptor repertoire Genome Biol vol 2(6), pp 1-12 [in pdf file] 12 Neurons & the Nervous System

human population in both intact and defective allelic forms. A small subset of identified OR, ORFs was discarded because of such defects as partial deletions of TM1 or TM7 regions. The argument can be made that these OR genes could encode functional receptors. In addition, it was hypothesized that odorant reception may also be mediated by receptors of a completely different class, such as guanylate cyclases. These caveats notwithstanding, we estimate that we have identified at least 90-95% of all full-length prototypical ORs in the human genome. Signal Generation & Processing 8- 13

There is no indication in the paper that Zozulya et al. looked for any receptor that was not a protein. Ronnett and Moon have provided additional information concerning the genes believed to support olfaction20. The inferential matching of genes to specific proteins believed to be found in the sensory neurons and assumed to relate to the sensory receptors remains in a primitive state. No data, or even sophisticated description, has appeared in the literature showing any specific protein participating in the actual transduction process.

8.1.1.2.4 Role of “Holes” in Genetics

Stewart has shown an interesting hydrogen bonding relationship between the nucleic pairs in DNA21. It stresses the multiple hydrogen-bond nature of the relationship between both thymine and adenine and between cytosine and guanine. As a result, these base pairs have almost identical stereographic shapes. The presence of multiple hydrogen bonds at every base pair along the DNA molecule offers a great deal on hole mobility along the molecule. 8.1.1.3 Transduction, neurotransmitters vs chemical signals

The term transduction has not made a successful transition from the physical sciences into the biological sciences. In their book of 2002, Gomperts et al22. review the definitions of the term found in the 2nd Edition of the Oxford English Dictionary. The dictionary notes, the only use of the term in the biological sciences related to external excitation relates to the visual modality, and a lament in the Journal of the Acoustical Society of America of 1947 that the mechanism has not been used in the theory of the hearing modality.

As is shown in this work, all of the external sensory modalities of the nervous system employ transduction of the stimulus into an electrical signal by one mechanism or another.

The lack of an adequate understanding of transduction in the neural system has led to the proliferation of alternate concepts relating a “first messenger” to a “second messenger” of chemical character. These second messengers are all ancillary to the fundamental transduction mechanism.

[xxx most of this section belongs in Chap 2 or 3 ] The biology community has drawn up a list of “chemical signals,” materials associated with communications between cells and larger components of the body. Communications is used here in the broadest possible sense. Figure 8.1.1-4 shows such a list from one author. It is interesting to note this list does not include many of the “chemical signals” generally associated with the nervous system. It is also interesting that nitric oxide is shown originating in endothelium, rather more explicitly as originating from the neural tissue in said endothelium. [xxx glutamic acid, etc. ]

20Ronnett, G. & Moon, C. (2002) G proteins and olfactory signal transduction Ann Rev Physiol vol 64, pp 189–222

21Stewart, I. (2011) Mathematics of Life: Unlocking the Secrets of Existence. London: Profile Books pp94-98

22Gomperts, B. Tatham, P. & Kramer, I. (2002) Signal Transduction. NY: Academic Press preface 14 Neurons & the Nervous System

Figure 8.1.1-4 Chemical signals within the body as described by one author. Add. The position of nitric oxide in this list is particularly interesting to a neuroscientist. From Johnson, 1998.

It is common in the biological community to bridge between chemical signaling agents and the more specific term neurotransmitter. This is frequently done without any formal definitions. In 1993, Hucho provided a remarkable discussion of general principles and nomenclature related to the neuroreceptors23. The paper is remarkably frank and skillfully worded. His opening sentences are enlightening, “Concepts evolve, and so did the receptor concept. Concepts are based on observations, on special experimental data that are generalized when somebody overlooks enough of them and is wise enough to see the general principle in the flood of numbers, plots and descriptions.” These sentences illuminate the fact that much of the framework of the neurosciences is based on concepts deeply rooted in philosophy and logic rather than rigorous application of the scientific method. Facts, plots and descriptions are relegated to secondary position behind “insights” that may be heavily weighted in the direction of the investigators prior training. These insights frequently take on a first order or linear tone in spite of the plots. Hucho proceeds to a definition of a receptor common in the fourth quarter of the 20th Century, “Receptors are proteins interacting with extracellular physiological signals and converting them into intracellular effects. Neurotransmitter receptors are integral membrane proteins; their physiological signals are neurotransmitters and neuromodulators.” No citations of any kind are given to defend

23Hucho, F. (1993) Transmitter receptors–general principles and nomenclature In Hucho, F. ed. Neurotransmitter Receptors. NY: Elsevier Chapter 1 Signal Generation & Processing 8- 15 these assertions. He goes on to find difficulty with this definition. “For example, many of the neuropeptides discussed in this chapter have not been classified as transmitters by the criteria summarized below. . . .On the other hand, it tacitly includes the so-called orphan receptors, receptors discovered by reverse genetics (the art of ‘pulling out’ clones with consensus probes homologous to sequences of well-known members of receptor families and super-families). Orphan receptor are proteins (actually in most cases just DNA sequence) for which the endogenous ligand, the physiological signal, has not yet been found” Finally he notes, “The molecular definition given here is different from a more biological receptor concept that names cellular entities like the rods and cones of the retina ‘photoreceptors’ and the cells of the muscle spindle ‘stretch receptors’.” Later on page 65 in the same volume, Otto notes, As discussed in Chapter 1, the term ‘receptor’ is differently and sometimes confusingly used in different contexts. In the field of signal transduction, the term ‘receptor’ stands for a protein structure, which activates a subsequent effector system while it is activated by binding an agonistic ligand. In the context of binding studies, this term has a much more general sense. There it is synonymously used fo the term ‘binding site’. Very often the terminology is mixed, thus bearing the danger of losing the awareness for the definition of binding specificity.” These citations highlight the considerable confusion found in the literature when terms are not precisely defined in the preparation of a published report. Hucho proceeds to note his text is based on concepts used in, and aimed at, the pharmacology community.

In fact, Hucho is far off track. None of the external sensory receptors of the neural system involved in transduction are proteins. This work will develop the details related to the chemistry of each external sensory receptor in turn. His concept of a transmembrane receptor is far too elementary to be useful. It will be compared to a more detailed description of individual receptors in Section 8.5.3.1.

In the pharmacology community, the high concentration of a substance near a neuron leads to its definition as a neurotransmitter or neuromodulator, even as in the case of GABA, the material is actually a residue (a waste product) of a reaction. In this case, the conceptual receptors known as GABAA and GABAB are actually the receptors for glutamate, which reacts electrostenolytically to form GABA with the release of both CO2 and GABA into the surrounding neural matrix. Functionally, it is not obvious that two receptors are required in this instance.

The host of defined receptor sites for glutamate is in even greater disarray. The TIPS (Trends in Pharmacological Sciences) prepares an ongoing summary of receptor nomenclature. As of 1993, this summary described eight (or nine) receptor sites for glutamate without defining the precise purpose of any of them. The 1993 supplement opens with a significant statement, “NO FORMAL system for the rational classification of receptor exists.” [Capitals in original] “Receptors are subtyped on the basis of different pharmacological profiles rather than on distinct second-messenger/signal- transduction pathways or primary structures.”

The “second messenger” concept has become the ultimate crutch in neuroscience. It arose in the 1950's based on research related to the liver by Earl Sutherland and his colleagues. It has been adopted by many research communities to provide a logical (if not scientific) explanation of nearly any transduction process. Figure 8.1.1-5, from Siegel et al., shows the extremes to which it is carried. An unspecified stimulant (the first messenger) elicits a second messenger (from an abbreviated selection of possibilities) after multiple unspecified processing steps by a series of proteins. The ultimate target is identified without regard to, or definition of, these intervening steps. The paradox associated with the conceptual hypothesis of the “second messenger” has been explored by Lichtstein & Rodbard24. While making significant assertions about the inadequacy of the concept in their paper, no followup discussions ensued at that

24Lichtstein D, Rodbard D. (1987) A second look at the second messenger hypothesis Life Sci vol 40(21), pp 2041-51. 16 Neurons & the Nervous System

time. Brezina & Weiss readdressed the subject ten years later25, “Neurons and other cells are regulated by a great multiplicity of neurotransmitters, modulators, hormones and other chemical messengers, which, through complex networks of extensively diverging and converging pathways, exert a multiplicity of effects. How do we analyze the functioning of such a complex network?” Unfortunately, their paper introduced even greater topological complexity by considering matrices of conceptual transmitters and introducing co- transmitters. They ultimately passed the questions they asked along to later investigators. They were unable to add preciseness to the overall discussion.

The above material takes on a Bayesian flavor. By philosophically limiting the range of phenomena considered (receptors are proteins, they create second messengers via other proteins) the empirical results are necessarily interpreted within that limited range. The likelihood that receptors of neurologically active stimuli could be other than proteins is eliminated in advance. Unfortunately, the data shows otherwise. First, most neuroreceptors related to chemoreception are actually specialized phospholipids integral to the lemma of the neurons. The initial visual receptors are carotenoids physically separate from any protein. Only the auditory receptors contain proteins, or protein-like materials, that are used as piezoelectric devices. Second, the transduction processes of the neural system do not involve any chemical process generating metabolic products. The laboratory community has clearly demonstrated that no experimental procedure has ever isolated a metabolic process directly traceable to transduction.

A major problem with texts and papers up through the turn of the Century has been their lack of attention to the types of Figure 8.1.1-5 The generalization of the “second quantum chemistry employed in the sensory messenger” concept. The concept can be used to modalities. The field of coordination explain anything. It has no physical foundation. PI; chemistry is typically missing from the index a variety (two are listed) of metabolic products of to these materials (Siegel, 1999, 2006). phosphatidylinositol, a phospholipid. AA; metabolic products (four are listed) of arachidonic acid, an unsaturated fatty acid. From Duman & Nestler in th Figure 8.1.1-6 provides a comparison of the Siegel et al. 6 Ed. 1999. historical chemical approach to neural sensing and the approach of the Electrolytic Theory of the Neuron. The historical approach is based primarily on logic and largely preconceived ideas. The electrolytic approach is based heavily on the observed performance characteristics of the neural system, architecture and tissue. The electrolytic approach provides a deterministic, modality specific description of the sensory process. It is based on a single “second messenger,” the electron, considered as a current that can be amplified, summed, differenced, exponentiated and interpreted. The signals related to the initial transduction current

25Brezina, V. & Weiss, K. (1998) Analyzing the functional consequences of transmitter complexity Trends Neurosci vol 20(11), pp 538-543 Signal Generation & Processing 8- 17 are transmitted over labeled-line neural paths. Probing of these paths electronically elicits signals that can be related (deterministically) back to the source stimulus. An important feature of the Electrolytic theory of the Neuron is that steps defined in this theory are readily quantifiable using conventional laboratory equipment. Much of the predicted performance has already been demonstrated in the laboratory by other investigators. The level of match between the predicted and measured performance is remarkable, regardless of the sensory modality involved. A second important feature is the laboratory data demonstrating that the synapse is electrically reversible, a difficult phenomenon to explain based on the unidirectional assumption of the chemical approach.

Figure 8.1.1-6 An initial comparison of the Electrolytic and chemical theories of the neuron and initial processes within the neural system. The quantifiable character of the Electrolytic Theory is remarkable compared to the largely conceptual character of the chemical theory after all of these years. See text.

The chemical theory of the neuron has been notoriously poor at providing any quantifiable explanations of sensory operation. This chapter will develop contiguous, detailed and mathematically quantifiable models of each of the sensory modalities, usually involving multiple parallel channels to the range of colors, scents and tastes discernible by the human. The model of each of these modalities will begin with a metric physically related to the stimulants unique to that sensory mechanism. The description of the gustatory modality will include a metric related to the various stimulants for the first time. The individual models will explain how the time constants related to that modality are independent of any chemical kinetics (except for the electrostenolytic process providing electricity to the sensory neurons. These models will also be contiguous with those in the following chapters related to signal projection and signal interpretation leading to cognition by the mind. 8.1.1.4 Chemicals of major importance in the chemical theory of the neuron 18 Neurons & the Nervous System

Johnson has discussed the importance of G-proteins and the “second messenger” concept. Most authors discussing the second messenger concept rely on the chemical cyclic AMP (or cyclic adenosine monophosphate) to fill this role. However, Takeuchi et al. have noted there are many sensory neurons that do not contain cAMP. In some cases they assign the second messenger role to another chemical, InsP3, Inositol 1,4,5-triphosphate. Inositol and its ability to complex with sodium, and potentially other alkali and alkali-earth ions, is of major importance in the gustatory modality (Section 8.4). The nomenclature of the very large number of isomers in the inositol family is confused, partly due to a major change in 1968. Parthasarathy & Eisenberg have described its history and usage as of 1991 and describe it as “in statu nascendi”26. Even while they were writing a subsequent change, in which they participated, was being negotiated in 1988. As a result, any representation of inositol in the literature, such as that in the special issue of the Biochemical Journal of 1986, must be questioned and placed in historical context27. The role of GABA in the chemical theory is that of a distinct neuroinhibitor (neurotransmitter) affecting the sensitivity of neurons to excitation. Soon after its discovery in the brain in the 1950's, one of the principles noted, “GABA probably was a metabolic wastebasket28.” The Electrolytic Theory of the Neuron provides its true and unique role, as the end product of the electrostenolytic process powering all neurons. In this process, it is an end-product that is recycled via a portion of the citrus cycle. It presence in excess in the neural matrix does in fact inhibit the operation of the nearby neurons. 8.1.1.5 A redefinition of the “messengers” of the neural system

The literature has been less than substantive when discussing the so-called 1st and 2nd messengers. Very little concerning the 1st messengers appears in the literature and the use of the term 2nd messenger is inconsistent. The term messenger appears to have entered the neural literature via attempts to explain what appears to be chemical communications between non-neural cells. It has been used in the neural literature in a largely conceptual context for years. A 1st messenger has been associated with any external medium impacting a neuron. A second messenger has been described as any agent found within a neuron, resulting from transduction of the 1st messenger, and supporting subsequent signaling within that neuron. A wide range of chemicals have beed defined as 2nd messengers based primarily on their presence in the internal chemistry of the neuron, regardless of its measured role in signaling. Based on these presences, elaborate conceptual reactions have been defined to explain the importance of these chemicals in the signaling process.

Lichtstein & Rodbard revisited the 2nd messenger concept in 198729. After suggesting there are a very large number of uncharacterized specific signals called “1st messengers,” they noted only a hand full of 2nd messengers had been proposed. Based on this situation they said, “Even allowing for the discovery of a large number of additional second messengers, there remains a paradox in terms of information-transfer within the cell: how can so many specific signals produce so many effects through so few relatively nonspecific intermediates?” They offer no rational solution to the stated paradox after considering multiple options based on the chemical theory of the neuron. Their only figure is a conceptual block diagram focused on hormones as the 1st messengers. The paper brings little new to the discussion of 2nd messengers. Brezina & Weiss addressed the subject again in 1997

26Parthasarathy, xxx & Eisenberg, xxx (1991) Bio-, stereochemistry, and nomenclature In Reitz, A. ed. Inositol Phosphates and Derivatives. Washington, DC: American Chemical Society

27Special Issue (1986) The phosphates of phosphatidylinositol in blood Biochem J vol 238(2)

28Martin, D. & Olsen, R. (2000) GABA in the Nervous System: The View at Fifty Years. NY: Lippincott Williams & Wilkins pg 7

29Lichtstein, D.& Rodbard, D. (1987) A Second Look at the Second Messenger Hypothesis: A Minireview Life Sci vol 40, pp. 2041-2051 Signal Generation & Processing 8- 19 with similar inconsequential results30. The Electrolytic Theory of the Neuron offers a clearer and more detailed description of these “messengers.” The Electrolytic Theory of the Neuron redefines the 1st messengers narrowly as external stimulants that are transduced by the sensory neurons into electrical signals that are subsequently relayed to the central nervous system (CNS). It defines a set of chemicals, the neuro-facilitators and the neuro- inhibitors, that provide electrical power to the neurons and/or reduce the power available to the neurons. It also defines a set of neuro-modulators, typically hormones, that change the operating characteristics of the neurons, independent of the power supplies, and thereby impact the electrical signals passed to the CNS. The only 2nd messenger recognized by the Electrolytic Theory of the Neuron is the electron. It exists in three operational forms, 1. a free electron moving within the conduction band of a medium, 2. an electron moving within the valence band of a semiconducting medium (and not considered free) 3. a free electron on the surface of an insulating material that participates in the electro-magnetic propagation of a signal along that insulating surface. Some of these mechanisms may be foreign to the experience of the reader. However, they are well known mechanisms in semiconductor physics and electro-magnetism.

The 2nd messenger of this theory (the electron in one of its forms defined above), created by the stage 1 transduction mechanism, is used exclusively for signaling between the stage 1 and stage 7 neurons.

As noted in Section xxx, the Electrolytic Theory of the Neuron recognizes that the original designations of currents in a neuron by Huxley and Hodgkin were euphemisms. Their label “sodium current” did not assert the current consisted of sodium ions, it referred to a conventional current entering through the lemma of a single compartment neuron. Likewise, their label “potassium current” referred to a conventional current flowing out of the lemma of the neuron. The inward current label was based on the higher concentration of sodium ions in the matrix surrounding the neuron. The outward current label was based on the higher concentration of the potassium ions within the neuron compartment. It has been repeatedly shown that the lemma of the neurons is impervious to both of these ions in their fundamental form. As in any electrolytic or electrical device, the primary current is in fact the electron (flowing in opposite direction to the “conventional” current defined erroneously by Benjamin Franklin based on a conceptual positively charged particle).]

The Electrolytic Theory of the Neuron recognizes the multi-compartment (and three-terminal) character of the neural portion of the neuron cell. As a result, two currents are recognized relating to the axoplasm. The first is the polarizing current of electrons entering the axoplasm of the neuron from the electrostenolytic power source on the outer surface of the axolemma. The second is the depolarizing current of electrons passing out of the axoplasm and into the podaplasm under the control of the voltage between the dendroplasm and the podaplasm.

A 3rd messenger can be defined as the substance(s) (typically but not exclusively hormones under most definitions of hormones) released by stage 7 neuron-affecters in order to influence muscle, neuronal and potentially other types of tissue. 8.1.1.6 Ultimate detection thresholds for the sensory neurons

Block has provided an evocative discussion of the potential sensitivity of the sensory neurons from a fundamental noise perspective that appears to be on track31. He even speculates on the threshold performance of poorly defined sensors such as magnetoreceptors and gravitational receptors.

30Brezina, V. & Weiss, K. (1997) Analyzing the functional consequences of transmitter complexity TINS vol 20(11), pp 538-543

31Block, S. (1992) Biophysical principles of sensory transduction In Corey, D. & Roper, S. eds. Sensory Transduction. NY: Rockefeller Univ Press Chapter 1 20 Neurons & the Nervous System

8.1.2 Comparative anatomy and histology in neuroscience

Johnson has provided a comprehensive clinical text including useful background on the neural system in humans32. An organism incorporates a variety of sensory neurons that support communications within its neural system. There are other means of communications within the organism that will not be addressed here, specifically chemical communication between cells and hormonal communications between the central nervous system and the extremities of the organism. Many authors have attempted to describe and organize a list of the sensory receptors of the neural system. The following list attempts to order them relative to their approximate importance to the human neural system.

NEURAL RECEPTORS OF THE EXTERNAL ENVIRONMENT Photoreceptors of vision. Phonoreceptors of hearing. Vestibular-receptors of spatial orientation. Chemoreceptors of olfaction and gustation. Odor-receptors of olfaction. Taste-receptors of gustation. Somatosensory neurons– heat, cold, pressure & physical damage.

NEURAL RECEPTORS OF THE INTERNAL ENVIRONMENT Kinesthetic receptors of spatial positioning and activity. Chemoreceptors of vascular system. Pressure receptors of the vascular system. Temperature receptors primarily of the vascular system.

The majority of the sensory neurons employ quantum-mechanical sensing mechanisms. These mechanisms are readily identified by the unique excitation/de-excitation (E/D) response function associated with these mechanisms. For convenience, this same E/D function (or equation) will be described as the photoexcitation/de-excitation equation (P/D) in vision, as the phonoexcitation/de- excitation equation (P/D) in hearing, and as the chemoexcitation/de-excitation equation (C/D) in olfaction and gustation.

The sensory receptors of vision and the external chemical environment will be seen to employ external transduction mechanisms that couple energy into the neural system by a quantum- mechanical mechanism. The sensory receptors of hearing and kinesthetics employ internal transduction mechanisms that deliver electrical charge to the neural system as a result of quantum- mechanical mechanisms, namely the piezo-electric effect.

Material reported in the fundamental research arena of the biological cell must be read very carefully to insure its relevance. As an example, the early paper of Bangham et al. have reported on a very professionally performed set of experiments on artificial phospholipid membranes using a material only described as ovolecithin33. Ovolecithin is generally described as lecithin obtained from egg yolks. While they stress the non-poroscity of artificial lemma to cations, compared to anions, the control of the purity of the presumed bilayer membrane is open to further review.

8.1.2.1 Areas of specialized lemma in sensory neurons

In the evolution of this work, it has been appropriate to define the plasma membrane forming the lemma of neural cells, and three specialized forms of plasma membrane. With the extension of this work to include olfaction and gustation, it is appropriate to define a fourth form.

32Johnson, L. ed.(1998) Essential Medical Physiology. NY: Lippincott-Raven

33Bangham, A. Standish, M. & Watkins, J. (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids J Mol Biol vol 13, pp 238-252 Signal Generation & Processing 8- 21 Plasma membrane--The outer membrane completely surrounding a cell and consisting of a double wall membrane of two leaves. Each leaf usually consists of a liquid crystalline film of biological phospho-glycerides. The cell is usually divided by internal membranes into at least three distinct functional sections in neurons. These sections are associated with the morphologically defined axons, dendrites and podites. These sections of the membrane may show further specialization. The character of the membrane is also divided into four functional types. The type 1 plasmalemma consists of a molecularly symmetrical continuous liquid crystalline bilayer that is impervious to transverse molecular and electron flow (it is a very good insulator). The type 2 plasmalemma consists of a molecularly asymmetrical continuous liquid crystalline bilayer that is impervious to transverse molecular flow but acts as an electrical diode with respect to electron flow. The type 3 plasmalemma consists of a liquid crystalline bilayer with many embedded proteins providing a transport path through the membrane. The type 4 plasmalemma consists of a molecularly asymmetrical continuous liquid crystalline bilayer that is impervious to transverse molecular flow but acts as an electrical diode with respect to electron flow and incorporates an external structure subject to chemical attack.

The type 5 plasmalemma consists of a continuous liquid crystalline bilayer of axoplasm that is impervious to transverse molecular flow but acts as an electrical conductor with respect to electron flow and incorporates an external structure acting as a receptor site for neuro-affector agent precursors. Upon formation of the neuro-affector agent, it is held at the receptor site until released under neural control (subject to a change in the axoplasm potential).

Type 1 membrane is the classical membrane that is impervious to molecular as well as electron flow. Type 3 membrane supports the commonly conjectured but not demonstrated flow of large molecules through the cell wall. Types 2 is an asymmetrical bilayer membrane supporting the electrostenolytic process providing negative electrical bias to the plasma of all cells relative to the surrounding matrix. Type 4 membrane is a more specialized version of a type 2 membrane where the outer leaf of the bilayer has been specialized to support olfaction and gustation.

Any time two type 2 plasmalemma are juxtaposed between plasmas of appropriate electrical potential, an active electrolytic device can be formed. 8.1.2.2 The transduction mechanism of sensory neurons

Squire et al. have provided a complex, and largely conceptual, table comparing the transduction mechanisms employed by the different sensory modalities based on the conventional chemical theory of the neural system. It was modified from Shepherd (1994) and focuses on the “second messenger” concept to explain how transduction is accomplished. Their figure 23.18 shows how truly conceptual the 2nd messenger actually is. The table hypothesizes a large number of proteins identified as G- protein-coupled receptor (GPCR) families as a primary participants in the transduction process. Their method of participation is largely unspecified. The table also includes a large number of question marks in place of pertinent information. Interestingly, it does not include the hearing modality. It will not be referred to subsequently in this work. The Electrolytic Theory of the Neuron provides a much simpler, more deterministic, and more compact table. It eliminates all of the largely conceptual, and frequently stochastic chemical processes previously associated with the neural system. Summarizing the work to follow, the transduction process employed by the various sensory modalities can be described using Figure 8.1.1-7. The steps in these processes are much fewer using the Electrolytic Theory of the Neuron. [xxx put brief version of this in Chapter 1 ] [xxx elaborate on my table ] 22 Neurons & the Nervous System

Figure 8.1.1-7 Summary of transduction mechanism in sensory modalities. All transduction mechanisms rely on quantum-mechanical processes except for the transduction of ionizable electrolytes that attack the chemical receptors via a different path.

8.1.2.3 Uniqueness of the transduction mechanism of sensory neurons

The transduction mechanism employs a process not previously presented in textbooks and journals of physical chemistry . The process differs from first and second order chemical reactions and from consecutive reactions and conventional chain reactions. The excitation/de-excitation mechanism of the sensory neurons employs what can be considered a chain reaction with replacement of the original reactants within a closed loop under the control of a stimulating event. The energy associated with this stimulating event may be derived from photons, phonons, odors and mechanical work. The mechanism typically employs a quantum-mechanical transition that is reversible under very controlled conditions. In the case of vision, the mechanism frequently involves a resonant oxygen Signal Generation & Processing 8- 23 molecular structure that can be described using many names, an enol structure, a conjugated oxygen structure, or a hybrid alcohol/aldehyde structure. The chromophores associated with the photoreceptors of vision, the Rhodonines, exhibit this structure. It will be important to differentiate between the stimulants and their internal constituents. In olfaction, these are called odorants and odorophores respectively. When speaking across odorant boundaries, the odorophores are described globally as olfactophores. Similar terminology is used in gustation but the use of gustaphore in both an individual and global context can be confusing. The individual odorophores and gustaphores of a particular stimulant, as a group, are described as the determinants of the stimulant in some papers. The molecular structure supporting transduction within a given modality are in intimate contact with the respective sensory neurons. These structures can be considered energy processing complexes (EPC’s). In the case of the chemoreceptors, they can be described as stimulant sensing complexes (SSC’s). The overall transduction mechanism in a given modality can be described as either an EPC/photoreceptor, an EPC/phonoreceptor or an SSC/chemoreceptor. The EPC/phonoreceptors are very similar to the combination used in other mechanoreceptors. The mechanoreceptors will not be discussed in detail in this work. The precise nature of the energy band gap found in the EPC’s is unusual. The width of the bandgap is much wider than normally associated with a single molecular species (as measured by flame chromatography as an example). It can be associated with a bulk crystalline material but is more appropriately associated with a single layer liquid crystalline structure in sensory processes. It is proposed that all of the EPC’s of photo-detection and chemodetection involve liquid crystalline surface layers. + All of the transduction mechanisms of the sensory neurons appear to conform to a single excitation/de-excitation equation. In the following sections, this equation will be labeled the P/D Equation for both photo and phono detection and the C/D Equation for chemo-detection. 8.1.2.4 Sensory neuron regeneration

The literature is controversial concerning sensory neuron regeneration. There are reports of regeneration of the sensory neurons of smell following laceration34 and possible regeneration of both olfactory and visual neurons on a routine basis. There is a conventional view that the sensory neurons of hearing are not replaced, and that damage to the cilia of hair cells is permanent. The question is difficult to resolve because of the static character of most histological investigations. In the case of hearing, it will be shown that the piezoelectric material within each cilium is replaced but the outer lemma of the cilium is not replaced. The catastrophic failure occurs when the cilium becomes bent over. The bud-like photoreceptor cells frequently found near the root of mature cells could be immature or replacement cells or they could be cells stunted during neurogenesis.

8.1.3 Sensory modalities addressed in this chapter

This chapter will focus on the external sensory modalities, with the exception of the sense of touch. Pribram, ‘91 has treated the tactile modality in considerable detail. His material can be related to the neural circuits of this work without difficulty. The internal sensory modalities, such as sensing oxygen content of the blood, will not be addressed here because of their range of functions and sparse information about their operation, and sometimes even location. The sensory modalities of vision and hearing will be addressed in summary form here because of

34Farbman, A. (1992) Cell Biology of Olfaction. Oxford: Cambridge Univ Press pp pp 210-211 24 Neurons & the Nervous System their extensive treatment in other materials35,36 by this author. 8.2 The photoreceptor neurons of vision EMPTY

[xxx Lam noted in 1978, that he was unable to identify any chemical neurotransmitter (of the half dozen known at that time, present in or released by the photoreceptors of the vertebrate retina37. “In conclusion, our chemical studies so far indicate that none of the known putative transmitters appears to be a likely candidate for the photoreceptor transmitter. Conversely, Lam provided good data on the specific activities and the concentrations of certain amino acids in the retina. Subsequently, Sarthy & Lam provided tentative identification of chemical neuro transmitters in the Newt38. They also reported a close association between these amino acids and the retinal neurons of goldfish39. ] Figure 8.2.1-1 Three terminal active biological device, the Activa. (A) A cross section of the junction between two 2.2.3.2 The two-terminal membranes. Equivalent chemical and electrical schematics biological transistor of the are shown. (B) Equivalent circuit of (A) if the two bases are photoreceptor cell MOVE to part of intimately related quantum-mechanically. Note the “A” Chapter 8 xxx above the base region in (B). The conventional currents are positive for a positive voltage Veb. (D) Output current Ic In the absence of electrical access to the versus the collector potential Vcb where Vcb is negative. region between the membranes, the two- membrane laminate has limited (but very important) application as an amplifier. Figure 8.2.1-2(A) and (B) illustrate this situation. Here again, the symbol “A” above the base region signifies an active biological semiconductor device. Note that if the collector is made negative with respect to the emitter, the bias requirements for “transistor action” is still obtained. The emitter to base region is forward biased and the collector to base region is reverse biased.

35Fulton, J. (2004) Biological Vision: A 21st century Tutorial. Bloomington, Indiana: Trafford and Processes in Biological Vision. www.neuronresearch.net/vision

36Fulton, J. (2008) Hearing: A 21st Century Paradigm. Bloomington, Indiana: Trafford and www.neuronresearch.net/hearing

37Lam, D. (1978) Physiology and biochemical studies of identified cells in the vertebrate retina In Osborne, N. ed. Biochemistry of Characterized Neurons. NY: Pergamon Press pp 239-260

38Sarthy, P. & Lam, D. (1983) Retinal regeneration in the adult newt, Notophthalmus viridescens: appearance of neurotransmitter synthesis and the electroretinogram Dev Brain Res, vol 6(2), pp 99-105

39Lam, D. Marc, R. Sarthy, P. Chin, C. Sul, Y. et al. (1980) Retinal organization: Neurotransmitters as physiological probes Proc Neurochem Internat vol 1, pp 183-190 Signal Generation & Processing 8- 25 If the Activa is fabricated without explicit electrical contact to the base, it is still possible to obtain transistor action by exciting the Activa by other means. If quantum-mechanical energy of sufficient strength (not intensity) is applied to the region of the device near the emitter-base junction, free charges of opposite sign can be generated in the base region. The charge of higher mobility will impact the operation of the device more significantly. Solving the equations applying to the current in such a device, it can be shown that the base current must be equated to the collector current minus the emitter current. Solving for the collector current, I(out) = I(free)/" or the output current is typically 50-100 times the current generated by the incident energy. In specially produced man- made transistors, this amplification factor can be as high as 5,000. Devices of this type are excellent photon detectors and can also be used as mechanical impact detectors, ionization gauges, etc. Frame (C) shows the output current as a function of a fictional input potential that cannot be measured, Ve-c. The input current remains equal to the output current. The transfer characteristic of such an Activa to energetic stimulation is given by the equation above and is independent of the collector-to-emitter potential.

8.3 The phonoreceptor and neurons of hearing & the vestibular system EMPTY

Carpenter & Sutin show a particularly useful graphic of the labyrinth40. Figure 8.2.1-2 The three-terminal active biological device, the Activa, with an open terminal. This is the configuration used in the initial circuits of the photoreceptor Figure 8.3.1-1 shows the middle and inner cell. Charges created in the base region are treated as if the ear from a pneumatic/mechanical came from the emitter. perspective.

This representation makes it clear that the round window is primarily a pressure relief port that responds to any pressure applied at the oval window. Similarly, the eustachian tube is a pressure relief port that responds to any pressure introduced into the middle ear by the motion of the tympanic membrane. [xxx appears in Ganong75, page 106. copy to ear book as well, I added words only to section 4.2.2.5.

8.4 The chemoreceptor neurons of taste and smell

Chemoreception in its broadest sense involves the sensing of both external stimuli and internal stimuli. The internal chemoreceptors are focused in two regimes, Figure 8.3.1-1 Pneumatic/mechanical those designed to sense the chemistry of the representation of the transmission of vibrations from the outer to inner ear. From Churchill, 1972.

40Carpenter, M. & Sutin, J. (xxx) Human Neuroanatomy, 8th Ed. London: Williams & Wilkins pg 366 26 Neurons & the Nervous System blood, the chemistry of the inhaled gases, and the chemistry of the ingested substances. The external chemoreceptors are focused in three regimes, those designed to sense distant stimuli and generally related to olfaction (smell), those designed to sense nearby chemical stimuli and generally related to gustation (taste), and a third category involving those designed to sense and react quickly to levels in the previous two channels that could cause damage to the organism (usually described as nocioreceptors). This section, and sections 8.5 & 8.6, will be limited to the external chemical sensory neurons associated with olfaction and gustation. The internal chemical sensory neurons, such as those associated with the enteric system, and with O2-level and CO2-level sensing, will be addressed superficially in Section 8.9. Laffort has defined three modalities to the area of external chemoreception41. “1. Olfaction or the sense of smell, characterized by a very acute ability to differentiate the most subtle molecular structures and to detect them at very low concentrations. It could be said that this is an analytical sense. 2. Gustation or the sense of taste, characterized by overall perception of four fundamental tastes; sweet, salty, acidic and bitter. It could be said that this in an integrative sense. 3. Common chemical sensitivity or trigeminal sensitivity (conveyed by the trigeminal nerve), characterized by perception of irritating, stinging and suffocating stimuli.”

This last modality is frequently described as utilizing nocioreceptors mixed in with the sensory neurons of the other modalities.

Cagan & Kare noted in 198142, “The infrequency of evident scientific interest in and concern for taste and olfaction in recent years stands in striking contrast to the earlier appreciation of the importance of these senses.” Relative to research in the visual and auditory senses, the statement appears to remain true today.

Documenting the human sensations of smell and taste have been difficult because investigations have been largely limited to psychophysical experiments. Neurophysical data has been available primarily from lower species. Finger, Silver & Restrepo have edited a recent volume with a provocative title but virtually no information concerning the physiology of the olfactory and gustatory systems and only the most conceptual neurological material43. At best, they provide a long list of genes where various investigators have “provided evidence of involvement in olfactory transduction (page 187).” The volume does not appear to have been edited critically for consistency. Their introduction (Chapter 1) makes a number of key assertions: C “Most animals have chemoreceptors for monitoring internal as well as external chemical conditions.” C “Humans, being air-breathing vertebrates, have a passageway connecting the nose and mouth.” C “Our language does not always make a clear distinction between stimulation of the gustatory and the olfactory receptor cells. C “Taste is used colloquially to describe any chemical sensation produced by food in the mouth.” Several expressions are shown in strikeout and call for explanation. Many aquatic air breathing vertebrates (Cetacea as an example) do not have a passageway connecting the nose and mouth. From a scientific perspective, sensation is a perception developed within the CNS. A sensation within the

41Laffort, P. (1994) Olfactory communications and facets of odorousness In Martin, G. & Laffort, P. eds Odors and Deodorization in the Environment. NY: VCH Chapter 5 pg 105

42Cagan, R. & Kare, M. ed. (1981) Biochemistry of taste and olfaction. NY: Academic Press pg xix

43Finger, T. Silver, W. & Restrepo, D. eds. (2000) The Neurobiology of taste and smell, 2nd Ed.. NY: Wiley- Liss Signal Generation & Processing 8- 27 CNS is not directly associated exclusively with the insertion of food in the mouth. It may result from a chemical reaction within the oral cavity. It appears to also include an olfactory and frequently a somatosensory component. They went on: C “Chemical stimuli play an important role in behaviors such as feeding, territorial recognition, and sexual or social activities.” C “Thus, the diverse chemosensory functions all occur in a single cell in single cell animals.” C “In metazoan forms, the different chemosensory functions are usually divided into distinct sensory modalities.” The insertion of the phrase “in single cell animals” is justified here by their following sentence, where the word chemosensory has been inserted for clarity. After noting, “All taxonomic groups of tetrapod vertebrates have at least two major divisions of the nasal chemosensory organs, the main olfactory system and the vomeronasal system,” and the paucity of data, Johnston (Chapter 5) has provided a chapter exploring whether the human has a vomeronasal organ, without drawing a firm conclusion44. “In humans, current evidence supports the existence of a pocket in the nasal cavity which is in a position where one would expect to see a vomeronasal organ if there were one. Anatomical evidence, however, does not show convincingly that this pocket has any sensory cells with axons, nor that any cells in this region make connection with the olfactory bulb or other part of the brain (page 121).” Personal experience would suggest otherwise. Schwanzel-Fukuda & Pfaff have explained the source of this controversy (Section 8.xxx). Takagi went so far as to illustrate the dual olfactory system of the monkey and other small mammals45. Monti-Bloch et al. have provided a review of these organs showing the location of the vomeronasal organ in humans, its ontogeny, generator waveforms and some discussion of human pheromones46.

Doty edited a handbook of olfaction and gustation in 2003 that provides both academic and clinical material47. It offers considerably more detailed information than Finger et al. It focuses on anatomy and the chemicals of olfaction but does not address the neurophysiology of olfaction or gustation at a significant level. Turin & Yoshii (page 280) suggest there are only two currently viable theories of olfaction, one based on molecular structure (odotopes) and one based on “vibration theory.” The remainder of their chapter is very useful, especially the discussion of small differences between odorants and non-odorants. The hard molecular weight limit of “less than 300" is a useful criteria.

Based on the thinness of the database and the character of the sensory neurons of vision and hearing, the premises of this work can be stated as;

1. The sensory neurons of taste and smell, while distinct types, follow the cytology and electrolytic circuitry of the visual and hearing sensory neurons.

2. The major difference between the sensory neurons of taste and smell compared to those of vision and hearing relate to the electrolytic structure in and around the fine dendritic structure of the cells. of the cells. 3. The low energy levels related to taste and smell leads to the proposal that the taste and smell sensory neurons operate in the amplification mode similar to the hearing sensory neurons.

44Johnston, xxx

45Takagi, S. (1980) Dual nervous system for olfactory functions in mammals In van der Starre, H. ed. Proceedings of 7th ISOT London: IRL Press pp 275-278

46Monti-Bloch, L. Jennings-White, C. & Berliner, D. (1998) The human vomeronasal system In Murphy, C. ed. Olfaction and Taste, XII. NY: New York Academy of Sciences pp 373-389

47Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker 28 Neurons & the Nervous System

4. The absence of any reaction products following sensing leads to the proposal that the transduction process in taste and smell sensory neurons relies upon coordinate chemistry rather than reaction chemistry. 5. The signal flow between the taste and smell sensory neurons and the CNS exhibits the same architecture as that of vision and hearing. The olfactory signal flow is similar to that of vision (independent of the spinal column). The gustatory signal flow is similar to that of hearing (signals proceed to the CNS via the lower brain stem). The following material will demonstrate these postulates to the extent possible within the available database. It will demonstrate the likelihood that two sensory channels, that for the organic acids and that for acrid chemicals (bitter in taste, pungent in smell) appear in both the oral and nasal cavities of mammals. - - - - -

8.4.1 Brief summary of the literature, glossary & overview

Michael Mann, of the University of Nebraska Medical Center, has provided a good on-line source of introductory information about taste and smell, but without any in depth presentation of the mechanisms involved48. It follows the conventional conceptual view that has evolved over many years. The framework developed here is quite different. However, the data is valuable. 8.4.1.1 Recent chemoreception literature

Squire et al. have discussed the information extracted by the gustatory modality. This information related to a chemical stimuli involves quality, intensity and hedonic value (where hedonic value refers to the perceived pleasantness or unpleasantness of a tastes sensation). They suggest the hedonic value is based on genetic, physiologic and experiential factors. The last factor can be heavily culturally-based. They also note the three factors are interrelated in developing a perceived flavor.

Moncrieff provided a broad text on these sensory modalities from the human perspective in 196749. His chapters 4 (Olfaction) and 5 (Gustation) followed by chapters 11 (taste and constitution) and 15 (flavour and food) provide a broad outline of the problem. Dodd & Squirrell provided a considerable amount of histological data and some performance data related to olfaction in 198050. Shepherd provided an overview of the problem in 198851.

Lawless & Lee attempted to describe the scheme leading to the concept of flavor as shown in Figure 8.4.1-1 The concept, based on sensations and not histology provides independent “irritation” channels that could be associated with putative nocioreceptors. It could also be expanded to include pungency and astringent sensory channels within the nocioreceptors. Hofmann et al. have provided some cursory conceptual material related to these modalities52. At the histological level, nocioreceptors remain largely notional.

48Mann, M. (marginally updated to 2011) The Nervous System in Action. http://www.unmc.edu/physiology/Mann/ Chapter 10

49Moncrieff, R. (1967) The Chemical Senses. London: Leonard Hill

50Dodd, G. & Squirrell, D. (1980) Structure and mechanisms in the mammalian olfactory system Symp zool Soc Lond vol 45, pp 35-56

51Shepherd, G. (1988) Neurobiology, 2nd Ed. NY: Oxford Press

52Hofmann, T. Ho, C-T. & Pickenhagen, W. eds. (2004) Challenges in Taste Chemistry and Biology. Washington, DC: American Chemical Society Chapter 1 Signal Generation & Processing 8- 29 This work will only consider flavor as a perception based on the combined sensations of taste, smell, texture, temperature and occasionally pain as presented to the saliency map. Teranishi has noted, “We can not adequately express, define, or explain our taste and smell sensations.” After alluding the sight and hearing, he goes on, “. . .but we cannot adequately define flavor either qualitatively or quantitatively53.” It is extremely difficult to definitively separate the sensations generated by the gustatory and olfactory modalities. Lim & Lawless have provided an interesting study of the qualitative difference in sensations for the same chemicals depending on whether the nasal passages are closed off54. Such differences may reduce the accuracy of much of the data in the journal literature. Besides the four historical primary tastes, and possibly umami, the terms pungency (associated with peppers), a cooling sensation, a tingling sensation, and an astringent sensation may be included in the description of a flavor. Whether these sensations are the result of the gustatory sensory channels or mechanoreceptor channels is frequently discussed. The field of flavor science has mushroomed since the 1980's, with many important texts and papers supporting the commercial preparation and storage of foods. The contribution of these works to the understanding of the transduction of stimulants ultimately into sensations has been largely lacking in these works.

Sensing associated with taste and smell has been under intense investigation ever since Axel & Buck were awarded a Nobel Prize in 2004 for their work reported in 199155. The real breakthrough has come beginning in 1995, particularly with the autoradiographic work of the Bret Johnson & Micheal Leon team at the University of California, Irvine (discussed in Section 8.6.3).

The taste and smell modalities are known to involve three distinct regions, the olfactory epithelium in the upper nasal cavities, the vomeronasal organ in the lower nasal cavities and the gustatory surfaces of the oral cavity.

Hudspeth & Tanaka provided a brief review that only touched on the chemical senses, taste and olfaction56. They note the classical concept suggests the taste sensations of sweetness, sourness, saltyness and bitterness. They quickly note the sensitivity of the human system to fat as well. Many alternates to this brief listing have been proposed. Nearly a century ago, Ikeda suggested an additional axis to the taste map involving glutamate (or more specifically monosodium glutamate)57. He named the sensation associated with glutamate umami. Sugimoto & Ninomiya have provided a review of more recent work related to umami58. Axel & Buck suggested a very large number of independent olfactory sensor types. They inferred several relationships between the olfactory system and over 1000 genes.

Pfaffman et al. showed that a single neural channel exhibits responses from a wide variety of taste

53Teranishi, R. (1999) Preface In Teranishi, R. Wick, E. & Hornstein, I. eds. Flavor Chemistry: Thirty Years of Progress. NY: Kluwer Academic/Plenum

54Lim, J. & Lawless, H. (2005) Qualitative differences of divalent salts: multidimensional scaling and cluster analysis Chem. Senses vol 30, pp 719–726

55Buck, L. & Axel, R.(1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition Cell vol 65, pp 175-187

56Hudspeth, A. & Tanaka, K. (2000) Sensory systems: editorial overview curr Opin Neurobiol vol xxx, pp 443- 446

57Ikeda, K. (2002) New Seasonings Chem Senses vol 27, pp 847-849 A reprint of original

58Sugimoto, K. & Ninomiya, Y. (2005) Introductory remarks on umami research: candidate receptors and signal transduction mechanisms on umami Chem Senses vol 30(suppl 1) pp i21-i22 30 Neurons & the Nervous System stimuli59. The effects were compared between a variety of rodents. An important confirmation involved the sensitivity to both chloride ion and quinine among other substances. The simplest conclusion was that the chloride ion concentration was not in a class by itself. In fact they found no correlation between the response to NaCl and NH4Cl. They also noted a correlation between fructose and both NaCl and NH4Cl in the squirrel monkey but not the hamster. Pfaffman et al. reported relative sweetness for the sugars at equal molar concentrations in the following order; sucrose > fructose > maltose > lactose > dextrose > galactose. Note that sucrose, maltose and lactose are disaccharides before their hydrolysis. Before hydrolysis, all are reducing sugars except sucrose. There was no indication that the subject could identify the specific sugars listed in this sequence. The situation at different concentrations is more complicated. For NaCl, it is reported to taste sweet at very low concentrations, then salty sweet, and then pure salty at a concentration of 0.2 M. “KCl is reported as sweet, bitter, bitter-salty and then salty, bitter and sour as concentrations increases.” They use a term “side bands” but do not define it explicitly. Pfaffman et al. also noted the nominal 7-day turnover of sense cells in the taste buds. The responses in the squirrel monkey were subject to prior adaptation, or pre-adaptation, commonly labeled priming in the psychological literature.

Kaissling & Thorson provided Figure 8.4.1-1 describing the olfactory sensillum of a moth as they understood it60. Their early paper is important. It asserts that one molecule of bombykol (a pheremone of the moth, Bombyx mori, is sufficient to cause a perceived neural response in that species. They also provided a passive electrical circuit overlay to this figure.

Squire et al. (2003, pg 613) have provided a similar image but stress the difference in color/contrast between the individual cells within a given taste bud. They also note the typical bud contains not a few, but typically 50-150 chemoreceptor neurons.

Figure 8.4.1-1 ADD The histology of the olfactory sensory neuron of the moth Bombyx mori. From Kaissling & Thorson, 1980.

59Pfaffman, C. Frank, M. Bartoshuk, L. & Snell, T. (1976) Coding gustatory information in the squirrel monkey Chorda Tympani, In Sprague, J. & Epstein, A. eds. Progress in Psychobiology and Physiological Psychology. NY: Academic Press. vol 6, pp 1-27

60Kaissling, K. & Thorson, J. (1980) Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization In Satelle, D. et al. eds. Receptors for Neurotransmitters, Hormones and Pheromones in Insects. NY: Elsevier Signal Generation & Processing 8- 31 Shepherd has provided a recent paper61 describing the olfactory process in the same context as will be developed in this work. He describes the sensory signals as being processed initially in the olfactory bulb, being passed through the thalamus, and generating complex signals (involving the high level concept of flavors) associated with the correlation and interpretation processes of stage 4 and 5. Unfortunately, his paper does not develop any theory of the transduction process at the detailed level in olfaction. Shepherd has introduced a postulate that requires additional discussion. He melds the perception of flavor with the ability of an animal to enunciate that perception. From that perspective, he implies that the human species, because it is most highly developed in the enunciation area, necessarily has the most highly developed flavor perception (2005, pg i5). Multi-dimensional scaling (MDS) has been applied to taste. Hellekant et al. have provided a broad paper developing a multi-dimensional space for taste in 20 rhesus monkeys using 26 stimulants including those related to umami62. They also provide an unusual cluster analysis that relates to the individual subjects rather than a broad list of chemicals. Rather than dissolve their stimulants in distilled water, they used an artificial saliva (which contained many of their stimuli in small amounts). Their figures 3 (related to the anterior tongue) & 8 (related to the posterior tongue) show the variation in sensitivity among their subjects to their chemicals. The need for large statistical samples in gustatory sensor research is obvious. Selected samples can provide very erroneous insights. Sensitivity was described in terms of the number of action potentials within five seconds over a given nerve. It is difficult to ascertain any average subject from figure 8. Figure 3 suggests distinctly different sensitivity groups within their subjects. These figures were ignored in the overall paper. They discuss the performance of monkey compared to humans and chimpanzee but not to orangutan. See Section 8.6.xxx for further discussion.

The task of this section is to put this material into the framework of the overall neural system.

Johnson & Leon have provided a summary of their extensive experimental work that is also consistent with this work63. The loss of the perception of taste (ageusic) and the loss of the perception of smell (anosmia) are ancient ones. However, they have not been studied comprehensively. Henkin has written extensively on the problems64. Farbman has also written in generalities on the subject, including some medical correlations65. Personal experience suggests a deficiency in the metal complexes (particularly involving zinc) leads to anosmia. Magnesium may also play a role in olfaction. Turin has explored the role of zinc in olfaction in developing his inelastic electron tunneling hypothesis66. Physical trauma to the head frequently causes shearing of the fasciculated neurons passing through the cribriform.

DeSimone discussed the difficulty of assigning a single transduction mechanism to the chemo-

61Shepherd, G. (2005) Outline of a Theory of Olfactory Processing and its relevance to humans Chem Senses vol 30(suppl 1), pp i3-i5

62Hellekant, G. Danilova, V. & Ninomiya, Y. (1997) Primate sense of taste: behavioral and single chorda tympani and glossopharyngeal nerve fiber recordings in the Rhesus Monkey, Macaca mulatta. J Neurophysiol vol 77, pp 978-993

63Johnson, B. & Leon, M. (2006) xxx Blass text

64Henkin, R. & Hoetker, J. (2003) Deficient dietary intake of vitamin E in patients with taste and smell dysfunctions Nutrition vol 19(11), pp 1013-1021

65Farbman, A. (1992) Cell Biology of Olfaction. Oxford: Cambridge Univ Press pp pp 115-116

66Turin, L. (1996) A Spectroscopic Mechanism for Primary Olfactory Reception Chem Senses vol 21, pp 773-791 32 Neurons & the Nervous System stimulus sensitive transduction neurons67. Achieving sensitivity to non-polar and both positively and negatively charged polar materials in one sensory neuron configuration is difficult and probably impossible. DeSimone discusses these problems in the context of his understanding of the applicable 1981 technology. The neural system appears to take a different path. The following discussion will develop different sensory neuron configurations to account for the different character of the stimuli based on the available laboratory evidence. Farbman discusses the individually identified chemo- sensitive sensory neurons of the very simple nematode, Caenorhabditis elegans as an introduction to the sensory neurons of other species68. He cites the report of Sengupta & Carlson (Chapter 3 of the same work). The two groups agree that individual chemo-sensitive neurons of the worm respond to a range of similar chemical stimuli. They report a large group of genes (on the order of 550) involved in encoding specific sensory neurons with a much smaller set of morphologically identifiable sensory neurons. They do not discuss the transduction mechanism per se. They do note, “Genetic and structural data implicate the cilia (microvilli) as the location of the primary signal transduction events in sensory signaling.” Farbman describes only four distinct sensory neurons behind the chemosensory pore of C elegans optimally sensing 11 different substances. 8.4.1.2 The problem of defining terms

In 2008, Erickson prepared a review of the gustatory research field spanning his long career69. He complained loudly about the lax scientific approach taken in this field, a subject he has addressed many times70. He was most concerned about the lack of definitions related to the “basic tastes.” He notes specifically the lack of clear definitions of the terms sweet, sour, salty, bitter etc. After presenting a 17 page paper followed by 16 pages of critique by 18 senior members of the community and a 9 page response, the community remained without a set of definitions of these terms. It is clear that the basic problem is the origination of the scientific concepts of taste in the world of human psychophysics and little actual human electrophysiology related to that world. Virtually all of the electrophysiology is on lower mammals, particularly rats.

Erickson can be easily criticized for raising a set of fundamental philosophical questions related to science in general and a lack of a set of definitions in particular. However, in doing so, he owes his audience his best shot at providing a framework for resolving most of those problems. He did not do that.

While Erickson makes many strong points that should be considered by any young researcher entering the field, he does not converge on a solution to these problems. Nor does he provide a set of working definitions for the parameters of gustation or olfaction.

Many have noted the ambiguity associated with the terms basic tastes and primary tastes in the taste literature (Breslin, page429). The literature is not clear when the terms are applied to one or more stimuli (stage 0), when they are applied to a sensory channel (stage 1), or when they are applied to a perceived condition (stage 5). Taste is primarily a (stage 5) perception based on information extraction from signals developed by the sensory neurons (stage 1) conveying information extracted by signal manipulation within stage 4. The stimuli eliciting such tastes are introduced into the oral cavity (stage 0). There appear to be four primary sensory neuron types, labeled H-best, N-best, S- best and Q-best. The relevance of these terms will be developed later. A major problem in olfactory research is the significant separation between the term aromatic as

67DeSimone, J. (1981) Physicochemical principles in taste and olfaction In Cagan, R. & Kare, M. ed. (1981) Biochemistry of taste and olfaction. NY: Academic Press page 225

68Farbman, A. ((2000) Cell biology of olfactory epithelium In Finger, T. Silver, W. & Restrepo, D. eds. The Neurobiology of taste and smell, 2nd Ed.. NY: Wiley-Liss Chapter 6

69Erickson, R. (2008) A study of the science of taste: On the origins and influence of the core ideas vol 31, pp 59–105

70Erickson, R. (2000) The evolution of neural coding ideas in the chemical senses PhysiolBehav vol 69, pp 3–13 Signal Generation & Processing 8- 33 used in perfumery and general language, and the term aromatic as it is now used in organic chemistry. In perfumery, the term denotes a sensation while in chemistry, it is a chemical containing a benzene ring (with its π-cloud of electrons).

8.4.1.2.1 Is the bitter tasting CaCl2 a salt? The definition of a “salty” flavor obscures a major problem. Salt is a very ancient term used in many languages around the world to define the common salt, NaCl. However, the technical community defines salt as the residue of a reaction between an acid and a base. By this definition, CaCl2 and 71 MgCl2 are salts, yet it is generally recognized they taste bitter, more like quinone than table salt . This work will develop the fact the archaic name “salty” as used in gustation really applies to the + sensation resulting to stimulation by the complex Na C(H20)n. where n is typically six. This is the gustaphore that will be described in this work as a “natrophore.” The sodium ion carries a plus one valence and is typically shown in column 1a of the periodic table. They critical aspects of this structure when it acts as a gustatory stimulant are the distance in Angstrom between the pairs of oxygen atoms complexed to the sodium ion, and the ability of these oxygen atoms to coordination bond to the sensory receptors.

The salts of CaCl2 and MgCl2 are technically salts. Their cations typically carry a plus two valence and are typically shown in column 2a of the periodic table. However, when placed in solution, the cations can form a variety of complexes with the water through coordinate bonding72. Most 2+ commonly, the magnesium atom is surrounded by six oxygen atoms (Mg[H2O]6 ). The critical aspects of these structures when they act as gustatory stimulants are the distances between the Magnesium ion and the coordinate bonded oxygen (2.07 Angstrom), and the pairs of oxygen atoms (2.9 Angstrom, the oxygen atoms are in contact with each other) complexed to the cations of these salts, and the ability of these oxygen atoms to coordination bond to the sensory receptors73. These distances are different from those of table salt and result in the bitter taste of these chemicals. Their bitter taste confirms their classification as picrophores, rather than natrophores. See Section 8.5.4.6 for more details.

Katz et al. have reported on the coordination chemistry of the calcium, magnesium beryllium and zinc ions74.

“Salty” is an archaic term that is misleading and should be discarded from the academic and food industries. It is not used in this work, except in quotations from old literature. 8.4.1.3 Brief Glossary

The following definitions of these gustatory terms are provided only for discussion purposes by an outsider. They are subject to continual review and revision.

Aromatic–1. (Perfumery) Eliciting a substantial pleasant sensation and associated with an “aroma.” 2. (Org. Chemistry) A chemical structure containing a benzene ring (and accompanying π-cloud of electrons). Basic tastes doctrine– A loosely defined proposal dating from the early 1900's that the gustatory

71Scott, T. & Mark, P. (1987). The taste system encodes stimulus toxicity. Brain Res vol 414(1), pp 197-203

72Bock, C. Kaufman, A. & Glusker, J.(1994) Coordination of water to magnesium cations Inorg Chem vol 33(3), pp 419-427

73Glusker, J. Katz, A. & Bock, C. (1999) Metal ions in biological systems Rigaku J pp 8-16

74Katz, A. Glusker, J. Beebe, S. & Bock, C. (1996) Calcium Ion Coordination: A Comparison with That of Beryllium, Magnesium, and Zinc J Am Chem Soc vol 118(24), pp 5752–5763 http://pubs.acs.org/doi/abs/10.1021/ja953943i 34 Neurons & the Nervous System modality can only perceive a limited set of independent or “basic” tastes. Basic tastes– (ne primary tastes) The major groupings of perceptual responses to human psychophysical taste studies as defined by multidimensional analyses of large scale studies involving the widest possible range of stimulants and subjects, and providing adequate statistical precision. Based on the number of basic functions resulting from these studies, the basic taste space exhibits three orthogonal dimensions suggesting these dimensions are presented to the cognitive functions of the brain as independent parameters and the pattern of these parameters is used to define specific tastes based on learning (including some pre-wired memory in infants). By considering the basic tastes as limits of the multidimensional taste space, all tastes can be represented within this space. Benzene– used as a suffix to describe a benzene ring combined with and any other moiety.

Phenyl– used as a prefix to describe any benzene ring of the form C6H5 combined by only one bond with another single moiety. Taste– (noun) A semantic descriptor of a perceived sensation derived principly from the gustatory modality. Sweet– A basic taste perception closely associated with the quest for food and commonly associated with a wide range of sugars and sugar like substances.

Sour– A basic taste perception closely associated with the desire to avoid ingestion of toxic substances and commonly associated with a range of acidic chemicals.

Salty– A basic taste perception closely associated with the desire to maintain an appropriate homeostatic condition with respect to the salinity of the fluids of the body, and necessarily associated with the fundamental saline solution of biology.

Bitter– A basic taste perception closely associated with limiting the ingestion of a variety of complex chemicals that are not easily processed by the digestive system.

The correlation between these perceptual responses and the electrophysiological signals emanating from individual sensory neurons has been demonstrated when the neurons have been stimulated by the principle chemicals associated with each of these perceived classes.

- - - - -

A variety of terms appear in the olfactory and gustatory literature that are not common to most of the neural literature. Some of these are described here. For a more extensive glossary, see the on- line glossary, http://neuronresearch.net/glossary.pdf {xxx put this up and be sure it is current.

Ab initio– From the beginning.

Ageusic– Lacking a sense of taste. Aliphatic compounds– Molecules containing hydrogen and carbon not in aromatic rings, although they may contain alicylic (non-aromatic) rings. Allosteric– of, relating to, or being a change in the shape and activity of a protein (as an enzyme) that results from combination with another substance at a point other than the chemically active site. Amiloride– A potassium-sparing organic diuretic (mol wt. = 229.6) used in the management of hypertension and congestive heart failure. Ampholytes– Molecules that exist in solution partly as neutral molecules and partly as positive and negative ions. Anomeric carbon– (or anomeric center)– Signal Generation & Processing 8- 35 1. A carbon atom attached to two oxygen atoms by single bonds. Typically found in cyclic monosaccharides. 2. An anomeric carbon is the new stereo-center created in forming the cyclic structure of a monosaccharide 3. The reducing carbon of a sugar; C-1 of an aldose, C-2 of a 2-ketose. 4. An example of anomeric carbon is that carbon in a monosaccharide (like glucose) about which rotation occurs. The anomeric carbon can be determined by the carbon (C) attached to two oxygen (O) atoms joined by single bonds. This rotation brings about two distinct configurations, a and ß -anomers. Carbohydrates can then change spontaneously between the a and ß configurations: a process known as mutarotation Anosmic– Lacking a sense of smell. Specific anosmia, lacking sensitivity to a specific stimulant. Apoptosis– The death of a cell due to internal mechanisms and events. “Cell death from within.” See also necrosis. Chorda tympani– A part of one of three cranial nerves, a branch of the facial nerve (the seventh cranial nerve) that serves the taste buds in the front of the tongue, runs through the middle ear, and carries taste messages to the brain. It branches from the facial nerve inside the facial canal, just before the facial nerve exits the skull via the stylomastoid foramen.

Confocal microscopy– The technique of using a pinhole aperture in the return path (and often in the illumination path as well) of the microscope to limit the field of view in both depth and cross- section. Important in scattered light rejection. The pinhole in the return path is frequently called a lyot stop in optics texts. Scanning of the restricted field is frequently employed to recover a useful field of view.

CLSM– Confocal laser-scanning microscopy.

Dipolar ion– A species bearing no net charge but exhibiting a large dipole or multi-pole moment. A dipolar ion is typically a super-polar molecule surrounded by an intense electrostatic field.

Fascicle– Small bundles of neurons, below the level of a nerve

Flavor– A perception based on sensations of taste, smell, texture, temperature and occasionally pain as presented to the saliency map.

Flavorant– An additive known to affect the perceived response to a gustatory stimulant. See flavor.

Glomerulus– a small convoluted or intertwined mass (as of organisms, nerve fibers, or capillaries). In neurology, a neuropil.

Haworth projection– A projection where the sugar ring is represented as a flat polygon, commonly a pentagon or a hexagon, instead of in proper geometrical conformation like a chair. The advantages of the Haworth projection are that it is easy to draw, and it is very easy to see whether substituent groups on the ring are on the "top" or the "bottom" of the ring (cis or trans). Heterodimer– A protein composed of two polypeptide chains differing in composition in the order, number, or kind of their amino acid residues Homologous– a : having the same relative position, value, or structure: as (1) : exhibiting biological homology (2) : having the same or allelic genes with genetic loci usually arranged in the same order . b : belonging to or consisting of a chemical series whose members exhibit homology. InsP3– (or IP3) Inositol 1,4,5-triphosphate, a proposed second messenger that causes the release of calcium from certain intracellular organelles. 36 Neurons & the Nervous System

Isoelectric point– The pH level at which the number of amino acids or peptides exhibiting positive charges is equal to the number exhibiting negative charges. As a result, no migration of these particles will occur under the influence of an electrical field. Kinetics, chemical– A branch of physical chemistry which involves the rates and mechanisms of chemical reactions Simplest, the long term average output-input relationship in a chemical reaction. Typical, the average output-input relationship in a chemical reaction. Advanced, the output-input relationship showing all transient elements of a chemical reaction Miraculin– A natural tetrameric glycoprotein. While it does not generate a sweet sensation itself, if applied to the tongue, it will make ordinary sour foods, such as citrus taste sweet for up to one hour. Gymnemic acid can reverse the sweet taste caused by miraculin, as it can the taste of sucrose. Mitral– 1. Relating to or resembling a miter worn by certain ecclesiastics. 2. Relating to a mitral valve. Motif– A distinctive usually recurrent molecular sequence (as of amino acids or base pairs) or structural elements (as of secondary protein structures). A simple protein motif consisting of two alpha helices.

MRR– Molecular receptive range. Describes the spatial receptive field of a specific odor on the olfactory epithelium. From Mori et al. 1992.

Necrosis– The death of a cell due to external mechanisms and events. “Cell death from without.” See also apostis.

Neural response functions– A term used to describe the variation in sensitivity of individual types of sensory neurons within the multidimensional taste space. Apparently a bell-shaped function exhibiting little overlap with the functions of the adjoining types of sensory neurons sampling the complete space.

Neuropil– An intertwining of a mass of axons and neurites forming large numbers of synapses.

Neurotrophins– Soluble proteins believed to support cell growth .

Odor map (or odor image)– The representation of the odor/taste environment in the saliency map of stage 4 and accessible by the stage 5 cognitive portion of the brain.

Odotope– The common molecular substructure responsible for interaction with a given stimulant sensing complex.

Olfactophore– A particular stimulant sensing complex associated with a chemoreceptor. Analogous to a chromophore in vision. Used differently here than suggested by Ham & Jurs, 1985. Parosmia– A perverted olfactory perception. Parametric measurement– A measurement made while the circuit is being stimulated abnormally, as in patch-clamp experiments or in the global application of pharmaceuticals to the surface of a cell. Parametric stimulation– The artificial excitation of a neuron by electrical means not associated with the neurite terminals via a synapse. Pheromone– A substance secreted to the outside of an individual and received by a second individual of the same species in which they cause a specific reaction, for example, a definite behavior or developmental process. Vertebrate pheromones remain largely operationally defined and are not identified chemically with a few exception. Peptides– Simple proteins consisting of only a few amino acid units. Signal Generation & Processing 8- 37 Piriform cortex– (sometimes prepiriform) An area in the anterior temporal lobe of each cerebral hemisphere forming part of the olfactory cortex, receiving nerve impulses from the olfactory receptors via the olfactory bulb without the prior involvement of the thalamus as in other sensory systems. It is not part of the neocortex but is a three-layered palaeocortex. Also spelt pyriform cortex, an etymologically incorrect but common form. From Coleman, A. (2001) A Dictionary of Psychology. Placode– A platelike structure, like the hard toothlike scales of sharks, skates, and rays. Pseudogenes– Genomic DNA sequences similar to normal genes but non-functional; they are regarded as defunct relatives of functional genes. See www.pseudogene.org QHCl– Quinine hydrochloride, a complicated molecule consisting of two fused aromatic rings, quiniline, the bicyclic quinuclidine, and a OH and a –O– separated by 5 carbons on the fused aromatic. The OH is separated by four carbons from a N in one of the rings.. Reducing sugars– Sugars which contain a free aldehyde (sometimes keto/alcohol) group and reduce indicators such as Cupric ion (Cu2+) complexes to the Cuprous form (Cu+). The reducing agent in these reactions is the open-chain form of the aldose or ketose. The reducing end of a sugar is the one containing a free aldehyde or keto group.

Umami– A putative taste sensation resulting from stimulation by monosodium glutamate.

Vomeronasal organ– Also known as Jacobson’s organ. Reported in the literature to provide specialized intra-species communications via pheromones.

8.4.1.4 Overview of chemoreception problem

Henning has provided a simple description of the basic requirements of chemoreception75. He lists;

“1. The graded transduction of a chemical into an electrical signal;

2. Some amplification should be allowed for;

3. The process should be fast enough to generate a potential within a few 100 ms;

4. The mechanism proposed must meet modest specificity requirements.”

As Henning notes, “Most of the existing theories on odor and taste satisfy only part of these criteria.”

Focusing on the ligand-receptor aspect of transduction, he has reiterated the five basic questions of Scatchard and added two of his own,

1. How many? (binding sites). 2. How tight? (KD). 3. Where? 4. Why? (Forces involved) and 5. What of it? 6. What are the kinetics of binding and disassociation? 7. Is there interaction among binding sites? Such simple questions may obscure the complexity of the problem. Rossiter has taken a different philosophical path and asked five different questions. (1) How do we recognize and discriminate between thousands of odors? (2) Which molecular properties determine the smell of a compound?

75Henning, G. (1971) Some general criteria for the receptor process in gustation and olfaction In Ohloff, G. & Thomas, A. eds. Gustation and Olfaction. NY: Academic Press pp 239-242 38 Neurons & the Nervous System

(3) Why, in some cases, do compounds which are completely different in structure have similar odors? (4) And conversely, why do compounds which are very similar in structure have dramatically different odors? (5) How can our sense of smell respond to chemicals which we have never encountered before and do so in a way that enables us to describe and categorize the odor? These appear to be the more important questions. It is the goal of this work to discover and present substantive first order answers to all of these question.

Kare76, and on occasion others, have noted an important fact, “Many illustrations of olfaction and taste have been given using the insect system as examples. Frogs eat flies, yet man does not relish them. Butterflies chemically attract one another but have no apparent chemical effect on me. Synthetic sweeteners, appealing to many humans, are offensive to many species. All the evidence supports the conclusion that species live in different sensory worlds, which may or may not overlap.” Care is called for in the field of comparative physiology. In the same 1971 discussion, Kare asked for estimates of how long it would take to achieve a general understanding of the gustation and olfaction modalities. The responses ran from five to fifty years. Clearly such an understanding did not occur in the first forty years. It is hoped this presentation will achieve most of the requirement at the end of forty years.

8.4.1.5 Attempts to establish a structure-odor or structure-response relationships

The chemoreception research field has suffered from a lack of an adequate model of the neurophysiological system involved. As a result, it has followed an approach long used in pharmacology.

Beets provided a clear description of the SAR (structure-activity relationship) developed in the field of pharmacology and applied to the field of olfaction77. The early work was primarily in the industrial development of synthetic structures mimicking natural pheromones (primarily musks). These are primarily multi-fused-ring structures. Thus, the field started by attacking one of the most complex areas of organic chemistry. Hansch, writing in 197378, recognized the extreme complexity of the task based on the staggering complexity of the biological system and noted that “any search for quantitative relationships between biological response and molecular structure, by means of classical methods of kinetics and thermodynamics impossible.” Based on his premise, he opens with, “The correlation of chemical structure with biological response produced by a set of related drugs is an impossible problem. Nevertheless, it is so important and so fascinating that, countless investigators have spent their careers on it and countless more will continue to do so.” This situation remains true today. However, better tools offer alternate paths to solutions in the sensory transduction area (devoid of kinetics).

Two books were found encompassing the SAR approach to chemorecetor exploration and published in the last 30 years. According to Dodd79, the purpose of a SAR study is to correlate the biological

76Kare, M. (1971) editorial comments at a symposium in Ohloff, G. & Thomas, A. eds. Gustation and Olfaction. NY: Academic Press pg 254-259

77Beets, M. (1978) Structure-Activity Relationships in Human Chemoreception. London: Applied Science Publ.

78Hansch, (1973) Quantitative approaches to pharmacological structure-activity relationships In Cavallito, C. ed. International Encyclopedia of Pharmacology and Therapeutics, Section 5, Vol 1, Structure-Activity Relationships. NY: Pergamon Press Chapter 3

79Dodd, G. (1976) Structure-activity relationships in chemoreception In Benz, G. ed. Structure-Activity Relationships in Chemoreception London: Information Retrieval Limited pp 1-9 Signal Generation & Processing 8- 39 effects of a chemical, e.g., its taste, with the molecular properties of the stimulant. The definition was followed by a 1-2 page conceptual illustration. Two years later, Beets spent 10-12 pages expanding on his interpretation of a SAR but did not offer a concise definition of the subject. After warming to his subject, he defined a set of properties, Sn of a chemical structure, S, as encompassing all relevant features of S that have been recognized by man. “When the structure of an unknown molecular species is drawn on a sheet of paper with complete indications of configurational details, all the properties, chemical, physical and physiological, which the species will have are at the same time irrevocably and fully defined.” This is clearly an inadequate Bayesian approach to science. A scientist in this position “doesn’t know what he doesn’t know.” He then defines a similar set of activities, Cn, related to the field of chemoreception and apparently associated with the larger set of sensations, C, presented to the cognitive elements of the brain (but more commonly associated with the motor responses used by the brain to describe these sensations). Avoiding the use of specific mathematical concepts, Beets appears to define a direct relationship as an exclusive path between a specific element of the structural set Sn and a specific element of the the activity set, Cn. He defines an indirect relationship as one where one activity, Cn, can be correlated with other activities in the set C, presumably in a traceable response to an element of S. The SAR approach has been described as pragmatic, without a close association with any theory.

In a 1996 review, Rossiter also employed two concepts to aid in organizing her olfactory research, the chemical structure-odor relationship (SOR) and the chemical structure-activity relationship (SAR). She reviewed odor properties of various chemicals in the perfume industry and noted, “In an attempt to predict such properties, chemists have been searching for correlations between molecular structure and odor for more than six decades. The fruits of this research are a wide and varied collection of postulated structure-odor relationships (SORs).” Rossiter described her attempts to move from the SOR regime to the SAR regime and even to a quantitative structure-activity relationship (QSAR). She noted the rapid conversion of both SOR and SAR studies to computerized methods but did not describe her precise understanding of the SAR methodology.

At the conclusion of the theory development section of this work, a significant amount of discussion will look at the framework defined by the theory and how it can solidify the concepts of the SOR and SAR in gustation and olfaction (Sections 8.5.xxx and 8.6.xxx). 8.4.2 The stimuli processed by the chemical sensors

The major chemical sensors of the neural system consist of the olfactory and gustatory modalities. They are very closely related and their perceived information, smells and flavors are closely related. Many investigators believe the olfactory subsystem is the dominant modality. Odorous chemicals must be volatile (mol. wt. below 300-400), minimally water-soluble and lipid-soluble to be sensed by the olfactory modality. Tastable chemicals need to be soluble in saliva at pH = 7.0. The sugars are not odorous but dissolve freely in water and are sensed by the gustatory system. The sugars are not soluble in non-polar solvents.

A key feature highlighted in the following discussions is that the stimulants are present not only in solution, but frequently in a hydrated state. when hydrated, it is the stereochemistry of their hydrated state which is important in chemical sensing. Squire et al. (2003, pg 607) has discussed the various terminologies in use to describe chemical stimulants. “The distinguishing features of an odor molecule have been called determinants or, alternately, epitopes.” The term epitope is drawn from immunology and implies a variety of features not necessarily relevant to the sensory modalities. “Theoretical studies suggest that odor ligands commonly comprise three or four determinants, which interact with two to six receptor sites.” The assertion, there are three or four determinants, which interact with two to six receptor sites,” is quite compatible with some of the results of multidimensional scaling applied to gustation (Section 8.6.3 xxx). However, multidimensional analysis leads to a much larger number of statistically independent dimensions in olfaction (Section 8.4.4.3 xxx). The chemical receptors react to a wide range of ionic, primarily inorganic, materials and non-ionic, primarily organic, materials. The difference between these large classes suggest the chemical receptors may utilize at least two distinct transduction mechanisms. 40 Neurons & the Nervous System

[xxx edit overall to show sugar is detected by stereoisomerism or by energy. ] The perceived tastes of the sugars have been studied in detail. However, the chemical differences between the sugars causing these perceived differences are very subtle differences in stereoisomerism. These differences suggest a stereographic approach to transduction. However, the ease of transition between the different isomeres of the sugars suggest this is not a reliable detection criteria. Other mechanisms relating to the reducing powers of the sugars must be considered. On the other hand, many of the ionic materials are quite capable of interacting directly with at least selective sections of the membranes forming the neurites of the receptor cells. A complication of understanding the olfactory modality is the requirement to use individual purified chemicals in the laboratory. Natural stimulants contain large numbers of molecules contributing to their odors as shown in Squire et al. (page 650) for the jasmine flower from Mori & Yoshihara (1995). Twenty one individual chemicals are shown. Only two are described as peculiar to the jasmine.

8.4.2.1 The sensitivity of the gustatory receptor neurons

The sensitivity of the gustatory neurons varies significantly with the duration of the applied stimulation, the chemical character of the stimulant, any recent prior stimulant and other stimulants applied simultaneously. Eyzaguirre & Fidone provided the following summary related to these parameters.

The perceived taste associated with a given stimulant also varies with its applied concentration. A weak concentration of NaCl (9-20 mM) is perceived as sweet by humans. However, if the concentration of NaCl is raised above 30 mM, a very definite salty sensation will result.

An alkaline stimulant (other than a salt) is only sensed at pH values above 10. The sensory neurons responding to an alkaline stimulant are believed to be sensitive to a variety of stimulants. Surprisingly, the taste receptors responding to acid have a very high threshold, the presence of a sour stimulant is not recognized until the pH of the solution falls to less than 4. It must fall to a pH of less than 3.5 before a definite sensation of sourness is perceived. Figure 8.4.2-2 places these numbers in context.

Organic acids frequently provoke a sensation that is a mixture of two independent sensations. Citric acid, for example, elicits a sensation that is a mixture of sweet and sour. Picric acid evokes a sensation that is a mixture of bitter and sour. The appreciation of sourness depends very much on the rate of application of the acidic substance and on the secretion of saliva. Saliva has a buffering capacity, which tends to neutralize the acid applied to the tongue. Figure 8.4.2-1 Threshold and recognition values versus pH of ionic materials. The sweetness of the sugars is particularly hard to quantify because they are non- electrolytes. The sweetest of all natural sugars is fructose, followed by sucrose, glucose and others–lactose being among the least sweet of all sugars. The artificial sweetener, trade-named saccharin is considered 700 more sweet than sucrose. For test purposes, the bitter taste is evoked by the application of quinine salts. Signal Generation & Processing 8- 41 Distilled water is sensed by the tongue since its introduction invariably changes the pH of the existing solution. Mixing of stimulants can result in unexpected perceptions. Eyzguirre & Fidone suggest individual stimulants are perceived separately. However, masking is a commonly observed effect. Masking suggests certain sensations are subtracted from each other within the neural channels projecting the taste sensations to the brain. Eyzaguirre & Fidone discuss both inhibitory and excitatory signals within the olfactory bulb. Eyzaguirre & Fidone provide a graph (page 147) showing the action potentials of a single neural path generated by NaCl as a function of concentration. Their figures on page 150 show the graded generator potential of an olfactory sensory neuron as well as a combination of generator potential and action potential stream resulting from stimulation with butyl alcohol. The generator waveforms shown are negative going, have an amplitude of a few millivolts, and are identical to those expected from the Excitation/De-excitation Equation of photoreception (ref. xxx). Because of the necessity for the stimulants to diffuse through the mucosa or saliva, the electrophysical chemical receptor responses tend to be measured in seconds. The figure on page 150 from Ottoson, 1956, shows a total rise time of 1 1/3 seconds and a tail of about two seconds to the 1/e point. The delay from time of stimulant application was not shown. The literature is unclear about the responses of individual chemoreceptor neurons to ionic material. Figure 8.4.2-2 shows the receptor potentials of a single neuron to a series of salts containing other positive ions than sodium80. The common constituent is the chlorine ion.

There is similar confusion concerning the response of specific neurons to non-ionic material. There are many reports discussing different sets of stimuli and the field of organics and biologicals that act as stimulants is very large. Leinders-Zufall (1998) provided data on four individual cells to a group of six organics. Figure 8.4.2-2 Receptor potentials of a single rat taste cell to salts. Stimulation was 0.1 M Adaptation and masking are particularly concentrations of NaCl, KCl, NH4Cl, CaCl2 and important mechanisms in the olfactory MgCl2 respectively. Water rinses were applied modality. between the stimuli. From Beidler, 1961.

8.4.2.1.1 Synopsis of the Chemical Theory of the Neuron

[xxx consolidate chemical theory comments. ] There are a variety of current chemical theories of the neuron. Ham & Jurs suggested the following in 198581; Perhaps the best mechanism is that proposed by Dodd and co-workers (Dodd and Squirrell, 1980; Dodd and Persaud, 1981) which explains all of the known features of olfaction and has precedence in other areas of biochemistry. According to this mechanism, olfaction is based on the interaction of molecules with membrane proteins. The odorant molecules act as allosteric regulators of enzyme activity. That is, the interaction of the odorant with the receptor involves the binding of the odorant molecule to the receptor and the change in the conformation of the receptor to accommodate the molecule. Therefore, the size, shape and electronic properties of the molecule are

80Beidler, L. (1961) xxx Progr Biophys vol 12, pp 107-xxx

81Ham, C. & Jurs, P. (1985) Structure-activity studies of musk odorants using pattern recognition: monocyclic nitrobenzenes Chem Senses vol 10(4), pp.491-505 [xxx pdf file ] 42 Neurons & the Nervous System

important in determining its nature as an odorant. The Dodd and Squirrell theory, labeled the Allosteric Membrane Enzyme (AME) Hypothesis, was expressed in 1985 using narrative in less than one page, and using no figures. They developed it during 1978-1985. “1. Binding sites for odorants. These may be proteins or phospholipids and may exhibit different degrees of selectivity to different odorants, with possibly high binding specificity to some odorants of biological significance, such as pheromones. 2. Receptor potential. This is brought about by ligand-initiated conformational change in an ion- gating protein, by any of the following mechanisms: (a) modulation of membrane fluidity, with consequent alteration in membrane phospholipid-protein interactions; (b) direct ligand induced conformational change of an allosteric protomer of a membrane enzyme with intrinsic ion-gating properties, such as a Na+, K+-ATPase or a Ca2+-ATPase; or modulation of a second messenger system through an enzyme cascade system or through non-covalent binding or covalent chemical modification of the protein. In the latter mechanisms it is not necessary for the binding protein to be part of an oligomeric transducer complex. Additional degrees of freedom are open if it is not, and the ligand-protein complex may diffuse on or off the transducer moelcule, allowing several receptors to share the same transducer. This may allow cooperative interactions between clusters of binding proteins.

3. Intensity coding. The fraction of sites occupied will mainly determine the magnitude of the receptor potential.

4. Quality coding. The degree of response of different primary neurons to identical stimuli may be determined by either membrane phospholipid composition or by relative distribution of receptor proteins. With the former, little difference may be expected for many congeneric odorants, while with the latter, greater differences may be expected.”

The mechanisms of transduction and chemical to electrical conversion are largely speculative in the Dodd & Squirrell (1980) paper. While the conventional wisdom, based primarily on long-standing pedagogy, is proteins play a major role in chemoreceptor transduction, the literature is virtually devoid of any data or plausible mechanisms involving proteins in transduction (particularly in the last twenty years).

This work will introduce the questions, “Can a dipeptide that has been modified to the point it cannot participate in a longer peptide chain be considered a protein? (Section 8.xxx)”

Turin & Yoshii have summarized the current “plausible theories of odor detection” based on the conventional chemical theory of the neuron82. They describe two classes of theories; shape-based theories and vibration theories. They suggest the shape-based theories are converging on the “across neuron pattern” concept with individual patterns defined as odotopes. They note the demise of the vibrational theories in the 1980's only to be resurrected recently based on a potential “inelastic electron tunneling spectroscopy” approach. This approach defines a series of energy levels associated with individual sensory neurons that provide piecewise coverage of the desired energy range. This approach is largely conceptual at this time. Turin has described it in depth83. Turin & Yoshii describe the difficulties with these theories (pp 289-292). They note the apparent role of zinc (and potentially other metallic ions) in olfaction and gustation. They conclude in 2003, “The fact that after several decades of experimental investigations, the basic mechanism by which odors are detected remains open to question shows that there is much work to be done.”

82Turin, L. & Yoshii, F. (2003) Structure-odor relationships: a modern perspective In Doty, R. ed. Handbook of Olfaction and Gustation. NY: Marcel Dekker pp 279-282

83Turin, L. (1996) A Spectroscopic Mechanism for Primary Olfactory Reception Chem Senses vol 21, pp 773-791 Signal Generation & Processing 8- 43 In summary, explanations of the operation of the chemoreceptors based on the chemical theory of the neuron suffer from; 1. A lack of specificity below the conceptual level. 2. No explanation of how the chemistry of the mucosa and saliva remain unchanged during and after stimulation. 3. No explanation of how chemical species return to the external surround after passing through the sensory neuron wall in order to cause a sensible change in the neural system. 4. No explanation of how a second messenger is generated without any chemical reaction involving enough energy to break a single chemical reaction bond. 5. No definitive description of any protein employed in transduction (resulting in an electrical signal at the pedicel of a sensory neuron). Parsimony suggests the eight processes conceptualized by Van der Heijden (page 70) for the transduction process under the Chemical Theory of the Neuron is less likely than the one step transduction process involved in the Electrolytic Theory of the Neuron. Wright has made an interesting observation that appears to apply to both the gustatory and olfactory villi/cilia. “The olfactory cilia are one micron in (outside) diameter and one to three hundred long.. It is difficult to imagine any chemical transformation going on in a pipe of these dimensions, so the primary process of stimulation must be a physical one not involving making or breaking of chemical bonds84.” 8.4.2.1.2 Electrolytic Theory of the Neuron & chemoreception

[xxx retitle or split section ] The photoreceptor neurons and the phonoreceptor neurons of the Electrolytic Theory of the Neuron provide a logical foundation for the proposed chemical receptor, or chemoreceptor, neurons. Squire et al. have noted the “dendritic” extremity of the olfactory neuron is virtually identical to the equivalent structure of the photoreceptor neuron85. However, their wording appears to misinterpret their source, Dodd & Squirrell. Dodd and Squirrell provide a more detailed view that provides more explicit terminology. They describe a conventional cilia emerging from the “knob” of the chemoreceptor, as the base of a chalice, that then subdivides into the individual microtubules (or dendrites in many texts) forming the staves of the chalice. Referencing a review by Steinbrecht (1969), Dodd & Squirrell note, “The proximal portion of the cilia has a diameter of about 0.25 μm and a normal 9(2) + 2 axonemal structure, but after a few μm there is a distinct narrowing of the cilium to a diameter of about 0.15 μm diameter and from here on the cilium gradually tapers to about 0.06 mm in diameter.

The above description surfaces two important features of neural cilia.

Neural cilia fall into two distinct classes, those formed of nominally solid protein in long extruded rods and those formed as fluid filled tubes supporting the flow of electrical currents toward the axoplasm of the neuron. The latter structures are frequently labeled villi.

9(2) +2 refers to the cylindrical arrangement of 9 pairs (sometimes sporadic triples) of microtubules encircling 2 central microtubules within, and near the base of, a neural cilia of the villi type. This level of detail can only be seen with electron-microscopy86. Diameters considerably below 0.5 micron are obviously challenging for a light microscope. The gradual tapering of the distal part of the cilium is accompanied by a progressive reduction in the number of microfilaments (microtubules); thus any cross-section through the mucus shows cilia with

84Wright, R. (1971) In Ohloff, G. & Thomas, A. eds. Gustation and Olfaction. NY: Academic Press p 26

85Squire, L. Bloom, F. McConnell, S. et al. eds. (2003) Fundamental Neuroscience, 2nd Ed. NY: Academic Press page 601

86Rhein, L. & Cagan,R. (1981) Role of cilia in olfactory recognition In Cagan, R. & Kare, M. eds. Biochemistry of taste and olfaction. NY: Academic Press pp 47-68 44 Neurons & the Nervous System varying numbers of microfilaments according to the point at which each has been cut.” They also note the difficulty in measuring the microtubules. “Lengths are likely to be underestimated.” Lengths vary from about 5-10 μm in humans to 80-200 μm in frogs. They also note, “Olfactory cilia are unlike the respiratory cilia of the surrounding tissues which maintain a constant diameter and retain a 9(2) + 2 structure of microfilaments throughout their length.” While Squire et al. note the microtubules contain no other organelles, Dodd & Squirrell note the microtubules are covered with “a large number of particles in the membranes of the olfactory cilia compared to corresponding respiratory cilia, in bovines. It is postulated that these particles might represent olfactory receptor sites.” The terminal elements of the olfactory neuron is analogous to those of the photoreceptor cell with the disk stack removed. This analogy provides significant clues to the transduction mechanism of the olfactory system used to sense organic chemicals. The photoreceptor microtubule elements are sensitive to quantum-mechanical shocks greater than 2.34 electron-volts. Figure 8.4.2-3 compares the proposed schematic of the chemoreceptor neuron compared to a micrograph of a human olfactory neuron. Morrison & Costanzo suggest the belt-like complex just below, or girdling, the knob (arrows) provides connection between adjacent neurons. In fact, the connections represent the connection to a reservoir of glutamic acid shared by adjacent cells and providing electrical power to the sensory neurons. The proposed chemoreceptor is based on the photoreceptor with the disks and associated apparatus removed. Relating this proposal is difficult because of the limited empirical data base available. In-vivo chemoreceptor neuron investigations are particularly difficult. XXX

Dodd & Squirrell note, “Despite the generally held belied that nerve cells in adult vertebrates are unable to be regenerated, there is a continuous process of cell renewal in the olfactory epithelium, with the basal cells acting as stem cells.” They reference Moulton (1975) who observed a turnover rate of about 29 days in mouse.

Teeter et al. have provided a very useful summary of the empirical database leading to the Signal Generation & Processing 8- 45 generation of the “taste potential87.” They note the graded potential encountered at the pedicle of the chemoreceptor neurons. They also suggest repeatedly the possibility that there are more than one class of chemoreceptor neurons. They note the frequent assumption that morphologically light and dark cells, as well as basal cells in some species, might employ different transduction mechanisms. Pfaffman et al. also describe the analog character of the chemoreceptor neuron responses88. Kaissling and Thorson have provided one of the few detailed histological descriptions of the chemoreceptors which are frequently found enclosed within a “hair wall” in the olfactory modality or a taste bud in the gustatory modality89. Their figure 5 associates the names outer segment and inner segment with the histology of the chemoreceptor (with the soma of the cell associated with the inner segment). The electrical overlay in that figure clearly shows the histologically bipolar cell exhibits more than just two electrical terminals. The overlay does not assume any active electrolytic device within the cell to provide electrical amplification. Nor does the overlay show any electrical output projecting along the axon. By extension, their electrical overlay is compatible with that shown in the figure shown here. Figure 8.4.2-3 The morphology of the olfactory [xxx discuss odor detection mechanism here chemoreceptor and proposed neural circuitry if appropriate ] compared. The proposed histology and neural circuitry is identical to that of the photoreceptor. A; the proposed histological configuration based on the 8.4.3 Architecture of the external photoreceptor of this work. B, the morphological chemoreceptor modalities image of an olfactory neuron. x18975. The lower stem of this image will be labeled the inner segment Two schools of thought have arisen in this work. It has sometimes been labeled the concerning the architecture of the external dendrite. The arrows in the image point to the chemoreceptors. One school has drawn podite bias terminal of the cell. Image from analogies with the immune system. The Morrison & Costanzo, 1990. other has drawn analogies with the visual and hearing systems.

The immune analogy supporters drew three conclusions (Hildebrand & Shepherd, 1997):

(a) There is likely to be a large family of receptor molecules (100-1000 members). (b) An olfactory receptor is likely to have a variable region of odor binding and a constant region for second-messenger transduction. (c) The receptor is likely to belong to the GPCR superfamily. The analogy has supported the presumption of a large family of protein-based odor-binding protein (OBP) receptors despite a paucity of evidence to support the presumption at this time. It also supports the description of the stimulant as a ligand whose set of determinants constitutes an

87Teeter, J. Funakoshi, M. Kurihara, K. et al. (1987) Generation of the taste cell potential Chem Senses vol 12(2), pp 217-234

88Pfaffmann, C. Frank, M. Bartoshuk, L. & Snell, T. (1976) Op. Cit.

89Kaissling, K. & Thorson, J. (1980) Op. Cit. 46 Neurons & the Nervous System olfactophore. A problem with this analogy is the failure of many stimulants to cause the production of cAMP within the chemoreceptor neuron (relying upon the chemical theory of the neuron) . This fact has required the development of an alternate theory involving selective activation of either cAMP or IP3 (Inositol 1,4,5-triphosphate). The vision analogy supporters drew similar but divergent conclusions: (a) There is likely to be a more modest number of receptor molecules capable of sensing a variety of molecules. (b) Each receptor neuron supports only one or a few stimulant sensing complexes (SSC) that may or may not be protein-based OBP’s. (c) The SSC is sensitive to the determinants of a given stimulant rather than the overall structure of the stimulant. (d) Stage 2 signal processing within the peripheral neural system and stage 4 signal feature extraction within the CNS extracts the pattern information needed to describe a specific stimulant or stimulant set. This work will rationalize these estimates. The Electrolytic Theory of Chemical Sensing shows that the gustation modality employs only four sensory receptor types (Section 8.5) while the olfaction modality employs less than 23 sensory receptor types, with about 15 accounting for most of the olfactory responses (Section 8.6). Two of the sensory receptor types appear in both the gustation and olfaction modalities; the type sensitive to organic acids and the type sensitive to bitter substances.. The individual sensory receptor is sensitive to a specific molecular geometry and employs coordinate bonding rather than reaction chemistry to perform transduction.. 8.4.3.1 Top level architecture of the olfactory modality

Only a few investigators have offered schematics of the olfactory modality. In this work, the top level architecture will be developed first to show how it fits into the framework of the overall neural system. Then, the architecture of the peripheral portion of the modality will be developed before developing the architecture of the sensory neurons.

One paper must be mentioned because of its novelty. Zou et al. presented a paper in 2001 that they felt duty-bound to retract in 200890. It purported to define an overall neural path from the olfactory sensory neurons to the CNS using what they described as a transneuronal tracer to travel that path. While a plausible result under the conceptual chemical theory of the neuron, such a transneuronal tracer traveling the length of the neural path is not compatible with the Electrolytic Theory of the Neuron.

Figure 8.4.3-1 shows the top level architecture of the olfactory modality employed in this work. It focuses on the mammalian orders as the fish and birds appear to employ different architectures and/or naming conventions.

The figure is based on the nominal sensory modality architecture discussed in Section xxx. The figure differs primarily in the identification of two distinct initial signaling channels associated with the main olfactory channel and an auxiliary channel known as the vomeral olfactory channel. The main channel is associated with the olfactory mucosa located in the upper nasal passages immediately below the main olfactory bulb within the cranium. The sensory neurons of the main channel pass through the cribiform plate of the ethmoid bone to reach the main olfactory bulb. The sensory neurons of the vomeral channel take a separate path to reach the auxiliary olfactory bulb. The vomeral channel can be considered analogous to the foveal channel of the visual modality. It is a high precision channel used to identify, with high chemical resolution, stimulants (pheromones) arising from other members of the same species, particularly members of that species of the opposite sex.

90Zou, Z. Horowitz, L. Snapper, S. & Buck, L. (2001) Genetic tracing reveals a stereotyped sensory map in the olfactory cortex Nature vol 414, pp 173-179 Retracted in Nature vol 452, pg 120 (2008) Signal Generation & Processing 8- 47 In the context of this work, both the stage 2 main olfactory bulb and auxiliary olfactory bulbs are considered parts of the peripheral neural system based on their function. They are concerned with signal manipulation leading to a compaction of the information derived from the sensory neurons prior to the projection of that information to the stage 4 circuits of the CNS, all of which are located within the cranium. The olfactory signal paths leading to the higher cortical centers have not been agreed upon by the histologists working in the field. Some assert the initial point of arrival of the signals is via the pons. Others assert the initial arrival point is at the inner surface of the temporal lobe (the uncus) of the cerebral cortex. Still others suggest paths centering on the thalamic reticular nucleus (TRN), the nominal arrival point for all sensory modality signals after initially passing via the brainstem (in the general region of the pons). The major path of olfactory signals appears associated with the fifth maxillary nerve as illustrated by Pansky (1988) in Section 8.6. Noback has provided several artistic representations of the signaling paths of the olfactory modality91. His text makes it clear that several questions remained open concerning the signaling paths. Noback clearly differentiates graphically between the fifth nerve and the olfactory tract (page 5). The uncus, TRN, thalamus and other proposed structures based on morphology are very close together in most species. Noback shows paths potentially proceeding to each of these bodies (pages 222-225). It requires additional physiological research to define the precise functional areas associated with these signal paths. Better understanding of the functional relationships should lead to more clarity in the morphological/histological understanding as well.

Figure 8.4.3-1 Top level architecture of mammalian olfactory modality ADD. The block diagram follows the architecture of other sensory modalities. It does exhibit a dual input structure, main and vomeral channels, similar to the visual modality.

Noback has also highlighted the bi-olfactory circuitry connecting the two main olfactory bulbs. These connections imply at least a crude directional sensing capability associated with the olfactory modality

91Noback, C. (1967) The Human Nervous System. NY: McGraw-Hill Chapters 1 & 15 48 Neurons & the Nervous System in many mammals. The olfactory modality plays a more important role in the limbic system than other sensory modalities. Thus, it contribution to both the emotional as well as the cognitive activities of the brain must be considered. As a result the outputs of both the limbic and cerebral subsystems of the CNS are extensive. At the most basic level, the motor outputs related to respiration (supporting sniffing) and to pointing (orientation relative to the surroundings) are clearly important. The other outputs related to olfaction are more diffuse, but no less important. [xxx Add words ] 8.4.3.2 Expanded architecture of the olfactory modality

Figure 8.4.3-2, from Graziadei, provides a comprehensive block diagram of the olfactory modality noting the three distinct function sensory structures, the olfactory epithelium, the septal organ and the vomeronasal organ. while discussing the developmental aspects of this modality92. He has described this as a simplified scheme of connections. A number of his functional elements have no orthodromic path to the primary CNS elements. Two points are of interest when he shows the “pyriform cortex” passing its output primarily to the mediodorsal thalamus on its way to the neocortex. First, the pyriform cortex is normally considered part of the neocortex. His reference to the neocortex may more properly be labeled prefrontal cortex. Second, the mediodorsal thalamus probably represents the TRN surrounding the thalamus acting in its role of a supervisor and routing control center. This discussion will suggest the primary functional path is from the olfactory epithelium through the olfactory bulb, to the pyriform cortex via the TRN and to the prefrontal cortex via the TRN again. It will suggest the vomeronasal organ supports a separate path involving less computational capability via the accessory olfactory bulb, the corticomedial amygdala, and the hypothalamus to the prefrontal cortex.

Many of the locations mentioned above fall outside the annotation provided by Brodmann. The septum is in area 25 and the pyriform cortex is generally in area 28. Note the septum (aka septal area or septal region) and the septal organ are separate entities in Graziadei. Most of the elements, other than the olfactory bulbs and sensory organs, are found within the limbic system.

Note the lack of a direct path from the olfactory bulbs to the thalamus or TRN. Merging of sensory data in a first pass through the TRN, before stage 4 information extraction, is a primary responsibility of the TRN. The olfactory signals do not contain spatial information about the external environment. Therefore, the stage 2 signals need not pass through the TRN on the way to stage 4 signal manipulation. Following stage 4 information extraction, they do pass through the TRN and medio-dorsal thalamus before insertion into the saliency map and cognition by the pre-frontal lobes of the neocortex.

92Graziadei, P. (1990) Olfactory development In Coleman, J. ed. Development of Sensory Systems in Mammals. NY: John Wiley & Sons Chapter 13 Signal Generation & Processing 8- 49

Figure 8.4.3-2 Schematic of the mammalian olfactory modality shown with incomplete paths. Graziadei describes it as highly simplified. This text offers additional comments on this figure. From Graziadei, 1990. 50 Neurons & the Nervous System

Figure 8.4.3-3 from Pansky shows the peripheral neural circuitry of the human at a coarse level. [xxx both olfactory and gustatory or what??? Drop this figure ]

Figure 8.4.3-3 Gross neural circuitry of the olfactory and gustatory systems REVIEW. The graphic appears incomplete and the symbology of the neurons appears inconsistent and the arrows should be relied upon. From Pansky, 1988.

Pansky notes “the area of the olfactory receptor cells in man are located in a small 2.5 sq. cm patch high up in each nostril. The total number of receptor cells is about 100 million in man. Using these coarse numbers, the diameter of the olfactory receptor neurons is approximately the same diameter as the photoreceptors. Each cell is embedded in a matrix of supporting (sustentacular) cells (page 279). The neurite portions of these receptor cells are embedded in the odor-absorbing secretion coating the mucosa.”

Dodd & Squirrell (1980) provided Figure 8.4.3-4 showing the variations in olfactory system among different vertebrates. The difference between the olfactory sensitivity of dogs (German shepherd) and humans is clearly due to the difference in surface area of the olfactory epithelia, not the sensitivity of individual chemoreceptors. Signal Generation & Processing 8- 51

Figure 8.4.3-4 Neuron density in olfactory epithelia of selected species. From Dodd & Squirrell, 1988.

The axons of the receptor cells are unmyelinated and very fine. Noback provides a summary of the peripheral neural system in rabbit. “There are approximately 50,000,000 receptor cells per nostril, and 45,000 mitral cells, 150,000 tufted cells, and 2,000 glomeruli in each olfactory bulb.” Mori et al. give the number of glomeruli as 3,000. In the rabbit, up to 25,000 axons converge on each glomeruli, the major stage 2 signal processing centers. A single glomeruli typically contacts about 24 of the 48000 mitral cells. The other neurons mentioned are found near the glomeruli in a structure reminiscent of the retina of vision. The axons do not branch until they enter the glomeruli of the olfactory bulb. Each axon makes 15 to 40 synapses with the orthodromic neurons within the olfactory bulb. As noted below, the mitral neurons are the stage 3 ganglion cells that project phasic signals to the CNS. 52 Neurons & the Nervous System

Dodd & Squirrell (1980) have stressed the high level of convergence leading to the glomeruli. Figure 8.4.3-5 suggests the complex structure of the neuritic tree(s) of a neuron required to achieve such high degree of convergence. The labeling has been modified to suggest the basal neuritic tree is poditic in order to provide a differencing capability within the input structure of this neuron. This figure from Cajal will be discussed further in Section XXX.

8.4.3.2.1 The vomeronasal organ and auxiliary olfactory bulb

Like the vision and hearing modalities, the olfaction modality has a specialized high performance sensing and signal processing channel (although it may be vestigial in humans93). The vomeronasal organ (VNO) is analogous to the foveola in vision. The VNO is also known as Jacobson’s Organ. The literature suggests the VNO forms in the early human fetus and then regresses in the neonate94. The VNO is a specialized olfactory epithelium, with a specialized stage 2 signal processor in the auxiliary olfactory bulb (AOB), and a distinct stage 4 signal processing area (the xxx ).

A problem in interpreting the human VNO is the fact the major cranial nerves were defined before the role of the VNO emerged. Schwanzel-Fukuda & Pfaff have noted the nerve associated with the VNO is now called the terminal nerve or zeroeth cranial nerve95.

The literature suggests the SSC’s of the VNO Figure 8.4.3-5 A Purkinje cell of the cerebellum. may be very specific to certain protein The dendritic tree is shown emanating from the stimulants. They may allow the identification apex of the cell. The (proposed) poditic tree is shown of specific members of a species via proteins in emanating from the basal region. The complexity of its urine. the cell is compatible with a very high order of signal convergence. Reproduced from Cajal (1899) McLean & Shipley have described the AOB by Dayan & Abbott, 2001. and compared the neural schematics leading from the MOB and the AOB to the CNS96. They note the AOB appears to project neurons directly to the amygdala. 8.4.3.2.2 Afferent neural paths to the main olfactory bulb

93Smith, T. Mooney, M. Burrows, A. et al. (2001) Prenatal growth and adult size of the vomeronasal organ in mouse lemurs and humans In Marchlewska-Koj, A. Lepri, J. & Muller-Schwarze, D. eds. (2001) Chemical Signals in Vertebrates 9. NY: Kluwer Academic pp 93-99

94Schmidt, H. & Beauchamp, G. (1992) Human olfaction in infancy and early childhood In Serby, M. & Chobor, K. eds. Science of Olfaction. NY: Springer-Verlag pg 380

95Schwanzel-Fukuda, M. & Pfaff, D. (2003) The structure and function of the nervus terminalis In Doty, R. ed. Handbook of Olfaction and Gustation. NY: Marcel Dekker Chapter 48

96McLean, J. & Shipley, M. (1992) Neuroanatomical substrates of olfaction In Serby, M. & Chobor, K. eds. Science of Olfaction. NY: Springer-Verlag pp 126-171 Signal Generation & Processing 8- 53 McLean & Shipley have also described the afferent neurons projecting to the olfactory bulbs. Nickell & Shipley have discussed the commissural and centrifugal connections between the two main olfactory bulbs97. 8.4.3.2.3 Schematic of the human main olfactory bulb

Johnson has provided the best conceptual schematic of the peripheral elements of the olfactory modality in humans (omitting any auxiliary olfactory bulb) in Figure 8.4.3-6. The figure is similar to a simpler ones in Pansky and in Imamua et al. showing only summation within the glomeruli. Note, there is no correlation between the glomeruli labeled A through D and the groups of ORN, labeled A through D, passing along an individual fascicle. The ORN within each group are drawn using different symbology to aid in tracing their axons to the apical dendritic trees within a given glomeruli. The poditic (basal dendritic) trees are shown synapsing only with the axons of granule cells. Shepherd provided a similar conceptual schematic in 2004 (page 167) Yokoi et al. have provided a simpler, yet still conceptual, schematic of the ORN and glomeruli that has been reproduced widely in introductory texts98. It is characterized by bidirectional flow at the synapses of the basal dendritic trees of the mitral/tufted cells with granule cells. Such signaling is not supported in this work.

97Nickell, W. & Shipley, M. (1992) Neurophysiology of the olfactory bulb In Serby, M. & Chobor, K. eds. Science of Olfaction. NY: Springer-Verlag Chap 6. pg 196

98Yokoi, M. Mori, K. & Nakanishi, S. (1995) refinement of odor molecule tuning by dendrodentritic synaptic inhibition in the olfactory bulb PNAS USA vol 92, pp 3371-3375 54 Neurons & the Nervous System

Figure 8.4.3-6 Neuronal schematic of the main olfactory bulb. The organization closely follows that of the visual retina. Four glomeruli are shown, each receiving three of the approximately 25,000 chemoreceptors that would converge on it. Two types of stage 3 neurons, mitral cells (mc) and tufted cells (tc) have their apical dendrites in the glomeruli, about 100 per glomerulus. Basal dendrites of these cells are located in deeper layers of the olfactory bulb. Their axons project to the brain along the olfactory tract. The periglomerular cells (pgc) of the external plexiform layer forms reciprocal synapses with a given glomerulus and spreads lateral inhibition to surrounding glomeruli. The granule cells (gc) form reciprocal synapses with the basal dendrites of the stage 3 neurons. From Johnson, 1988. Signal Generation & Processing 8- 55 There is very little evidence in the literature suggesting differencing by the periglomeruli cells. The mitral and tufted cells correspond to the ganglion cells of the retina and most other peripheral neural tissue. In fact, Gershon labeled the type 1 ganglion neurons of the enteric system mitral neurons99. Griff et al have expanded on the similarity of mitral and tufted neurons100, “Mitral cells differ from tufted cells by the location of their cell bodies, the distribution of their lateral dendrites, and their cortical and subcortical projections.” Though these anatomical differences are well known, physiological differences between these neurons are less well understood.” They are clearly similar cells with only subtle differences in functional assignments. Yokoi et al. and Wilson101 group these two types in their discussions as mitral/tufted cells. Nagayama et al. explored the operational differences between these two cell types102. Xxx was it significant? Note most investigators combine mitral/tufted category. ] See Section 8.9.2 for the topography of enteric system neurons.

8.4.3.3 Top level architecture of the gustatory modality

Spector & Travers have developed a top level model of the gustatory system that is a simplified version of that being presented here103. They note, “This neural coding process involves at least three stages. They define a first stage that is conceptually the same as stage 1 of this work, a “stimulus interacts with the receptors and triggers related transduction events in taste bud cells.” Not mentioned explicitly in their formulation is the signal processing achieved in the peripheral gustatory system, stage 2 of this work, before forwarding the signals to the central nervous system. Their second stage corresponds to stage 3 of this work. It “involves the activation of afferent fibers that transmit the peripheral signal to the brain.” Their third stage conceptualizes the combined stages 4 & 5 of this work. “It represents the processing of the peripheral signal that occurs in the central nervous system (CNS).” This work separates this processing into the information extraction function in stage 4 and the cognition related to processing that information in a more global context combining all sensory modalities in stage 5. Figure 8.4.3-7 has been expanded from that of Spector & Travers to incorporate these additional features. It is likely that the signals ascending from the parabrachial nucleus pass through the control function of the thalamic reticular nucleus on the way to both the VPN and the amygdala/hypothalamus. Squire et al. (2003, pg 635) has described the projection of the gustatory signals to the CNS, including projections to elements of the thalamus. The means by which signals from the limbic system are passed to the prefrontal cortex remains unclear. However, Spector & Travers do suggest signals from the gustatory neurons of primates are recorded from the orbitofrontal cortex area of what this work calls the prefrontal cortex.

The Spector & Travers paper is comprehensive but contains a wide range of puzzles and paradoxes. These challenges will be discussed below as appropriate. They use the term tuning in a very broad manner to describe the selectivity of sensory neurons to inadequately described classes of stimuli. The stimuli were the conventional; sucrose, NaCl, HCl and quinine-HCl. The stereochemical and quantum-mechanical properties of these substances was not developed.

99Gershon, M. (1981) The enteric nervous system Ann Rev Neurosci vol 4, pp 227-272

100Griff, E. Mafhouz, M. Perrut, A. & Chaput, M. (2008) Comparison of identified mitral and tufted cells in freely breathing rats: I. Conduction Velocity and Spontaneous Activity Chem Senses vol 33, pp 779-792

101Wilson, D. (2001) Receptive fields in the rat piriform cortex Chem Senses vol 26, pp 577-584

102Nagayama, S, Takahashi YK, Yoshihara Y, Mori K. (2004) Mitral and tufted cells differ in the decoding manner of odor maps in the rat olfactory bulb. J Neurophysiol vol 91, pp 2532–2540

103Spector, A. & Travers, S. (2005) The representation of taste quality in the mammalian nervous system Behav Cogn Neurosci Rev vol 4(3), pp143-191 56 Neurons & the Nervous System

Figure 8.4.3-7 Top level architecture of the gustatory modality of the rodent. Only ascending pathways are shown. CT(VII); chorda tympani branch of the facial nerve. GSP(VII); greater superficial branch of the facial nerve. LT(IX); lingual-tonsillar branch of the golssopharyngeal nerve. SLN(X); superior laryngeal branch of the vagus nerve. The mechanism for collecting information from the limbic system on the left for presentation to stage 5 is unclear. See text. Modified from Spector & Travers, 2005. Signal Generation & Processing 8- 57 Noback has presented an alternate, and less complete graphical description of the gustatory modality in Figure 8.4.3-8. It shows a clear path from the peripheral neural elements of the gustatory system to the thalamus and on to the medial surface of the parietal lobe of the cerebral cortex.

Figure 8.4.3-8 ADD The taste (gustatory) pathway. From Noback, 1967.

8.4.3.4 The path of stimulants to the external chemoreceptors

The gross path of stimulants to the external chemoreceptors is the same. The stimulant is brought into contact with the fluid environment surrounding the microtubules of the outer segment of the neuron. At the detail level, the paths for the olfactory and gustatory receptors are different. The chemical content of the mucus and saliva are fundamentally different. The saliva is charged with beginning the digestion process and it includes a variety of enzymes not relevant to gustation. Whereas the mucus of the olfactory surface is nominally continuous and surrounding all of the chemoreceptors, the saliva transports the stimulants to semi-enclosed taste buds that tend to isolate 58 Neurons & the Nervous System the (potentially different) fluid environments of different types of chemoreceptors. Johnson has provided the concentration of the major electrolytes of saliva (page 446-449) as a function of flow. Figure 8.4.3-9 shows the artificial saliva used by Hellekant.

In either the gustative or olfactory situation, the stimulant is required to diffuse from the air/fluid environment through the fluid, to the exposed elements of the chemoreceptors. The diffusion rates through the fluid environment may be significant to the concentration of the stimulant at the chemorecptors as a function of time. Figure 8.4.3-9 Artificial saliva used by Hellekant et al., 1997. Bradley & Beidler have listed all of the “major” proteins and peptides (60 out of at least 200) they have found in the saliva of the human and the mouse104. They did not rank these materials by importance or function. 8.4.3.5 Common elements of the chemoreception modalities

Figure 8.4.3-10 presents a composite dendrogram of the chemoreception modalities. It is drawn to highlight the fact that it is the combination of the sensations of taste and smell, as well as other modalities that combine to create a flavor experience. It also highlights the likelihood that the same acidophore receptors are probably used in both the gustatory and olfactory modalities. The diagram emphasizes the presence of significant stage 2 signal processing in the olfactory modality signal paths but virtually none in the gustatory paths. [Incorporate features from the draft figure dendrogram proposal.ai ]

104Bradley, R. & Beidler, L. (2003) Saliva: its role in taste function In Doty, R. ed. Handbook of Olfaction and Gustation. NY: Marcel Dekker pg 642 Signal Generation & Processing 8- 59 Besides sharing the organic acid receptors, the two modalites employ essentially identical sensory neurons, varying only in the specific molecule used to form the receptor of transduction (Sections 8.xxx & 8.xxx). These molecules are used to interface with the gustophores and olfactophores appropriate to the individual sensory channels illustrated. Most of the groups designated on the right consist of large numbers of molecules providing different gustatory and olfactory stimulants. Many stimulants incorporate more than one odorophore or gustophore. The generator waveforms of the sensory neurons exhibit different time constants due to the properties of the neural matrices in which they reside. This theory and the available data confirm there are four independent sensory channels in the gustatory modality. Signals from these channels encounter little signal processing until they reach stage 4. The olfactory data base is inadequate to determine the precise number of sensory channels in olfaction, however the number is surely less than nine (as opposed to some geneticist claiming nearly one thousand). The geneticists have based their assertion on a total gene count where the purpose of the individual genes is largely unknown. Figure 8.4.3-10 A proposed dendrogram of the A few chemicals that have previously been chemoreceptors from the receptor perspective. The considered odorants (the inorganic acids in gustatory modality can be described in a three- particular) are more properly described as dimensional space and its stage 2 is rudimentary. nociophores (irritants to the nocioreceptors). Olfaction involves an n-dimensional space and the glomeruli play a major role in stage 2 signal Laffort has addressed the subject of protein processing. based receptors as recently as 1994. “Unfortunately, all attempts carried out to date have failed, at least as far as the isolation and identification of highly specific proteins are concerned105.”

8.4.3.5.1 Binding sites on chemoreceptor membranes

Cuatrecasas and Hollenberg have provide the definition of a binding site (quoted in Brown et al., 1977) 106, “By definition, a binding site on a membrane surface can be called a "receptor" only if it exhibits the following seven characteristics. The binding of the small molecule (i.e., ligand) to the putative receptor must 1. exhibit steric specificity,

105Laffort, P. (1994) Relationships between molecular structure and olfactory activity In Odors and Deodorization in the Environment; Martin, G., Laffort, P., Eds. NY: VCH Publishers pg 163

106Cuatrecasas, P. & Hollenberg, M. (1976) Membrane receptors and hormone action. in Anfinsen, C. Edsall, J. & Richards, F. eds. Advances in Protein Chemistry, NY: Academic Press pp 251-451 60 Neurons & the Nervous System

2. pharmacological specificity, 3.. and target-cell specificity. The ligand 4. must bind to the putative receptor when the ligand is present at physiological concentration, 5. and it must be possible to saturate the available receptors on the membrane. 6. The binding must be reversible. 7. The most crucial test is that the interaction of the ligand with the receptor must elicit a physiological response.

Pogni et al. have also provided some background material and parametric data107 They obtained the EPR spectra of several copper complexes including those formed with histidine and glycine. They explored di-tri- and tetra-peptides of these materials with the different amino acids in different relative positions. 8.4.3.5.2 Transduction response of chemoreception

The sensory neurons of the chemoreceptor modalities (in both mammals, reptiles and insects) show the same transduction characteristic as do the quantum-mechanical sensory neurons of the visual and auditory modalities. This characteristic is shown in Figure 8.4.3-11. Boecke et al. made special efforts to use a continuously flowing air stream and probe the plasma of the sensory neurons of the 108 rabbit . The waveforms all exhibit a finite time delay (tDA) associated with the attack portion of the waveform. The waveform exhibits a first order departure from the baseline when recorded using a well balanced oscilloscope probe of sufficient bandwidth. The shape of the waveform is critically dependent on the product of the intensity of the stimulus and the capture cross section of the individual sensory neurons. At high values of this product, the waveform exhibits an “overshoot” that is fundamental to the mechanism. Note the time delay (tDD) associated with the decay characteristic after cessation of the stimulus. The value of tDD is determined by the response level at termination of the stimulus. The decay characteristic following the decay delay is a first order decay with a time constant, τD. This time constant is intrinsic to the transduction process of the sensory neuron. It is typically a few tenths of a second.

Laboratory procedures frequently involve slow and poorly timed application of various stimulants to the chemical receptors, resulting in loss of detail concerning the intrinsic response of the sensory receptor neurons. 8.4.4 Global aspects of potential stimulant sensing chemistry

Beginning early in the 20th Century, investigators assumed the SSC’s of olfaction had to be protein based. Although this position has yet to be confirmed, the assumption is taught as fact to this day. Cohn Figure 8.4.3-11 The observed transduction and Edsall edited a major volume in 1943 characteristic of the chemo-sensory neurons. Upper describing the state of the art related to waveform, transduction mechanism response. Lower waveform; timeline of stimulus application. Note rise and fall times of stimulus application are shorter than the time delays of the response.. See text. From Boeckh et al., 1963.

107Pogni, R. Baratto, M. Busi, E. & Basosi, R. (1999) EPR and O2A — scavenger activity: Cu(II)—peptide complexes as superoxide dismutase models J Inorg Biochem vol 73(3), pp 157-165

108Boecke, XXX from ottoson in Beidler, pg 107 Signal Generation & Processing 8- 61 proteins, peptides and amino acids109. It remains a valuable compendium to this day. After approximately 100 years of research, Ko and Park noted the following concerning the role of proteins in olfaction, “The recent progress in a study on the olfactory receptor genes is expected to allow for the identification of the unknown function of the receptor proteins in the near future110.” Infrequently, investigators have asserted the SSC’s could be lipids. However, this assertion has never become a viable option. It is possible the field of chemistry has not advanced sufficiently to understand the chemistry of the SSC’s. The field of coordinate chemistry (involving a metallic ion bonded to a non-carbon atom of an organic ligands) and the closely related organometallic chemistry (involving a metallic ion bonded to a carbon atom) are now maturing. They may hold the answer to the SSC problem. - - - - This work will show that the transduction involved in chemoreception relies upon coordination chemistry (or coordination complex chemistry) involving the phospholipids of specialized regions of the sensory neuron lemma. While coordination chemistry plays a critical role in many stable compounds required by life (hemoglobin as an example), it plays a similar role in olfaction and gustation involving short lifetime coordination complexes. In his preamble, Lawrance notes111, “What we can conclude is that metal coordination chemistry is a demanding field that will tax your skills as a scientist. Carbon chemistry is, by contrast, comparatively simple, in the sense that essentially all stable carbon compounds have four bonds around each carbon center. Metals, as a group, can exhibit coordination numbers from two to fourteen, and formal oxidation states that range from negative values to as high as eight.”

The coordination chemistries of the metalloproteins, metalloenzymes are well explored at this time, The coordination chemistry of the metallophospholipids is basically unexplored, particularly for the lighter metals of groups 1s and 2s. Most texts do not address the complexes of sodium and calcium. In the 2004 (2nd ) edition of Comprehensive Coordination Chemistry112, volume 3 contained a chapter on the coordination chemistry of the 1s and 2s elements that hardly addressed sodium and calcium.

- - - -

- - - -

Oncley provided a comprehensive, but very early, discussion of the electric moment and relaxation times associated with many proteins, peptides and amino acids that may play a role in olfaction113.

As noted below, many of the more complex biological molecules, such as hemoglobin, are best described in the coordinate chemistry context.

This section will develop the possibility that the SSC’s are metallic ions bonded to simple peptides instead of more complex proteins. Lehninger notes the reaction of CuSO4 with either a peptide or a protein gives a purple complex of Cu2+ and the peptide, which can be measured quantitatively in a

109Cohn, E. Edsall, J. eds. (1943) Proteins, Amino Acids and Peptides. NY: Reinhold

110Ko, H. & Park, T. (2006) Dual signal transduction mediated by a single type of olfactory receptor expressed in a heterologous system Biol Chem vol 387, pp. 59–68

111Lawrance, G. (2010) Introduction to Coordination Chemistry. NY: Wiley

112Parkin, G. vol ed. (2004) Comprehensive Coordination Chemstry II, Vol 3: Coord. Chem. of the s, p and f Metals. NY: Elsevier

113Oncley, J. (1943) The electric moments and the relaxation times of proteins as measured from their influence upon the dielectric constants of solutions In Cohn, E. Edsall, J. eds. (1943) Proteins, Amino Acids and Peptides. NY: Reinhold Chapter 22 62 Neurons & the Nervous System spectrophotometer. Others have noted, as described below, the spontaneous assembly of many zinc- based metal complexes. The mucosa of animals is frequently noted for its distinct color. This color is suggestive of the presence of a Cu2+ or other transition element complex with a peptide. A peptide is a minimum molecular weight protein. 8.4.4.1 Proteins as potential SSC’s in olfaction

The mention of protein to a biochemist usually invokes the image of large complex molecules of amino acids. Proteins are conventionally associated with the following tasks within an animal organism; 1. as structural elements, 2. as muscle tissue, 3. as chemical transporters, 4. as enzymes, 5. as hormones, and related generally to chemical decomposition (both digestion and phagocytosis) and synthesis within the organism. While their role in sensory neural transduction has been conceptualized generally, such roles have not been previously defined at the detailed cytological and specific reaction levels. Such detailing is a major goal of this volume. Similarly, their role in neural system signaling and function has been described largely conceptually. Many of the commonly accepted roles of proteins within the neural system have never been demonstrated in the laboratory. Discussions relating to this situation will occur frequently throughout this volume.

The generation of large molecular weight proteins in accordance with the DNA code by mitochondria within a cell is generally considered the normal mode of protein production. Proteins are usually segregated into simple (containing only amino acid ligands) and conjugated proteins (those containing non-amino acid ligands). There are also small molecules of simple proteins consisting of only amino acids, such as the peptides. They typically contain less than a dozen amino acid units. Some peptides, such as the dipeptide carnosine (mol wt. 226), are not derived from proteins encoded by DNA. They are primarily ingested (or assembled from amino acids ingested) in food. Many other peptides do not derive from DNA encoded protein structures but are important biologically114. The proteins and peptides as a group do not have the low excitation energy associated with taste and smell. However, more specific complex molecules incorporating these peptides may. The C–N bond of the peptide linkage cannot rotate freely, a property of supreme importance with respect to the three-dimensional conformation of polypeptide chains. This rigidity assures a higher degree of specificity in steric unions.

Isolating individual proteins from a neurological environment is a technical challenge because of the variety of proteins in that environment. If a protein of interest is present in an organelle, it is typically best to isolate the organelle in quantity before attempting to isolate the protein.

[xxx edit ] It appears a family of small chelating peptides, including carnosine and its N-methylated derivative, anserine (β alanyl-N-methyl-L-histidine, mol wt. = 240), may be part of the molecules acting as SSC’s in olfaction. The larger family may include a variety of histidine derivatives. They appear to coordinate with the transition group metals, Cu, Zn, etc. easily. The combined molecules offer many unique properties. Price has discussed “Olfactory Receptor Proteins” as SSC’s without demonstrating the proposition115. His subtitle is more interesting, “Are olfactory receptor sites proteinaceous? Section xxx presents a plausible demonstration that the olfactory receptor sites are peptide-aceous but not necessarily

114Lehninger, A. (1970) Biochemistry. NY: Worth pg 89-91 and Chap 70

115Price, S. (1981) Receptor proteins in vertebrate olfaction In Cagan, R. & Kare, M. ed. Biochemistry of taste and olfaction. NY: Academic Press pp 70-72 Signal Generation & Processing 8- 63 proteinaceous.

8.4.4.1.1 The role of histidine-containing dipeptides

A family of chemicals, based on the positively charge amino acid, histidine, were studied extensively in the last quarter of the 20th Century. The results of investigations of carnosine and anserine were predominantly negative with respect to the role of these chemicals in chemical sensing by the neural system. However, the study of the coordinate chemistry of these materials provides a good foundation for understanding how the actual sensory receptors of chemical sensing actually operate. Trombley et al. observed, “In spite of the supportive evidence, the lack of a direct effect of carnosine on the afferent targets of carnosine-containing olfactory sensory neurons casts doubt on its status as a neurotransmitter. Discussions continued in 2000 concerning where carnosine is formed and released in the olfactory mucosa. Bakardjiev & Bauer note “Rats produce carnosine after stripping the neurons from the flat cell layer. The data suggest that this type of cell but not olfactory neurons synthesize carnosine116.” They later conclude, “A century after the isolation of carnosine we have learned many details about carnosine and related dipeptides. Nevertheless, the biological functions of these peptides in different tissues remain obscure, and a unifying concept to answer this question has not yet emerged.” De Marchis et al. have surveyed the role of carnosine in the cells of the neural system. Like others in 2000, De Marchis et al. conclude117, “In the last decade, several studies have been performed to understand the biological functions played by carnosine-related dipeptides in the nervous system. Although many theories have been proposed about a wide range of properties that enable them to act as antioxidants, metal chelators, free radical scavengers, inhibitors of protein glycosylation, and neuromodulators, their precise physiological role remains unknown.” Ronnett & Moon stated in 2002118, “Thus, in the absence of compelling functional studies the sum of these data does not support the role of carnosine as the neurotransmitter of the olfactory receptor neurons.”

Additional information on these and other studies of carnosine and anserine have been accumulated in Appendix N.

- - - - 8.4.4.2 Lipids as potential SSC’s in olfaction

The family of phospholipids, found in the bilayer walls of neurons, is gigantic. Weissmann & Claiborne provided a “bible” on this subject in 1975119. Even this work does not go far enough to describe all of the features of the family.

Figure 8.4.4-1 describes the basic features of the phospholipids of interest. The family is based on the triglyceryl framework of (a). The polar phosphate group of (b), along with two fatty acid groups, are added to form the generic arrangement in ( c). The basic phospholipid is shown in more detail on the right. The two fatty acid chains need not be of the same length, nor need they both be saturated as shown by Chapman (Weissmann & Claiborne, page 22).

116Bakardjiev, A. & Bauer, K. (2000) Biosynthesis, release, and uptake of carnosine in primary cultures Biochem (Moscow) vol 65(7), pp 779-782

117De Marchis, S. Modenal, C. Peretto, P. Migheli, A. Margois, F. & Fasolo, A. Carnosine-related dipeptides in neurons and glia Biochem (Moscow) vol 65(7), pp 824-833

118Ronnett, G. & Moon, C. (2002) G proteins and olfactory signal transduction Ann Rev Physiol vol 64, pp 189–222

119Weissmann, G. & Claiborne, R. eds. (1975) Cell Membranes: Biochemistry, Cell Biology & Pathology. NY: HP Publishing 64 Neurons & the Nervous System

Figure 8.4.4-1 Basic structural features of the phospholipids. See text. From Weissmann & Claiborne, 1975.

A more complex level of non-saturation in a phospholipid is a “conjugated fatty acid” forming one leg of the two chains. This is a relatively rare form not recognized in many texts (Lehninger 1970, page 190). Cook has provided a long list of conjugated fatty acids along with a set of rules for describing Signal Generation & Processing 8- 65 them120. When found in a phospholipid, this feature contributes electrical conductivity along the length of the chain. Zhou et al. found increases of 3-4 orders of magnitude121. As a result, when used in a neuron, this class of phospholipids exhibits a polar head attached to a conductive leg protruding through the bilayer of the membrane. The dipolar moment of the polar head is effectively extended (without increasing the lever arm of the moment) to the electrolytic environments on each side of the bilayer. The precise geometric form of the conjugated fatty acids depend on their cis and trans configurations. Figure 8.4.4-2, reproducing the Chapman figure, illustrates how multiple phospholipid molecules can exist adjacent to each other in a very small region of a single bilayer of a lemma. The kinks in the fatty acids are indicative of cis– double bonds along the chain. This is common in actual practice.

120Cook, H. (1991) Fatty acid desaturation and chain elongation in eucaryotes In Vance, D. & Vance, J. eds. Biochemistry of Lipids, Lipoproteins and Membranes. 4th Ed. NY: Elsevier Chapter 5

121Zhou, Q. Yang, Y. & Sun, A. (1998) Effects of molecular structures on the olfactory responses of phospholipid membranes to four alcohols Chem Physics Lipids vol 95, pp 1–9 66 Neurons & the Nervous System

Figure 8.4.4-2 Drawing showing the complexity of a real lemma. The size shown is about 30 x 30 Angstrom. It contains six cholesterol molecules, five phospholipid molecules of three different types, and four sphingolipid molecules of two different types. From Chapman, 1975.

If the dipolar moment of the polar group was to change, due to a modification of the polar group, such as through a coordinate chemical arrangement with another substance in solution, the electrical potential at the other end of the phospholipid would change relative to the electrolytic environment of the polar group. This change can be sensed by the appropriate electrolytic amplifier. Bangham shows a similar situation (Weissmann & Claiborne, page 30) using a reconstituted “black lipid membrane” and involving a change of over 100 mV in potential. A similar response is shown for a portion of neural tissue from a frog (demonstrating its electrolytic conductivity). Signal Generation & Processing 8- 67 In an excellent review, Bangham noted122, “The tight and spatially oriented packing of phospholipids as a smectic mesophase or black lipid membrane gives rise to a plane or planes of fixed ions at a (two-dimensional) concentration equivalent to a bulk uni-univalent concentration of 5-10 mole/liter!” His earlier 1968 review contains a wealth of valuable information about phospholipid bilayer membranes and their electrical interaction with sodium in aqueous fluids123. 8.4.4.2.1 Dipole potential and moment of phospholipids in lemma

The dipole moments associated with the various polar groups in phospholipids have been indicated in figure 10-3 of Lehninger (1970). However, no precise potentials or dipole moments are given. Sherer & Seelig give a value of 19 Debye for the dipole moment of phosphocholine. Klymchenko et al. have noted the absolute dipole potential for phosphatidyl choline bilayers are large relative to the input operating range of the common emitter circuit of the Activa developed here (~20 mV)124. They suggest it is difficult to measure these with precision but values from Ψd = –280 to –500 mV appear in their work. Thus, only a small change in the dipole potential would be needed as a result of a coordinate chemistry arrangement to be sensed by the neural system. See Section 8.5.5.1.1. While dipole potentials are difficult to measure for molecules in solution, because the molecules are free to rotate randomly in the absence of an applied field, this is not the case for the phospholipids when in liquid crystalline form in a monolayer. In this case, the dipole potential is easily measured. However the presence of the phospholipid in a bilayer complicates the measurement procedurally.

Petrov & Sachs addressed the subject of dipoles in phospholipids from a mathematical perspective125. Their theme was, “In view of the well-established charge and dipolar asymmetry of the two leaflets of a native membrane, the theory of flexoelectricity (and curvature elasticity) is extended to take into account this asymmetry.” They defined the direction of the dipole moment incompletely as, “dipoles are regarded as positive if pointing inward toward the hydrophobic core.” It can be assumed they relied upon the common definition of a dipole moment, “ the electric dipole moment points from the negative charge towards the positive charge, and has a magnitude equal to the strength of each charge times the separation between the charges.”

The dipole potential is most easily measured using the Langmuir film technique–floating a monolayer film of the material on a (typically) distilled water surface and measuring the potential difference between its upper and lower surfaces. The thin film must constitute a continuous liquid crystalline monolayer to provide the desired potential.

The dipole potential of a molecule is the vector summation of the dipole potential of each of its constituent asymmetrical chemical ligands. When a chemical entity is coordinately bound to another chemical entity, their net dipole potential can be calculated from their individual values. It is proposed that the change in dipole potential between the unbound and bound states of the phospholipids forming the receptors constitutes the principle parameter sensed in the transduction process of the olfactory and gustatory sensory neurons. 8.4.4.3 Coordination chemistry fundamentals

Conventionally, coordination chemistry is the study of compounds formed between metal ions and other neutral or negatively charged molecules. It specifically exempts compounds where the metal

122Bangham, A. (1972) Lipid bilayers and biomembranes Annu Rev Biochem vol 41, pp 753-776

123Bangham, A. (1968) Membrane models with phospholipids Prog Biophys Mol Biol vol 18, pp 29-95

124Klymchenko, A. Duportail, G. Mely, Y. & Demchenko, A. (2003) Ultrasensitive two-color fluorescence probes from dipole potential in phospholipid membranes PNAS vol 100(2), pp 11219-11224

125Petrov, A. & Sachs, F. (2002) Flexoelectricity and elasticity of asymmetric biomembranes http://www.sachslab.buffalo.edu/pdf/asymm-flexo-final.pdf 68 Neurons & the Nervous System binds directly to carbon. That field is addressed in the next section. More important to this work is the coordinate chemistry of the hydrogen bond. The hydrogen bond is very weak and typically temporary, depending on the environment of the bonding. materials. The temporary nature of these bonds is particularly relevant to the study of olfaction and gustation. The field of coordination chemistry is vast and its details complicated. A broad synopsis of the field has been assembled in Appendix N of this work. Only a narrower synopsis is included in this section. The nomenclature of coordinate chemistry is necessarily complex. Lawrence has provided an introduction focused on the terminology involved126. A brief description of the IUPAC rules on nomenclature is provided on Wikipedia127. Basolo & Johnson have provided multiple pages of nomenclature related to these compounds128. They differentiate between the primary valence (or oxidation state) of a metal ion and its secondary valence (or coordination number). “Every element tends to satisfy both its primary and secondary valence.” The primary valence tends to be satisfied with ionic bonds to other atoms. The secondary valence is frequently satisfied by more complex organic molecules attached to the “coordination sphere” of the metal ion. They discuss hemoglobin as an example of a metal in biology as an introduction to the field of bioinorganic chemistry (page 131).

Geometry Structure in complexation chemistry is first described by its "coordination number", the number of ligands attached to the metal (more specifically, the number of s-type bonds between ligand(s) and the central atom). Usually one can count the ligands attached, but sometimes even the counting can become ambiguous. Coordination numbers are normally between two and nine, but large numbers of ligands are not uncommon for the lanthanides and actinides. The number of bonds depends on the size, charge, and electron configuration of the metal ion and the ligands. Metal ions may have more than one coordination number.

Typically the chemistry of complexes is dominated by interactions between s and p molecular orbitals of the ligands and the d orbitals of the metal ions. The s, p, and d orbitals of the metal can accommodate 18 electrons (see 18-Electron rule; for f-block elements, this extends to 32 electrons). The maximum coordination number for a certain metal is thus related to the electronic configuration of the metal ion (more specifically, the number of empty orbitals) and to the ratio of the size of the ligands and the metal ion. Large metals and small ligands lead to high coordination numbers, e.g. [Mo(CN)8]4-. Small metals with large ligands lead to low coordination numbers, e.g. Pt[P(CMe3)]2. Due to their large size, lanthanides, actinides, and early transition metals tend to have high coordination numbers.

Different ligand structural arrangements result from the coordination number. Most structures follow the points-on-a-sphere pattern (or, as if the central atom were in the middle of a polyhedron where the corners of that shape are the locations of the ligands), where orbital overlap (between ligand and metal orbitals) and ligand-ligand repulsions tend to lead to certain regular geometries.

Isomerism The arrangement of the ligands is fixed for a given complex, but in some cases it is mutable by a reaction that forms another stable isomer. There exist many kinds of isomerism in coordination complexes, just as in many other compounds. Geometric isomerism Geometric isomerism occurs in octahedral and square planar complexes (but not tetrahedral). When two ligands are opposite each other they are said to be trans, when mutually adjacent, cis. When three identical ligands occupy one face of an octahedron, the isomer is said to be facial, or fac. If these three ligands and the metal ion are coplanar, the isomer is said to be meridional, or mer. For example, in

126Lawrence, G. ( Introduction to Coordinate Chemistry. NY: Wiley

127- - - - (2011) Coordination geometry. http://en.wikipedia.org/widi/Coordination_geometry

128Basolo, F. & Johnson, R. (1986) Coordinate Chemistry. London: Science Reviews Signal Generation & Processing 8- 69 an octahedral compound with three of one ligand and three of another, there are two geometric isomers: the mer in which each set of three same ligands is in a meridian and the fac in which each set of three is on a face of the octahedron. Structural isomerism Structural isomerism occurs when the bonds are themselves different. Linkage isomerism is only one of several types of structural isomerism in coordination complexes (as well as other classes of chemical compounds). Linkage isomerism occurs with ambidentate ligands which can bind in more than one place. For example, NO2 is an ambidentate ligand: it can bind to a metal at either the N atom or at an O atom. Color Metal complexes often have spectacular colors. These colors are caused by electronic transitions caused by the absorption of light. Most transitions that are related to colored metal complexes are either d-d transitions or charge transfer bands. In a d-d transition, an electron in a d orbital on the metal is excited by a photon to another d orbital of higher energy. A charge transfer band entails promotion of electron from a metal-based orbital into an empty ligand-based orbital (Metal-to-Ligand Charge Transfer or MLCT). The converse also occurs: excitation of an electron in a ligand-based orbital into an empty metal-based orbital (Ligand to Metal Charge Transfer or LMCT). These phenomena can be observed with the aid of electronic spectroscopy; also known as UV-Vis.[1] For simple compounds with high symmetry, the d-d transitions can be assigned using Tanabe-Sugano diagrams. Increasingly, these assignments can be confirmed using computational chemistry.

Many organic coordination compounds occur naturally. For example, hemoglobin and myoglobin contain an iron center coordinated to the nitrogen atoms of a porphyrin ring; magnesium is the center of a chlorine ring in chlorophyll. The field of such inorganic compounds is known as bioinorganic chemistry.

Coordination number

There are many definitions of the term coordination number, but there is no one simple unambiguous definition that works in all cases. For simple monodentate and chelating ligands, the coordination number can be defined as the number of atoms or ligands directly bonded to the metal atom.

The total number of points of attachment to the central element is termed the coordination number and this can vary from 2 to as many as 16, but is usually 6. In simple terms, the coordination number of a complex is influenced by the relative sizes of the metal ion and the ligands and by electronic factors, such as charge which is dependent on the electronic configuration of the metal ion.

Coordination Number 4 Two different geometries are possible, as shown in Figure 8.4.4-3. The tetrahedron is the more common while the square planar is found almost exclusively with metal ions having a d8 electronic configuration.

Tetrahedral The chemistry of molecules centered around a tetrahedral C atom is covered in organic courses. Similar arrangements are found where the central atom is a metal. Replacing the carbon in a tetrahedron by a metal, such as cobalt, is quite common. There are large numbers of tetrahedral Cobalt(II) complexes known.

Square Planar Figure 8.4.4-3 Two forms of coordination number 4 This is fairly rare and is included only because chemistry. some extremely important molecules exist with this shape. Additional definitions related to coordination chemistry and organometallic chemistry are available at http://www.ilpi.com/organomet/coordnum.html . These may not be sanctioned by any organization. 70 Neurons & the Nervous System

Garnovskii & Kharisov have edited a book on the fundamentals of coordination and organometallic chemistry129. They discuss the methods of direct synthesis of these types of compounds. However, they do not investigate such chemistry involving amino acids or involving specific metallic ions. . 8.4.4.3.1 Coordinate chemistry of the transition elements

The coordinate chemistry of copper appears to be the most studied of the biologically active metal ions. The endogenous presence of trace amounts of both copper and zinc in the CNS and particularly in association with the olfactory neurons is well documented130,131. Spiro noted that measuring these trace elements in biological tissue has been extremely challenging until recently, even though among the transition elements, zinc is second only to iron in abundance (page 3). Spiro has edited several volumes on metal ions in biology including an entire volume on copper proteins132. The latter included detailed information and structural diagrams on a number of copper proteins. He notes, “Metal ions are essential to life as we know it. This fact has long been recognized, and the list of essential ‘trace elements’ has grown steadily over the years. . . Vast stretches of this terra incognita remain uncharted.” He develops the importance of electron transitions between the various d orbitals (page 34). This work has not found the role of the coordinate chemistry of the metals, other than the alkaline and alkaline earth metals, significant in the first order theory of chemical sensing. 8.4.4.3.2 Chelation and the formation of metal complexes

Chelation is the binding or complexation of a bi- or multidentate ligand. These ligands, which are often organic compounds, are called chelants, chelators, chelating agents, or sequestering agent. Chelating agents form multiple bonds with a single metal ion. Chelants, according to ASTM-A-380, are "chemicals that form soluble, complex molecules with certain metal ions, inactivating the ions so that they cannot normally react with other elements or ions to produce precipitates or scale." The ligand forms a chelate complex with the substrate. The term is reserved for complexes in which the metal ion is bound to two or more atoms of the chelant. Virtually all biochemicals exhibit the ability to dissolve certain metal cations. Thus, proteins, polysaccharides, and polynucleic acids are excellent polydentate ligands for many metal ions. 8.4.4.3.3 Organometallic chemistry fundamentals

Organometallic chemistry is the study of chemical compounds containing bonds between carbon and a metal. Since many compounds without such bonds are chemically similar, an alternative may be compounds containing metal-element bonds of a largely covalent character. Organometallic chemistry combines aspects of inorganic chemistry and organic chemistry.

Organometallic compounds are distinguished by the prefix "organo-" e.g. organopalladium compounds. Historically, organometallic compounds have been known as organo-inorganics, metallo-organics and metalorganics. The metal-carbon bond in organometallic compounds is generally of character intermediate between ionic and covalent. Primarily ionic metal-carbon bonds are encountered either when the metal is very electropositive (as in the case of the alkali metals) or when the carbon-containing ligand exists as a

129.Garnovskii, A. & Kharisov, B. eds. (2003) Synthetic Coordination and Organometallic Chemistry. NY: Marcel Dekker

130Trombley P. Horning, M. & Blakemore, L. (2000) Interactions between Carnosine and Zinc and Copper: Implications for Neuromodulation and Neuroprotection Biochemistry (Moscow) vol 65(7), pp. 807-816.

131Spiro, T. (1983) Zinc Enzymes. NY: John Wiley & Sons

132Spiro, T. (1981) Copper Proteins. NY: John Wiley & Sons Signal Generation & Processing 8- 71 stable carbanion. Organometallic compounds with bonds that have characters in between ionic and covalent are very important in industry, as they are both relatively stable in solutions and relatively ionic to undergo reactions. Two important classes are organolithium and Grignard reagents. In certain organometallic compounds such as ferrocene or dibenzenechromium, the pi orbitals of the organic moiety ligate the metal. Electron counting is key in understanding organometallic chemistry. The 18-electron rule is helpful in predicting the stabilities of organometallic compounds. Organometallic compounds which have 18 electrons (filled s, p, and penultimate d orbitals) are relatively stable. This suggests the compound is isolatable, but it can result in the compound being inert

8.4.5 Extending the lock & key concept of stereochemistry

The following material will extend the framework of coordinate chemistry to incorporate the AH,B concept of coordinate chemical bond pairs (generally associated with Shallenberger) and the extension of that concept to the AH,B,X, or three-point, concept (generally associated with Kier). The AH,B,X concept involves the original coordinate bond pair to hold an additional element of the stimulant in a specific physical position relative to the sensory receptor moiety, so that it impacts the overall electrical dipole potential of the receptor moiety. These concepts are readily described using the key and lock analogy of stereochemistry. In the AH,B concept, two parameters are critically important. First, the distance between the AH (typically an hydroxyl ligand, OH) and the B (typically an oxygen ligand) elements of the stimulant. Second the magnitude of the dipole potential of the stimulant molecule. The first parameter corresponds to the distance between the tumblers of the lock, or the notches on the key. The second corresponds to the depth of the notches in the key and the corresponding critical length of the tumbler in the lock. The first parameter is critically important in determining what stimulants will coordinate with sensory receptor molecule of a specific sensory channel, and the second parameter determines the strength of the signal generated in the selected signal receptor channel.

The extended AH,B,X concept is almost exactly analogous to the (anti-theft) lock and key system used on many modern automobiles. In this analog, the parameters analogous to the AH,B concept must be satisfied, but an additional parameter must be satisfied. An additional electrical potential of specified amplitude must be applied to an additional element of the lock, usually provided by a resistor mounted in or on the key. [xxx need a figure or reference to later figure ]

These same lock and key concepts are used in both gustation and olfaction. 8.4.7 A structural shorthand to describe odorophores and gustaphores

Janssen has used a shorthand to describe the structure of various pharmacological preparations in a SAR context, based on a simple model of a chemical structure where each pair of shared electrons is indicated by a line between the two atoms133. In his application, he sought to define more complex functional groups by alternate simplified designations. Thus, he defined the structure of a complex chemical containing Ar = benzene or an isosteric aromatic ring; N = basic nitrogen and C = carbon as; Ar–C==C–C–C–N–C Janssen also defined a moiety as AA’= isosteric aromatic fused ring structure (the rings share at least two carbons). In the following discussion, it will be useful to describe the gustophores and odorophores of a given substance using this notation. It will become clear that a typical gustadant and odorant incorporate multiple gustophores and odorophores each requiring a description of the defined form. More

133Janssen, P. (1973) Structure-activity relationships (SAR) and drug design as illustrated with neuroleptic agents In Cavallito, C. ed. International Encyclopedia of Pharmacology and Therapeutics, Section 5, Vol 1, Structure-Activity Relationships. NY: Pergamon Press Chapter 2 72 Neurons & the Nervous System importantly, an adequate description of a gustophore or odorophore requires specification of its conformation, not just its constitution. Both the bond lengths between atoms and other entities as well as the bond angles associated with adjacent bonds are required. Furthermore, the association of hydrogen with some of these structures is a critical feature. Describing the required features frequently requires a true three-dimensional approach that goes beyond the conventional xxx diagrams. The symbology will be extended beyond that of Janssen, along the lines of that associated with Shallenberger & Acree (Section 8.5.3) AH = a moiety capable of sharing additional pairs of electrons while closely associated with hydrogen. The AH moiety may be OH, NH, NH2 or even CH in halogenated compounds. B = a moiety capable of sharing additional pairs of electrons. The B moiety may be O, N, an unsaturated center, or even the π-bonding cloud of the benzene ring.

X = a moiety capable of influencing the electronic energy density in a region of a receptor when brought into close proximity to that region. Even this terminology will be extended to include sulfur (and rarely phosphorus) in these sets.

In many situations, it will be the distance in three-dimensional space, or the d-value in Angstrom, between the A of the AH and the B that is of primary interest in a particular instance. In this work, the d-value can be considered the valence of the odorophore/receptor interaction. This use of the term is feasible because the conventional usage describing the state of oxidation of an atom plays a negligible role in olfaction.

Figure 8.4.6-1 illustrates the expanded concept.

1. The distance between the left-most oxygen and the nominal center of the π-cloud associated with the aromatic ring is of interest, AH–C+Ar. Note the distance to the center of the ring is beyond the first carbon from the AH moiety and is accounted for in this notation by the plus symbol.. 2. The distance between the NH2 group and the center of the aromatic ring is of interest, AH–C–C–C+Ar. Note the distance to the center of the ring is beyond the third carbon from the AH moiety and is accounted for in this notation by the plus symbol. 3. The distance from the A of the AH of the carboxyl group, specifically the OH group, to the center of the aromatic ring is of interest, AH–C–C–C–C+Ar. 4. The distance from the A of the AH of the carboxyl group, specifically the OH group, to the O of the same carboxyl is of interest AH–C–B.

All of the distances are calculated as the direct path (the hypotenuse of a triangle) between the two end points in three-dimensional space. Each of the resulting distances relate to a distinctly separate gustophore of tyrosine. Only some of these distances may be relevant to the gustatory modality of a specific species. That species may only have receptors sensitive to a subset of these distances. Some of the potential gustophores may also include structural features that interfere with an adequate stereochemical bonding between the gustophore and the sensory neuron receptor.

The centroid of the π–cloud of the aromatic ring is assumed to be well defined at the geometric center of the ring in this presentation. In fact, it may not be well centered due to the presence of ancillary ligands. Defining the electronic field of fused rings and more complex structures will require Figure 8.4.6-1 Tyrosine and the relevant additional investigation. gustophores. See text. Signal Generation & Processing 8- 73 The literature reports additional situations where only a double bond between two carbon atoms can be considered an unsaturated center able to act as a moiety of type X. A fully conjugated carbon chain is also presumed to be capable of acting as a moiety of type X.

8.4.7.1 Initial definition of one-dimensional taste and smell spaces

The calculated distance-values, d-values, associated with a series of gustatants and/or odorants can be plotted on a linear graph. Such a graph will be valuable later in developing various multi- dimensional interpretations of taste and the smell spaces. Taste and smell spaces of various orders have been used in the past to define various primary classes of gustatant and odorants. However, the typical gustatant and odorant is represented by a group of gustophores or odorophores. These groups can be directly related to various chemical geometries within the overall molecule. It is these chemical geometries (frequently described only by their d- values, that constitute the only classes of interest here. 8.4.7.2 Complex structural groups within chemical stimulants

Such odorants as musk are much too complex to be considered primary odorants or belonging to a primary class of odorants.

[xxx check this ] The simplest potential odorophore appears to be the highly polar carbonyl group. In its simplest form it could form a coordinate bond pair with the oxygen acting as B and the carbon and an associated hydrogen acting as AH. Beets (1978, pp 187-188) has suggested a variety of alternate configurations depending on the specific conditions of the sensory receptors.

The first two members of subclasses of this class are formaldehyde and acetone. Another first member is phenol of the primary class, aromatic alcohols. The other classes will be defined below. More complex odorophores within a class take on much more complex conformations.

Odorophores are characterized by their structural conformation, not just their configuration. Changing the conformation of an odorophore can change the intensity of the elicited sensation significantly.

Hydrogen bonds play a major role in the stimulant-receptor relationship. However, intramolecular hydrogen bonds are largely irrelevant to the chemoreception mechanism. It is the intermolecular hydrogen bonds that are significant. 8.5 The gustatory modality

[xxx change all H-best, Q-best etc to new terminology after definition in start of gustatory ] [xxx consolidate on angstrom or on nm throughout ]

Quoting van der Heijden in 1993, “Humans can perceive four tastes, of which sweet and bitter have received the most attention from scientists.” This will become evident when the sparse information concerning the elicitation of the acidic and salty sensations are reviewed. The literature fails to note the acidic sensation is based primarily on the sensing of organic acids. The sensation of salty is elicited almost exclusively by the hydrated sodium ion. While based on very fragmentary data, Boudreau has provided the most complete conceptual description of the overall gustatory modality134. Inconveniently, he uses the term ganglion in place of the more conventional nerve when speaking of the major nerves serving the oral cavity.

134Boudreau, J. (1989) Neurophysiology and stimulus chemistry of mammalian taste systems In Teranishi, R. Buttery, R. & Shahidi, F. eds. Flavor Chemistry: Trends and Developments. Washington, DC: American Chemical Society Chapter 10 74 Neurons & the Nervous System

Cagan & Kare edited a comprehensive volume with a focus on olfactory transduction in 1981135. The individual paper authors explored a broad range of potential transduction mechanisms. Some focused on the role of proteins on the surface of the sensory neuron membrane. Others focused on the potential for the lipids of the membrane to be involved in the primary mechanism. No conclusions were drawn. Kurihara, Miyake & Yoshii explored the work of Kamo et al136 in 1980. Kamo et al. attempted to mimic the sensory response of the gustatory neurons much as Hodgkin did for the visual modality sensory neurons (pages 249-286). Their equation 2 expresses the two-way operation of the proposed fundamental chemical reaction of the transduction process in abstract form. They then formulated a set of second order differential equations based on the Law of Mass Action (which requires a totally reversible reaction in solution). Unfortunately, there are a vast array of possible mechanisms that are satisfied by a second order differential equation. These include the quantum- mechanical mechanisms such as the production of nuclear isotopes. In their analysis, they ignored the rapidly rising attack transient and concentrated on the decreasing transient and the steady state value (prior to cessation of stimulation). However, their boundary conditions necessarily included the rapidly rising attack transient in their ultimate solution. While their particular solution of the differential equations is complete and includes the pulse condition (as opposed to just the impulse condition), there are two problems with their interpretation of it. First, they apparently did not treat the off-response properly by applying new boundary conditions. The off response is a direct and independent measurement of the second exponential in equation 2 and in the E/D equation of this work. Second, Kamo et al. did not treat the singularity within their equations properly. Their particular solution does not apply at w1 = w2. Removal of the singularity results in a different mathematical form that this author has labeled the Hodgkin condition (Section xxx). They noted Kashiwagura et al contained additional material on their investigation137.

Scott & Mark explored coding within the taste system in 1987138. Their analysis relied upon the chemical theory of the neuron. Their abstract opens with;

“Attempts to define the organization of the taste system in terms of the physical characteristics of stimuli have been largely unsuccessful.”

They noted specifically, “Molecular weight and pH did not relate to the total organizaton of the system. . . “ Their work, employing multidimensional scaling (MDS)will be addressed below.

Fisher & Scott reviewed the subject of food flavours in 1997139. The work is entirely conceptual, with few substantive sketches, and based on behavioral studies. It makes the conventional assumptions that taste involves either ion channels passing molecules through the sensory lemma or proteins on the surface of the sensory lemma. It does not demonstrate either of these concepts is correct, nor does it provide primary data relative to these concepts. Their table 3.1, from Kinnamon & Getchell, illustrates the continuing conceptual character of their thesis. Pages 85–87 provide a comprehensive list of conceptual gustatory features that suggest four basic taste sensory channels.

Kashiwagura et al. show the measured responses at the glossopharyngeal nerve of the bullfrog, Rana catesbeiana, for comparison with the mathematical analysis of Kamo et al. They varied both the flow

135Cagan, R. & Kare, M. ed. (1981) Biochemistry of taste and olfaction. NY: Academic Press Parts II & III

136Kamo, N. Kashiwagura, T. Kurihara, K. & Kobatake, Y. (1980) A Theory of dynamic and steady responses in chemoreception J theor Biol vol 83, pp 111-130

137Kashiwagura, T. Kamo, N. Kurihara, K. & Kobatake, Y. (1980) Interpretation by theoretical model of dynamic and steady components in frog gustatory response Am J Physiol (gastrointest.) Vol. 238, pp G445- G452

138Scott, T. & Mark, G. (1987) The taste system encodes stimulus toxicity Brain Res vol 414, pp 197-203

139Fisher, C. & Scott, T. (1997) Food Flavours: Biology and Chemistry. Cambridge, UK: The Royal Society of Chemistry. Signal Generation & Processing 8- 75 rates of the stimulants and the temperature of the experiment. The experiments were carried out in- vivo at 18°C except at reduced temperatures, 2.5°C, where an in-vitro condition allowed easier cooling of the tongue and stimulants. They noted the dynamic portions of their waveforms were sensitive to flow rate but the steady state values generally were not. Gustatory experiments are traditionally carried out under conditions convenient to the investigator. Stimuli are applied by pouring or brushing the material onto areas of the tongue that are only coarsely delineated. Typical time intervals are measured in tens of seconds with initial application frequently reported using voice records from the investigator. The records are generally action potential streams taken at the stage 3 neurons orthodromic to the taste buds, amplified and integrated. The integrated records faithfully represent the waveform of the gustatory sensory neurons prior to encoding by the stage 3 neurons. However, the rapidly rising attack portion of the waveform is frequently poorly reproduced. This makes determination of the precise shape of the attack waveform difficult to determine. The subject of taste modifiers (additional chemicals applied with the stimuli) and enhanced stimuli (stimuli with an additional ligand not found naturally) have not been introduced into a comprehensive theory of gustatory sensing. However, a few enhanced stimuli have been documented having perceived intensities as much as 30 times greater than the strogest natural materials. The measured waveforms of Kashiwagura et al. are very descriptive. Unfortunately, the amplitude of their waveforms, such as Figure 8.5.1-1 are only relative. This figure exists in several variants in the literature. The original shows a small step before the major rise in both waveforms. It appears this step is the absolute delay expected in these waveforms as a function of temperature. If so, the time lines have been drawn in for illustration purposes and should be moved to the left by 2-5 seconds depending on temperature. The off-response is not well defined in these waveforms for two reasons. The figure is the result of the stage 3 encoding which is largely asynchronous to the stage 1 sensory waveform. The time scale of these figures is too compressed to show the response well. Scaling from frame A suggests an attack time constant of less than 100 msec while the decay time constant is on the order of 20 sec. Pre-adaption with a NaCl wash had a significant impact on the transduction process, even though the stimulus contained no sodium. Note the difference in the baseline prior to stimulation for the two cases. These waveforms can be compared to those in Section xxx. Kashiwagura et al. offered no explanation for the effect of preadaptation on the waveforms shown. It is necessary to adjust the boundary conditions in some of the E/D equation to account for the preadaptation conditions in some of their other waveforms.

The Kashiwagura paper provides considerable data concerning the change in time constants with temperature in the frog.

- - - -

Although dated, the discussion of the Figure 8.5.1-1 The E/D waveforms of gustation in gustatory modality in Noback remains the bullfrog ADD. The pH of the stimulating CaCl2 was 5.7. A; sensory neurons were preadapted to 5 mM NaCl. B; sensory neurons were preadapted to a Ringer’s Solution. The leading edge of both waveforms are slightly curved. The off-response is not well defined at the scale of this figure. See text. From Kashiwagura et al., 1980. 76 Neurons & the Nervous System useful140. The description of the taste buds as bowl shaped features located behind pores in the lingual epithelium and containing on the order of 25 or more distinct gustatory sensory neurons is clear. He also notes the seldom reported high turnover rate among the sensory neurons, as opposed to just the sensory hair of each cell. “Each mature sensory neuron is replaced every 200 to 300 hours.” This turnover rate is similar to that of just the outer disks of the visual sensory neurons. It is also similar to the turnover rate of the piezoelectric proteins within the auditory sensory neurons. Like the retina of the visual system, the taste buds contain “sustentacular cells” that develop and replace the older sensory neurons. This replacement is associated with a transfer of the synapse with the orthodromic neurons from the old to the new sensory neuron. Noback also notes the neurons immediately orthodromic to the sensory neurons emerge from the taste bud and become myelinated immediately (therefore stage 3 neurons). As in the case of the auditory neurons, this suggests the encoding of the analog signals occurs at the first Node of Ranvier and not within the soma of these stage 3 neurons. This node of Ranvier occurs in what is conventionally described as the dendrite of the signal projection neuron. However, the myelinated neuron component directly after the encoding node of Ranvier is an axon segment. The number of gustatory sensory neurons is small, estimated at about 10,000 in human babies and the sensitivity of the modality is low relative to the olfactory modality (as many as 20,000 times the number of molecules are required for gustation as olfaction). However, it is probably the method of delivery more than the sensitivity of the receptors that is limiting. The quantum-mechanical character of the response of the gustatory sensory neurons suggest they are highly efficient at sensing molecules delivered to their immediate vicinity.

Glendinning et al. summarized the conventional wisdom concerning gustation in 2000. They noted,

“Among the sensory systems, the taste system is unusual in its capacity to respond to a large number of stimuli that vary greatly in molecular size, lipophilicity, and pH (e.g., salts, amino acids, sugars, acids, vitamins, fatty acids, and many toxic compounds). To accommodate this structural diversity, taste cells utilize a diverse array of transduction mechanisms. However, only a fraction of these mechanism occurs within any given taste cell, different subsets of taste cells appear to express different transduction mechanisms, and hence different molecular receptive rages.”

All of Glendinning et al’s. discussion is dependent on the chemical theory of the neuron, and the concept of pores through the dendrolemma of the sensory neurons or the presence of proteins inserted into and traversing the dendrolemma (the conventional G-protein hypothesis). Their figures are limited to simple conceptual sketches of a dendrolemma. No differentiation of dendrolemma into special classes is considered. The figures depend on the porosity of the hydraulic barrier (tight junction) between the sensory neurons for the sensing of simple positive ions (illustrated as Na+ & H+). They note, the hydraulic barrier is known to be impermeable to amiloride in vertebrates.

In summary, the Glendinning et al. material is an excellent source of laboratory results but provides no detailed theory of gustatory neuron operation. They do not address the time duration of the sensory mechanisms or explain the form of the C/D waveform. The internal conflicts within their models are characterized by the discussion of G-protein versus cAMP involvement in “sweet” sensing on page 325. There is virtually no detailed discussions of the precise role of proteins in the gustatory modality, other than the assertion that such a role must exist and subsequent Bayesian inferences derived from analyses of the genetic code. McManus et al. made the strongest presentation in a two page communication in 1981141. They asserted the most likely site along a protein to act as a sensory receptor was where proline residues disrupted the normal helical structure and exposed two hydroxyl groups to provide an AH,B bonding site. Their subsequent paper focused on polyphenols, rather than

140Noback, C. (1967) The Human Nervous System. NY: McGraw-Hill pp 118 &136-139

141McManus, J. Davis, K. Lilley, T. & Haslam, E. J Chem Soc Chem Commun vol. 7, pp 309-311 Signal Generation & Processing 8- 77 sugars, interacting with proteins. Smith and Davis made an important assertion in 2000 (page 362) that they refuted almost immediately, “A universal characteristic of mammalian gustatory neurons is their responsiveness to stimuli representing more than one of the classic four taste qualities.” This statement suggests the major perceptions of taste do not correspond to any prior categorization of individual stimuli into classes. It also suggests that, like in the case of yellow in vision, the perception of a prominent taste may not be related to an individual sensory channel. Such a perception may be the result of signal manipulation within stage 4. The many multidimensional analyses of taste perceptions appears to support this assertion. However, Smith and Davis follow immediately with the statement conflicting with their above view (page 363). Speaking of the moth, Bombyx mori, they say, “The most sharply tuned fiber is depolarized exclusively by specific isomers of the sugar alcohol, inositol, the other three fibers are less sharply tuned . . .” - - - -

The common protocol of recording the number of action potentials in a five second interval obliterates any information regarding the shape of the excitation/de-excitation function in gustation. It is known that most animals can discriminate or identify a gustaphore in less than one second.

- - - -

Spector & Travers (page 173) note the low likelihood of a chemotopic organization in the gustatory modality based on considerable investigation by the community.. Travers et al. have provided a paper showing how complex the gustatory signaling paths are and the difficulty of preparing a set of stimulants and a protocol for evaluating this modality unambiguously142. Their figure 3(lower right) is difficult to interpret, however it may show cases of a nominal pulse rate being reduced in the presence of a second stimulant, a feature of a differencing channel. It is common to find examples of summing among at least two channels in the neural paths of the chorda tympani. Pfaffmann said as an example, “Three types of fiber were found : those stimulated (1) by acids, (2) by acid and by sodium chloride, and (3) by acid and by quinine143.” He also noted, “Since some of the fibers respond to such a surprisingly diverse group of stimuli, one is reminded that the single fiber of these experiments is inferred from the appearance of the potential record.”

- - - -

Figure 8.5.1-2 is a schematic/block diagram of the proposed gustatory system. It shows the taste bud schematically followed by a preliminary block diagram of the modality through stage 4. The synapses within the taste bud are not shown in detail. However, the neurons leaving the taste bud are known to become myelinated immediately. Lacking any data showing analog signal differencing of the initial gustatory signals, no connections are shown to the potential midget ganglion (difference) encoding neuron. The exemplar signal path is shown proceeding to a parasol ganglion neuron wherein the first Node of Ranvier performs the encoding (modulation) function. The nucleus solitarius is the “1st relay” and the parabrachial nucleus is the “2nd relay” in the gustatory signaling chain. Signals from these elements divide and proceed to both the ventral posteromedial thalamus (PVM) and to the amygdala/hypothalamus of the limbic system. The signals from the PVM are known to proceed to the region of the operculum of the parietal lobe (BA 2), the adjacent insula and inner surface of the temporal lobe (potentially BA 27). Aggleton & Passingham have provided more specific information

142Travers, S. Pfaffmann, C. & Norgren, R. (1986) Convergence of Lingual and Palatal Gustatory Neural Activity in the Nucleus of the Solitary Tract Brain Research, vol 365, pp 305-320

143Pfaffmann, C. (1941) Gustatory Afferent Impulses J Cell Comp Physiol vol 17, pp243+ 78 Neurons & the Nervous System on these locations in the macaque144. Rolls & Baylis have provided some data on the nearly parallel olfactory and gustatory tracts in the primate orbiotfrontal cortex145. Signals from the amygdala/hypothalamus are known to connect to the orbitofrontal cortex area of what this work calls the prefrontal cortex.

Figure 8.5.1-2 Proposed schematic of the gustatory system ADD. The nucleus solitarius and parabrachial nucleus may be the same element as defined by different investigators. See text.

Each sensory neuron exhibits a small number of microvilli emanating from the neuron and into the cup formed by the taste pore in the lingual epithelium. This is illustrated more clearly in [Figure xxx ] The figure shows the sensory neurons delivering signals to nerves VII, IX and X depending on their location within the oral cavity. These neurons all enter the brain stem at the interface between the pons and the medulla. The afferent neurons within the taste bud frequently connect to multiple sensory neurons. However, this may be to support the rapid turnover of the sensory neurons with the afferent neurons connecting to only one functional neuron at a time. The literature does not suggest any differencing of neural

144Aggleton, J. & Passingham, R. (1981) Stereotaxic surgery under x-ray guidance in the rhesus monkey Exp Brain Res vol 44, pp271-276

145Rolls, E. & Baylis, L. (1994) gustatory, olfactory and visual convergence within the primate orbitofrontal cortex J Neurosci vol 14, pp 5437-5452 Signal Generation & Processing 8- 79 signals occurs within the taste bud. Therefore, the figure shows a neuron from the taste bud connecting to a parasol (summing) ganglion neuron, P. The potential for a midget (differencing) ganglion neuron, M, has been retained for discussion purposes only.. The neurons emanating from the taste bud become myelinated immediately and prior to reaching the soma of the neurons. This myelination suggests the sensory neurons of the gustatory modality are much like those of the auditory modality in that the first Node of Ranvier appears prior to the soma and acts as the encoding element within this stage 3 neuron. The nucleus of solitarius is known to sum signals from multiple sensory neurons but whether on a spatial basis or a stimulus basis remains unclear. This nucleus does not show an organization similar to a glomeruli or any recognizable chemotopic organization. Figure 8.5.1-3 is an schematic of the neural paths of mammals based specifically on the anatomy of sheep. The figure offers considerable definition of the separate neural paths and way points. It also shows the lack of a common focus of the gustatory signals as they pass through the rostral brainstem.

Figure 8.5.1-3 Schematic of mammalian neural paths in gustation. Based on the sheep but applicable to any mammal. Mistretta noted, the nasoincisor duct with taste buds, schematically indicated on the anterior hard palate and innervated by the greater palatine nerve, has not yet been reported in the sheep. From Mistretta, 1990. 80 Neurons & the Nervous System

The role of the PBN remains unclear. Some authors suggest it is missing, or inconsequential in humans146. Others sketch it in to conceptual drawings147. Whether the nucleus solitarius and parabrachial nucleus should be considered stage 2 signal processing elements or merely relay points is yet to be resolved. Their forward connections suggest they should be considered elements of stage 4, signal manipulation. Many signal paths emanate from the nucleus solatarius and the parbrachial nucleus, with the majority believed to connect to the ventral posteromedial thalamus (VPM) and the amygdala/hypothalamus. Beyond those elements, the neural pathway of the gustatory modality is poorly documented. The VPM appears to connect to an area of the cerebral cortex known as the insula with spillover onto the operculum of the parietal lobe (BA 2) and the upper medial surface of the temporal lobe (?BA 27). This region may constitute a portion of the saliency map devoted to gustatory and olfactory representations available for review by the cognitive functions of stage 5. The amygdala/hypothalamus appears to connect to the orbitofrontal portion of the prefrontal cortex.

Anatomical data on the tongue of many species appears in Bradley148. - - - -

Pfaffman et al. provided some early gustatory information using squirrel monkeys149. They recorded action potentials from the chorda tympani, a branch of the VII facial nerve. They introduced the concept of labeled lines with little detail. They did note, “Two-thirds of our sample of taste units fall readily into one of the four classic taste categories with a peak at one basic taste stimulus. ‘Side bands’ around such peaks produce a certain degree of multiple sensitivity. One-third of the responsive fibers, however, cannot be classified by a single ‘best stimulus’ but appear to have broad multiple sensitivity. All of their reported waveforms suggest parasol type encoding channels.

Teeter et al. have provided an interspecies review of the sensations generated in the gustatory modality150. However, their interpretations are all based on an undefined linear circuit network for the sensory receptors. No circuits or equations were presented. They generally assumed a change in plasma membrane impedance resulted from stimulation.

Recently Mattes has introduced data showing the gustatory modality of humans is also sensitive to a series of free fatty acids151. [xxx add words ] Lim & Lawless explored the taste of a variety of metal

146Mistretta, C. (1990) Taste Development In Coleman, J. ed. Development of Sensory Systems in Mammals. NY: John Wiley & Sons Chapter 14

147Rolls, E. & Scott, T. (2003) Central taste anatomy and neurophysiology In Doty, R. ed. Handbook of Olfaction and Gustation, 2nd Ed. NY: Marcel Dekker page 680

148Bradley, R. (1971) Tongue topography In Beidler, L. ed. Taste: Handbook of Sensory Physiology, Vol IV, Part 2, Chap 1

149Pfaffman, C. Frank, M. Bartoshuk, L. & Snell, T. (1976) Coding gustatory information in squirrel monkey chorda tympani In Sprague, J. & Epstein, A. eds. Progress in Psychobiology and Physiological Psychology. NY: Academic Press. vol 6, pp 1-27

150Teeter, J. Funakoshi, M. et al. (1987) Generation of the taste cell potential Chem Senses vol 12(2), pp 217- 234

151Mattes, R. (2009) Oral Detection of Short-, Medium-, and Long-Chain Free Fatty Acids in Humans Chem Senses vol 34(2), pp 145-150 Signal Generation & Processing 8- 81 salts152. [xxx add words ] Rogers et al. have offered a limited tree focused on bitter taste relationships153. [xxx add words ] Lundy & Norgren have provided results of a complicated set of experiments suggesting feedback of signals from the amygdala/hypothalamus and gustatory cortex to the parabrachial nucleus. However, while they located their stimulation and recording sites adequately for anatomical purposes, they did not identify the precise location of their sites with respect to the inputs and outputs of various elements containing millions of individual neurons. - - - - The mechanism of gustatory transduction has long been an unsolved problem. The likelihood of four distinct perceived tastes, sweet, sour, salty, bitter & have been proposes since ancient times. Umami became a potential fifth flavor during the early 1990's. Many, generally unsuccessful, efforts have been made to explain these perceived tastes based on the chemical structure of the stimulants154. - - - - Schifferstein has discussed the steps in gustation from a food technology perspective155. His totallly psychophysical steps appear remote from the physiological steps identified in this work.

Figure 8.5.1-4 shows a provisional block diagram of the gustatory modality. Frame A shows a simple linear representation of the gustatory modality that parallels that of xxx. However, the functional descriptions at the gaps in the line segments have been changed.

Frame B expands the conceptual character of Frame A to illustrate xxx.

Frame C shows the probable character of the gustatory modality based on its close analogy to the visual modality. A common question is whether the neural signals recorded at the output of the sensory neurons correlate well with the perceived tastes of the subject. Frank has provided a comparison of the sensed and perceived responses using factor analysis that

Figure 8.5.1-4 Provisional block diagram of the gustatory modality ADD. See text.

152Lim, J. & Lawless, H. (2005) Qualitative Differences of Divalent Salts: Multidimensional Scaling and Cluster Analysis Chem Senses vol 30(9), pp 719-726

153Rodgers, S. Busch, J. Peters, H. & Christ-Hazelhof, E. (2005) Building a Tree of Knowledge: Analysis of Bitter Molecules Chem Senses vol 30, pp 547–557

154Shallenberger, R. & Acree. T. (1967) Molecular theory of sweet taste Nature vol 216, pp 480-482

155Schifferstein, H. (2003) Human perception of taste mixtures In Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker Chap 38 82 Neurons & the Nervous System shows this is a close correlation in hamsters156. Her imposing a spherical overlay on her data leads one to believe the distance from the axes of various stimulants is more limited as their appearance on the graph moves toward the perimeter. A more open three-dimensional taste sensation space is suggested in this work (Section 8.5.3 xxx). Frank’s sensory recordings were at the chorda tympani nerve. The conclusion can be drawn that the stage 2 through stage 4 activity is designed to project and optimize the presentation of sensory neuron output data to be analyzed by the prefrontal cortex of stage 5. As noted in Section, the stage 2 differencing does not occur in the taste buds or the oral cavity. It is likely this signal processing occurs in the NST or possibly the parabrachial nucleus. The differencing function is strongly suggested by the basis functions of the multidimensional analyses presented in the literature. No data has been found in the literature describing the signals from the temporal lobe o f cerebral cortex describing the form of the data presented to the saliency map.

156Frank, M. (2000) Neuron types, receptors, behavior, and taste quality Physiol Behav vol 69, pp 53–62 Signal Generation & Processing 8- 83

8.5.1.1 Chemical families pertinent to gustatory receptor identification

The following discussions will focus on modifications to the outer bilayer of the cilia and villi of sensory neurons as leading to the likely receptors of gustation. The chemistry in this area can be extremely complex, and involve mechanisms not familiar to the majority of researchers. The complexity in the literature of potential lemma chemistry is partially due to the different time periods in which various investigators have worked. It is also due to the difficulty in controlling and identifying and avoiding uncontrolled chemical reactions involving constituents present in very complex molecules. When discussing complex organic chemistry, it is common in textbooks and journal articles to address the degree of saturation in the bond structure in a cavalier manner. A more refined approach is needed in this work. Figure 8.5.1-5(A) discusses the major possibilities. The case of a saturated hydrocarbon is a boundary condition. The molecule contains no double or triple bonds. However, an unsaturated hydrocarbon remains largely undefined. An unsaturated hydrocarbon with only one double bond among many double bonds appears to retain most of the properties of its fully saturated sibling. However, molecules with alternating single and double bond patterns can exhibit significantly different electronic properties. The brain and neural system is known to contain many molecules exhibiting a repeating isoprene structure where carbon-carbon double bonds are separated by two carbon-carbon single bonds, a structure associated with the squalenes Alternately, .visual sensory organs of the neural system are known to contain large numbers of molecules exhibiting a repeating isoprene structure where carbon-carbon double bonds are separated by one carbon-carbon single bond. Such molecules are known as terepenes and are referred to as conjugated hydrocarbons over a significant and defined portion of the entire molecules. They include the very important retinal derivatives, the rhodonines, which exhibit significant intra– and inter–molecular electronic conductivity when present in the liquid crystalline state. In the case of the rhodonines, the conjugation even extends to the O– and OH– ligands of the structure and explains the multiband spectral performance of the visual system.

Figure 8.5.1-5(B) attempts to normalize the terminology used to describe the chemistry involved in sensory neuron receptor identification. The goal is to define traceable names to the chemical receptors providing transduction of the presence of stimulants into graded electrical signals. To this end, the figure attempts to rationalize the words and figures of Lehninger (1970) with those of Benjamins et al. (2006). It employs an indenting style used to save space when using long descriptors. The chemicals of interest are at the convergence of two very large general families known as the phospholipids (phosphoglycerides) and the sphingolipids (containing no glycerol). They both contain two long chain hydrocarbons and a polar head group. The polar head groups typically exhibit a dipole potential and dipole moment because of their electrical asymmetry at the atomic constituent level. As normally conceived, the hydrocarbon chains of both families are nonpolar and electrically insulating. However, a thesis of the Electrolytic Theory of the Neuron is that at least one of the chains in the above phospholipids and sphingolipids can be present in a conducting (or semiconducting, i.e. directionally conducting) form. In this form, a dipole potential can be exhibited between the two ends of the overall molecule (particularly when the molecules are present in the highly oriented liquid crystalline state found in lemma bilayers). 84 Neurons & the Nervous System

Figure 8.5.1-5 A normalized etiology of chemicals relating to proposed gustatory sensory receptors.

Electrically conductive hydrocarbon chains in the liquid crystalline state are well known, and are widely used in this age of the liquid crystal display technology. The conductivity of hydrocarbon chains is primarily a function of the degree of saturation (unsaturation) of each chain. Fully saturated and minimally unsaturated chains are electrical insulators. However, unsaturated chains, particularly of the fully conjugated form, exhibit significant conductivity because of their sharing of excited electron π-bonds along the length of their chains. In an appropriate electrolytic circuit configuration, these conductive chains are able to transfer a potential at their interface with their polar head to the extreme end of their hydrocarbon chain. It is proposed that the value of this potential is used within the neural system to identify and quantify the stimulants used in gustation. Signal Generation & Processing 8- 85 Textbooks tend to show all phospholipids and sphingolipids as consisting of fully saturated fatty acid chains as a matter of simplicity and convenience. Benjamins et al. in particular show a range of saturated and unsaturated fatty acids of neurochemical interest. Their tabulation focuses on products of the isoprene rule, the squalenes that exhibit conjugate carbon-carbon bonds at every third carbon. A similar product of the isoprene rule, the terpenes exhibit conjugate bonds at every other carbon- carbon bond. The terpenes are known to be conductive and their may be other configurations providing conductivity, possibly involving cross-linking between the two parallel long chains. Four conductive phosphoglycerides have been identified as likely receptors in gustation. They have been identified on the right as being the receptors in of the four primary sensory channels of gustation. Frame C: The literature contains discussions of cerebrosides, of both the glycophospholipid and glycosphingolipid (defined as containing no glycerol) types containing one sugar ligand. Cerebrosides containing oligosaccharide complexes have been named gangliosides and globosides (the former containing sialic acid while the latter does not. As one travels down this figure, it is not always clear how precisely and exclusively the terms have been defined. The term gangliosides in particular seems to be used to describe both galtanophospholipids and galtanosphingolipids in the journal literature. Benjamins et al. have noted “galactocerebroside, in which galactose is β-glycosidically linked to ceramide, constitutes about 16% of total adult human brain lipids.” Such a high concentration suggests it is a structural component rather than a sensory or signaling component. They note the galactoseebroside appears primarily in the white matter, especially in myelin, a passive electrically insulating material.

The routinely reported presence of the cerebrosides and gangliosides at high concentrations within the brain does not necessarily indicate they are functional within the neural signaling system. There is considerable potential for these molecules to participate as structural components of the neuron walls. The reduced presence of oxygen in the case of the sphingolipids also suggests they are not as electrically important as the phospholipids.

Benjamins et al. have provided a set of shorthand labels for many of the chemicals that will be discussed in detail later in this chapter157. Most of the phosphoglycerides will be described using their more specific labels; the carboxyphosphoglyceride of interest is described as phosphatidyl serine or PtdSer.

The aliphatic alcohols are missing from the above table. It is generally believed that these molecules do not stimulate the gustatory sensory receptors of humans. However, they apparently do stimulate the olfactory sensory receptors. This situation suggests frequent experimental difficulties in isolating the sensory neurons of the two modalities in the laboratory. The difficulties frequently lead to confusion in the literature over the role of these molecules in gustation. 8.5.1 Overview of the gustatory modality

The following material will develop the theory of the gustatory modality as an explanation of the empirical data in the literature based on the Electrolytic Theory of the Neuron and transduction via a coordinate chemistry mechanism for each of the sensory channels of taste. The theory is comprehensive. It encompasses the sensing of all four classical sensations of taste. The theory of gustatory sensing recognizes a three dimensional taste space with the four classical sensations occupying four of the available six vertices. The space is expandable in either of two ways, if additional data appears supporting a fifth channel related to the label “umami.” This sensation could occupy an available vertex in the three dimensional space or it could require expanding the space to a fourth dimension (as is the case in the visual modality when the sensing of ultraviolet light is recognized– Section xxx). Moving from a conceptual understanding of gustation to a more formalized theory has provided new

157Benjamins, J. Hajra, A. & Agranoff, B. (2006) In Siegel, G. et al. eds. Basic Neurochemistry, 7th Ed. NY: Elsevier Chapter 3 86 Neurons & the Nervous System insights. These insights can be grouped as follows. Group 1 [xxx subdivide the following into groups with rational names. ] C Gustatory transduction involves a coordination chemical process of very low energy that is totally reversible. No chemical “reaction” is involved in the transduction process. C Transduction occurs totally external to the sensory neuron lemma. No stimulant components pass through the cell wall. C The transduction mechanism uses a set of phospholipids, widely recognized as being present in neural tissue but of previously unknown utility, as the sensory receptors. C The transduction mechanism employs a coordinate chemical union between the sensory receptors and the stimulant that is stereo-chemically specific. C The transduction mechanism results in a change in dipole potential of the phospholipids that are known to be polar. This change in dipole potential is sensed by the first Activa of the sensory neuron. C The histology and physiology of the sensory neurons following the transduction mechanism is the same as that of other sensory neurons. They lack an external axon like the auditory sensory neurons and share a microvilli structure similar to the visual sensory neurons. C The sensory channels of the gustatory system exhibit the same excitation/de-excitation function and the same adaptation functions as other sensory modalities. Only the time constants vary to accommodate anatomical and vascular conditions. C The gustatory modality exhibits the same neural architecture as all other sensory modalities but the stage 2 signal processing occurring outside the cranium appears to be minimal. C The gustatory modality uses a small group of sensory receptors (nominally four) to sense a wide variety of chemical structures (in the thousands). C The small number of sensory channels operate independently and can therefore be represented as statistically independent and therefore orthogonal, resulting in a three-dimensional taste space within the neural system. This space can be unfolded into a single dimensional space equivalent to the spectra of the visual and auditory modalities. C The unfolded representation of the taste space highlights the common feature wherein many stimuli are able to stimulate multiple sensory channels. C The “salty” sensory receptor is in fact only sensitive to the fully hydrated sodium ion; the chlorine ion plays no role in the transduction mechanism. C The “sour” sensory channel is primarily sensitive to the carboxyl group of organic (Lewis) acids. C The response of the gustatory modality to inorganic acids appears to be primarily via nocioreceptors. C The “sweet” sensory channel is primarily sensitive to the glucol group, consisting of either oxygen or hydroxyl oxygen at positions 1 and 2 of a cis-glycol. C The “sweet” sensory channel exhibits an overlay mode of operation, involving a three-point stereochemistry, that is supersensitive to man-made sweeteners. C The “bitter” sensory channel is primarily sensitive to organics with two orbitals capable of sharing an electron-pair and separated by a distance of 4.2 Angstrom. C The frequently proposed independent “umami” sensory channel cannot be confirmed based on the available empirical data and rigorous multidimensional analysis procedures. The dominant chemical associated with this designation, monosodium glutamate, is unusual in its ability to stimulate the salty, sweet and acid channels of gustation simultaneously (and potentially interfere with the Signal Generation & Processing 8- 87 electrical supply to the sensory neurons). Eliel et al. have discussed the different kinds of molecular models available in both physical and computerized form158. No effort will be made here to standardize on one type of diagram or physical representation. Simple line diagrams of chemicals leading up to include Haworth models will be used, augmented by computer models of specific chemicals prepared using Molecules-3D Pro, v2.10 from Molecular Arts. The specific geometric relationships between stimulants and receptors may not be shown faithfully because of the author’s limited familiarity with the program. However, the critical dimensions will be specified as clearly as possible. Figure 8.5.1-6 describes the sensory receptors related to taste. They are all phosphatidyl lipids derived from the normal lipids forming the outer bilayer of the microvilli of the sensory neurons. They differ primarily in the spacing between certain atoms in the structures that allow coordinate bonding with a variety of stimuli in the AH,B or AH,B,X configuration (Section 8.5.3). The figure is complex. However, by following the logic of Dowhan, it provides a variety of answers. It begins at upper left describing the chemical structure of a typical phospholipid and its ability to bond with a variety of terminal groups through esterification. The diagram has been annotated here to define one of the lipid molecules to be electrically conductive, most likely through complete conjugation of its fatty acid chain, like the isoprene chain of Vitamin A. The ligand in the box is labeled on the right with the trivial name choline, or formally phosphatidylcholine (PtdCho). This ligand plus the next one below it, ethanolamine, are the principle phospholipids of the lemma of all cells. The examples of ligands below these two are those commonly found in the lemma of neurons and particularly sensory neurons of the gustatory system. There are four of these ligands, each supporting a separate sensory channel of gustation leading to the sensation of sourness (presence of an organic acid or acidophore), sweetness (presence of a glucophore), saltiness (presence of a hydrated ion of sodium or natrophore) and bitterness (presence of a picrophore).

Each of the ligands exhibits a distinctive spacing between two or more orbitals susceptible to coordinate bonding with stimulants exhibiting a similar spacing between elements supportive of coordinate bonding. In the case of the sourness channel, the most important orbitals of phosphatidylserine (PtdSer) exhibit a spacing of 2.34 Angstrom when nonresonant (2.07 Angstrom when resonant). This is the same spacing as the two oxygen atoms of the carboxylic acid family. Thus, any carboxylic acid is amenable to coordinate bonding to PtdSer and eliciting a sensation of sourness. The proposed location of this bonding is shown by the small rectangular bracket next to the two oxygen atoms. PtdSer is therefore defined as the receptor of the sensory neuron initiating the sourness, or acid, channel of gustation.

Carboxylic acid is only mildly acidic, indicating it is not highly ionized in water. The dominant species is the nonresonant carboxylic acid form with a complete hydroxyl group. The spacing between the oxygen atoms in this configuration is 2.34 Angstrom.

Phosphatidylgalactose (PtdGal), also known as galactocerebroside (CerGal) exhibits a spacing of 2.6 Angstrom between OH–3 and OH–4. This spacing is compatible with a very large group of sweeteners containing a glucol group, or glucophore that can coordinate bond with this receptor and elicit a sensation of sweetness. A glucol group is defined more stereo-specifically than the similar glycol group. Glucol is a 1,2 cis-glycol or a partially dehydrogenated derivative. PtdGal is defined as the receptor of the sensory neuron initiating the sweetness channel of gustation. The ligand is shown in its standard notation that places the atoms of interest at the lower left. A bracket is shown to represent the stimulant bonding with the specified oxygen atoms. The PtdGal receptor is shown twice in a dashed box because of an additional phenomenon that has been documented. Besides the common two-point coordinate bonding arrangement illustrated with a spacing of 2.6 Angstrom in the top half of the box, it was found that an enhanced sensation of sweetness could be elicited by some stimulants if they employed a three- point rather than two-point bonding strategy. This super sweet stimulant situation is shown in the lower half of the box.

158Eliel, E. Wilen, S. & Doyle, M. (2001) Basic Organic Stereochemistry. NY: Wiley Interscience pg 25 88 Neurons & the Nervous System

Phosphatidylinositol (PtdIns) exhibits a spacing of 3.3 Angstrom between the two rightmost hydroxyl groups in the figure. This spacing is uniquely matched to the spacing between the oxygen atoms of adjacent water molecules hydrating with the sodium ion. Thus, this phospholipid is specifically attractive to coordinate bonding to the hydrated sodium ion and eliciting the sensation of saltiness. Note that this natrophore relates only to the sodium ion. The Signal Generation & Processing 8- 89

Figure 8.5.1-6 Summary: sensory receptors of gustatory modality based on the Electrolytic Theory of the Neuron and a coordinate chemistry mechanism. The glycol receptor is able to sense a variety of man-made “super sugars” using a tripartite coordinate union when they exhibit an electron rich area a prescribed distance from the two oxygen atoms of their AH,B structure. The bitter (quinine) receptor may also operate in a tripartite mode to sense a variety of “super bitter” stimuli. See text. Built using the style of Dowhan, 2002. 90 Neurons & the Nervous System anion of the salt plays no role in eliciting the salty sensation. PtdIns is defined as the receptor of the sensory neuron initiating the salty channel of gustation. Phosphatidyl 3'-O-aminoacyl glycerol (Ptd3'Og) is the fourth unique phospholipid of the gustatory modality. It exhibits a spacing of 4.2 Angstrom between the doubly bonded oxygen and the amine group of this amino acid based ligand. This spacing is compatible with a very large group of chemical that can coordinate bond with it and elicit a sensation of bitterness. Ptd3'Og is defined as the receptor of the sensory neuron initiating the bitter channel of gustation. Based on the similarity in the stereochemistry of the bitter receptor and the sweetness receptor, the bitter receptor has been reproduced twice within a dashed box. It appears likely that some of the extremely bitter stimulants that have been found may employ a three-point coordinate bonding arrangement like the super sweeteners do. This potentiality is shown in the lower half of the box using the dashed triangle to suggest the bonding arrangement.. The coordinate bonding between the various stimulants and receptors on the external lemma of the sensory neurons involves a very low energy (5kcal/mole) and does not constitute a chemical reaction in the conventional sense. No reaction products are formed and the original species reappear when the hydrogen bonds are disrupted. The stimulants do not pass through the lemma of the sensory neuron.

The major difference between the sensory receptor for sweetness and saltiness involves the steric crowding of the hydroxyl ligands of the polar molecular structure. The galactose molecule exhibits considerably more steric crowding than does the highly symmetric inositol molecule. This results in a smaller dimension, d, between positions 3 and 4 of the galactose molecule than in inositol.

Figure 8.5.1-7 summarizes the performance of the gustatory modality and presents a rectilinear graph for positioning stimulants in sensation space for the first time. As noted in Section 8.5.xxx, the signals passed to the brain by the sensory channels are treated as independent, and therefore orthogonal, by the brain. This orthogonal feature supports the presentation of the sensory data in a three-dimensional format, as suggested by the “basis factors” of multi-dimensional scaling applied to the gustatory modality.

An alternate d-value of 4.36 is shown for the bitter sensory receptor. It will be shown in Section 8.5.4.6 that this value is unlikely based on the accumulating data and particularly the d-values of the gustaphores of the alkaline earth salts. Signal Generation & Processing 8- 91

Figure 8.5.1-7 Summary: performance of the gustatory modality based on the Electrolytic Theory of the Neuron and a coordination chemistry mechanism. Top; stereo-geometry of the transduction process. Middle; a tabulation of pertinent labels and parameters. Bottom; a rectilinear sensation vs stimulus space describing the first order operation of the gustatory modality. Mean values are calculated or reported. Distributions are conceptual. Dashed line shows potential alternate bitter channel at d = 4.36 Angstrom. See text.

The discovery of a large number of man-made sweeteners, and obscure natural sweeteners, with immensely more perceived sweetness than the natural reference, sucrose, has changed the character of the search to understand the S-best sensory channel of gustation. van der Wel has provided a comprehensive list of these sweeteners159. He also noted the propensity of these sweeteners to exhibit multiple AH,B sites and potentially multiple AH,B,X sites (possibly with multiple X dimensions).. Figure 8.5.1-8 shows the first-order form of the gustaphores of taste. The titles are arbitrary but designed to emphasize the character of the gustaphore. Acidophore suggests the Lewis acid character of the acidophore, as opposed to a simple hydrogen ion. The first order acidophore is invariably derived from a carboxyl group and could be labeled a carboxylophore. The glucophore suggests a sweetness, but could just as readily be labeled an ethylophore to indicate the two carbon backbone.

159Van der Wel, H. (The role of organic chemistry in taste perception In van der Starre, H. ed. Proceedings of 7th ISOT London: IRL Press pp 13-19 92 Neurons & the Nervous System

The term glycophore is infrequently used and does not describe the general case. In the extended world of sweetness, at least one oxygen orbital can be replaced by another electron-pair sharing element. In gustation, the term orbital will be used to designate oxygen, or sulfur and potentially phosphorus atoms with their outer electrons in hybridized form and exhibiting shareable electron-pairs. In the more general case, the oxygen can be replaced by nitrogen, sulfur or an electronegative feature such as an unsaturated carbon bond or the π-bonding system of the benzene ring. In the more limited case, the term glycol is also appropriate as it describes two hydroxyl groups separated by two carbons. The controlling feature is the dimension, d, between the two orbitals. Trans– glycol configurations, do not exhibit the required d-value of about 2.6 Angstrom. The natrophore describes the configuration of the hydrated sodium ion giving what is commonly called the salty sensation. The anion of the salt plays no role in this sensation. The natrophore exhibits a 90 degree angle between the two orbitals due to the octahedral form of the hydrated sodium ion. The gustaphore contributing to the bitter taste has been labeled a picrophore to suggest its bitter taste. It could be described as the propophore to suggest the three carbon backbone of the structure.

An electronegative feature also appears to be important in a special situation leading to super sweetness, and potentially super bitterness. An electronegative feature a specific distance from the AH,B group appears to bond to a site on the receptor. This tripartite AH,B,X arrangement results in a sweetness sensation orders of magnitude (as great as 4000:1) greater than that of the dipartite arrangement (Section 8.4.xxx).

After the presence of two electron-pair sharing orbitals and the hydrogen bond associated with one of them, it is the distance, d, between the orbitals supporting the AH,B, or AH,B,X coordinate union that is the important feature of the gustaphores. The distances shown in the figure are nominal as little statistically precise data is available. There has been confusion previously in the case of the glucophore as to whether the distance measured was between the orbitals or the greater diagonal distance between one orbital and a hydrogen.

Figure 8.5.1-8 The first-order gustaphores of taste. The spacing, d, between the oxygen atoms (or alternate orbitals) is the critical dimension in determining the effectiveness of the gustaphore. See text. Signal Generation & Processing 8- 93 Figure 8.5.1-9 shows a three-dimensional taste space based on the above and Section 8.5. The figure shows the four basic taste sensations at four of the six vertices of a cubic space. The potential for a fifth basic taste, umami, is present. However, the subsequent analyses will show the major ligands of stimuli related to umami are a sodium (N-best) channel stimulant and a glucol (S-best) channel stimulant.

- - - - - All of the phospholipids defined above are known to be associated with the sensory neurons of gustation. They all exhibit a dipole moment that is a function of their state of coordination. When present with one conjugated lipid in the liquid crystalline form, as found in a bilayer, they exhibit a dipole potential that can be measured. This potential is also a function of the state of coordination of the molecule. - - - - -

Activity related to the gustatory modality began in antiquity. During the 19th and 20th Centuries, that activity has been primarily within the psychophysical and food science communities. No significant theory of gustation appeared. As a result, the current terminology has evolved from the earliest psychophysical observations. The concepts of sweet, sour, salty and bitter evolved into a Figure 8.5.1-9 Proposed taste sensation space for a formalism of S(sweet)-best, H(acid)-best, mammalian species exhibiting four primary taste N(salty)-best and Q(bitter)-best sensory sensation. See text. channels. Unfortunately, this formalism does not correspond to the operation of the neural system. Specifically, the neural system does not sense the hydrogen ion level present in the saliva ( a Lowry-Bronsted acid concept, 1923), it senses the presence of the carboxyl ligand of the organic acid anion. It senses the carboxyl ligand according to the Lewis (1916) definition of an acid, The situation is more complex, the sensing involves two coordinate bonds, each involving the sharing of an electron pair, but no net charge transfer between the stimulant and the receptor. Thus, no chemical reaction is involved. The coordinate bonds are fragile and temporary in character.

Similarly, the system does not sense the presence of a salt, or even the presence of sodium per se. It + senses the presence of the specifically hydrated form, Na(H20)6 of the sodium ion only. It specifically does not sense the presence of the chlorine ion in solution. The operation of the modality explains a critical aspect related to mono-sodium glutamate. This compound interacts with the hydrated sodium receptor and potentially both the glucophore and organic acid receptors. It does not interact with an independent umami sensory receptor. Figure 8.5.1-10 provides a necessary conversion process if one is to transition from a behavioral to a more fundamental understanding of the gustatory modality. No chemical reactions are involved in gustation; no residues are formed. Only temporary coordinate chemical bonds are involved. The “acid” sensory channel does not sense a proton; it operates only in the Lewis acid sense in order to sense a carboxyl ligand. 94 Neurons & the Nervous System

Figure 8.5.1-10 The transition from behavioral to fundamental perspectives in gustation. All the gustaphores exhibit an AH,B molecular configuration and suitable stereographic configuration capable of forming a dual hydrogen bond with a receptor.

To be clear, the hydrogen ion, H+, is not sensed by the gustatory modality. The gustatory modality senses an AH,B stereochemical configuration capable of forming a dual hydrogen bond with a d = 1.4 Angstrom dimension. This is the natural configuration of the carboxyl group.

Similarly, the “sweet” sensory channel does not sense the features typically associated with a sugar; it senses the glucol group associated with a wide variety of organic compounds. The “salty” sensory receptor does not sense a salt; it senses only the fully hydrated sodium ion. The “bitter” sensory receptor does not sense any feature uniquely related to the quinine molecule; it senses the picrophore, a pair of oxygen atoms separated by a three-carbon chain. [xxx confirm this, important thing is distance of 4.2 Angstrom, not # of carbons per se ] There is no “umami” sensory receptor, the typical umami stimulant, stimulates more than one of the above sensory receptor channels.

The gustatory section concludes with:

The realization that the gustatory receptors depend on specific steric distances between orbitals capable of sharing electron-pairs and not upon the functional groups within a molecule or the interconnection of those groups.

The hydrated state of any cation is critically important to the gustatory transduction process.

When monosodium glutamate is ionized and hydrated in solution, it is capable of stimulating three of the four sensory receptors, the acid, the sweet and the sodium receptors. There is no evidence for a separate and unique umami sensory channel. It has been reported by Beets that sodium benzoate can elicit sensations of sweet, bitter, sour, salty and/or tasteless160. However, the elicited sensations appear to vary significantly among individuals. The following discussion does not surface how sodium benzoate could elicit a bitter or tasteless response. 8.5.1.1 Extending the chemoreception concepts of Shallenberger, Kier and Beets

After Shallenberger et al. and Kier explored the coordinate bond pair concept in the context of the elicitation of a sweet sensation, Beets (1978, pg 188) suggested this concept could be extended to

160Beets, M. (1978) Op. Cit. pg 175 Signal Generation & Processing 8- 95 include all of the organic taste stimulants. This work will go farther and show that the concept also explains the major inorganic stimulant class, those eliciting a “salty” sensation. It will show the salty sensation is actually the sensation elicited by the hydrated form of the sodium ion acting as a stimulant. The salty sensation is actually elicited by both bases and salts of the sodium ion in hydration. However the activity of most bases containing sodium are too active chemically to be used in gustation. In section 8.6, it will be shown that the same concepts apply to the modality of olfaction, with similar constraints related to the chemical activity of the phenols. While the phenols can cause injury if employed in olfaction experiments, their derivatives play an important role in the field of perfumery.

8.5.1.2 The anatomy of the peripheral portion of the gustatory modality

A problem related to taste is the variation in sensitivity to different stimuli with location on the tongue. Age is known to play a significant role in taste, with many senior citizens experiencing a loss in taste sensitivity. Children are believed to exhibit an enhanced taste sensitivity possibly related to sensory cells covering a larger areas of the oral cavity. Eyzaguirre & Fidone have described the sensitivity of the human tongue using Figure 8.5.1-11 161. The sour taste is located at both edges of the tongue, salt and sweet at the tip of the tongue and bitter at the base of the tongue. Squire et al. (2003, pages 632-635) provide additional detail concerning the location of the gustatory sensory neurons. Johnson has provided more details of the human tongue and its connection to the main nerves. Johnson also provides a comprehensive posterior view of the palo- and neo-cortex that places the various neural paths in the brain associated with the gustatory modality in perspective.

Bartoshuk has questioned diagrams such as this based on its probable origination in 1901

Figure 8.5.1-11 Semi-schematic representation of the tongue that is archaic (see text). The location of different sensory modalities and the areas innervated by different cranial nerves (V, VII & IX) are shown. The vallate papillae are located in the region marked by the dashed lines. From Eyzaguirre & Fidone, 1975.

161Eyzguirre, C. & Fidone, S. (1975) Physiology of the Nervous System, 2nd Ed. Chicago, Il: Yearbook Publishers page 146 96 Neurons & the Nervous System and on their experiments162. He noted the presentation suggests an all-or-nothing response for different areas. He also notes that was not the intent of the original author. “If taste qualities were arranged chemotopically on the tongue such that sweet receptors were found on the front, then damage to the chorda tympani nerve would selectively impair one’s ability to taste sweet. Not only does this not occur, damage to the chorda tympani often produces virtually no change in the subjective taste world of the patient.” He provided an alternate figure describing the sensitivity of the tongue showing the high degree of overlap between the sensations throughout the surface of the tongue. The rough character of the tongue is due to the multiple papillae covering the forward sector of the tongue. Each papilla contains about 250 individual taste buds. Each taste bud forms a cavity containing a number of individual gustatory sensory neurons. The sensory neurons in each bud may sense different stimulants.

- - -- Mistretta studied the effects of age on the morphology of gustation and the number of gustatory sensory neurons in humans and other mammals163. She concluded, “The recent data from quantitative studies of taste buds in old humans, rhesus monkeys, and rats complement neurophysiological data on taste responses from aged rats and lead to the general conclusion that the peripheral taste system is maintained structurally and functionally across the life span.

The number of the taste buds varies immensely with species. Kare has compiled a simple table illustrating this variation164.

8.5.1.3 The morphology of the taste bud and sensory neuron

Figure 8.5.1-12 shows a modified figure from Mistretta (page 288), who modified a figure from Murray based on his photomicrographs165. Mistretta eliminated the myelination of the stage 3 neurons and labeled the hydraulic seals as tight junctions (which could be misinterpreted). She also annotated the Murray figure. The figure has been further annotated to show the presence of a somatosensory neuron as added to the Murray figure by Finger & Simon166. The figure as reproduced here is at a magnification of about 2100x.

The typical taste bud contains 5-50 sensory neurons and a number of supporting cells. It is believed that some of these supporting cells are responsible for the phagocytosis of non-functional sensory neurons at the end of their life expectancy (nominally 200 hours)167.

Murray began the practice of numbering the sensory neurons based on their reflected appearance

162Bartoxhuk, L. (1993) Genetic and pathological taste variation: what can we learn from animal models and human disease? In Margolis, F. ed. The Molecular Basis of Smell and Taste Transduction. NY: John Wiley & Sons pp 251-267

163Mistretta, C. (1989) Anatomy and Neurophysiology of the Taste System in Aged Animals Annal NY Acad Sci vol 561, pp 277-290

164Kare, M. (1971) Comparative study of taste In Beidler, L. ed. Taste: Handbook of Sensory Physiology, Vol IV, Part 2 Chap 13

165Murray, R. (1973) The ultrastructure of taste buds In Friedmann, I. ed. The Ultrastructure of Sensory Organs. NY: American Elsevier Chapter 1

166Finger, T. & Simon, S. (2000) Cell biology of taste epithelium In Finger, T. Silver, W. & Restrepo, D. eds. (2000) The Neurobiology of taste and smell. NY: Wiley-Liss Chapter 12, page 293

167Suzucki, Y. Takeda, M. Obara, N. & Nagai,Y.(1996) Phagocytic cells in the taste buds of rat circumvallate papillae after denervation Chem Senses vol 21, pp 467-476 Signal Generation & Processing 8- 97 after uranyl acetate staining; some stained cells appeared darker than others, Mistretta chose not to adopt this notation.

Figure 8.5.1-12 Morphological features of the mammalian taste bud. The analog waveforms from the sensory neurons are converted to stage 3 projection waveforms at the first node of Ranvier where the myelination begins. A second node of Ranvier is shown schematically as labeled. Note the hydraulic seals that protect the neural system from potential contamination. Note also the proximity of a somatosensory neuron. See text. Modified from an original in Murray, 1973.

Murray has provided a variety of actual micrographs of mammalian sensory neurons. However, they are difficult to interpret without considerable experience. A Figure 8.5.1-13 from Finger & Simon168,

168Finger, T. & Simon, S. (2000) Cell biology of taste epithelium In Finger, T. Silver, W. & restrepo, D. eds. The Neurobiology of Taste and Smell. NY: Wiley-Liss Chapter 12, page 290 98 Neurons & the Nervous System modified from Singh169, shows the same cytology of a gustatory sensory neuron but from a fly Drosophila. The importance of this figure is that it shows internal lemma as expected to support the electrolytic circuit of the neuron proposed by the Electrolytic Theory of the Neuron. It also shows the interstitial cells supporting homeostasis, and probably phagocytosis of the no longer functional sensory neurons.

The “outer dendritic segment” is not shown in enough detail in the figure for the purpose of this discussion. Figure 8.5.1-14 provides a more detailed view of mature mammalian dendritic segments in perspective view from Murray (page 55). Each dendritic segment ends in one or more structures called a colax. Each colax contains a group of 9(2) + 2 microvilli that emanate from its terminal surface just as they do in photoreceptor sensory neurons. The two in parenthesis indicates an individual pair of clsoely spaced cylindrical structures. The nine represents the arrangements of these cylindrical pairs around the center pair. Murray shows actual electron micrographs of these structures at 40,000x (in plan view).

The size of the microvilli remains open to discussion because of the difficulty in measuring them precisely. Murray (page 8) suggests they are about 100-200 millimicrons (nanometers) in width and 1-2 microns long. He notes others have set a minimum diameter of 50 millimicrons. Molecular structures do not exhibit hard edges at these scales. Murray has estimated the total number of microvilli at 30-40 with another investigator suggesting 5-9 microvilli from each colax. Murray notes Sclazi (1967) reported the microvilli were coated with a material identified as polysacchride in nature. More recently, the coating has been described as present in “rafts” on the surface. Figure 8.5.1-13 Tracing of a gustatory sensory neuron showing internal lemma. Cell is from the Beets (1973, pg 229) has given similar fly, Drosophila. See text. From Finger & Simon, dimensions for the microvilli, 0.12 microns 2000. wide and 2 microns long projecting into the pore. The axons of the sensory receptor cells are even more difficult to measure, as many have diameters at or below the resolution limit of light microscopy. Many originating at sensory cells have been

169Singh, R. (1997) Neurobiology of the gustatory systems of Drosophila and some terrestrial insects Micro Res Tech vol 39(6), pp 547-563 Signal Generation & Processing 8- 99 measured with diameters in the 0.05 to 0.5 micron diameter range. An average diameter is about 0.2 microns. 100 Neurons & the Nervous System

Figure 8.5.1-14 Two views of the colax and microvilli of a gustatory sensory neuron of rabbit. The individual microvilli emanate from individual colax structures in groups of up to nine. See text. From Murray, 1973. Signal Generation & Processing 8- 101 8.5.1.3.1 The structure of the “sweet” lemma of the microvilli

Glendinning et al. have discussed the sweeteners (page 322), “These taste stimuli include sugars, sugar alcohols, D-amino acids, and some proteins.” They also note, “There are no generally agreed upon molecular features that are essential for a compound to elicit sweet taste.” This work takes a different view based primarily on the detailed analyses of Shallenberger & Acree and of Kier. Beidler has provided a highly conceptual view of the surface of a microvilli and a proposed mechanism of transduction170. The microvilli, or “curly hairs” of the sensory neurons is approximately xxx in diameter and xxx microns long. At the Angstrom level, the wall of the microvilli can be considered a flat surface. The microvilli wall is specialized and consists of a sandwich of the outer or plasmalemma and the reticulolemma of the typical dendrite in close proximity. The total wall thickness is on the order of 160 Angstrom. Figure 8.5.1-15 shows the cross section of the proposed microvilli lemma of the gustatory sensory neuron. Dowhan & Bogdanov reproduce a figure remarkably similar to the right half of this figure171.

The polar heads of the globosides are the presumed receptors of the S-best sensory neurons. They are known to concentrate in “rafts” on the surface of the outer lemma. A better analogy is a “bunch of grass sprouts” emanating from the space between the bilayers of the lemma. Each exposed blade of grass consists of the polar head of the globoside molecule of the outer bi-leaf of the plasmalemma. The globosides consist of a pair of lipid chains occupying a cylinder about 9.7 Angstrom in diameter. The polar heads are much larger due to the array of sugars they contain. The diameter of the heads are unknown until their detailed structural orientation is determined. The polar heads Figure 8.5.1-15 The cross section of “sweet” are shown piercing the nominal surface of the microvilli EDIT lemma. REPLACE OR microvilli into the pore space and consisting of CONSOLIDATE WITH FIG FROM 8.5.5. The type three galactose sugars. Each galactose sugar 2 lemma forms an Activa within the local area of the provides one AH,B coordination site (shown as lemma. The type 4 lemma forms a diode connecting a white disk). The precise stereographic to the liquid crystalline water in the interstitial arrangement of the polar heads of the space acting as an electrical conductor. See text. globoside molecules relative to the lipid body is unknown at this time. However, the polar heads are known to form “rafts” on the external surface of the outer bilayer of the lemma. The glycophores are negatively charged at pH 7.0 and attract positively charged ligands in solution. The definition of a raft used in this work differs from that in Vance & Vance (2002, page 29). The raft of interest here consists of only the phosphatidyl molecules forming the sensory receptors of the various sensory channels that are embedded in the outer bilayer of type 4 lemma associated with the microvilli of the sensory neurons. The globosides are also known to have surfactant properties (Merck Index), although this may not be relevant when the nonpolar portion of the molecule is embedded in a liquid crystalline leaf of the microvilli lemma. As noted, the globosides are a major constituent of the type 4 lemma area of sensory neurons. The type 2 lemma area shown is capped by the electrostenolytic area providing power to this portion of

170Beidler, L. (1971) Taste receptor stimulation with salts and acids In Beidler, L. ed. Handbook of Sensory Physiology, Vol 4/1: Olfactory Sensing. Chap 11

171Dowhan, W. & Bogdanov, M. (2002) Functional roles of lipids in membranes In Vance, D. & Vance, J. eds. Biochemistry of Lipids, Lipoproteins and Membranes. 4th Ed. NY: Elsevier Chapter 1 102 Neurons & the Nervous System the sensory neuron. It is intimately associated with an Activa formed within the type 4 area. This Activa is in intimate electrical contact with the type 2 area via the liquid crystalline water shown shaded. The electrolytic aspects of these structures will be discussed in more detail below. The microvilli of the gustatory sensory neurons, like the microvilli and cilia of all sensory neurons do not contain any pores for the passage of positive alkali or alkali ions. Nor do they contain any GPCR’s involved in the transduction process. Signal Generation & Processing 8- 103 Figure 8.5.1-16 shows the electrophysiology of the “sweet” lemma shown above in greater detail. The microvilli lemma leaf closest to the dendroplasm is proposed to be primarily insulating except for the region of type 2 lemma where it is acts as an electrolytic diode. The leaf contacting the mucosa of the lingual pore is insulating in the type 1 areas and acts as an electrolytic diode in the type 2 and 4 areas. The bilayer acts as an active electrolytic device, an Activa, in the type 2 region when biased by the electrostenolytic process shown at the top of the type 2 column. The current through this Activa is controlled by the electrical potential of the hydronium of the type 2 area via the horizontal shaded area from the type 4 lemma column. In this instance, hydronium refers to the liquid n crystalline form of hydrogen-bonded water (H20) , and not the simpler ionic form of hydrated hydrogen + (H3O) discussed in many introductory textbooks. The current that passes through the diode of the outer leaf is determined by the chemical change in the polar head of the globoside (or cerboside) forming the leaf in this region. In the absence of any attack by a stimulus, the surface of the globoside in this area is polarized by the electrical potential of the hydronium at -24 mV and the potential of the polar head of the globoside combined. This net potential may have a significant impact on the stimulus/globoside interaction. The high charge gain of this configuration will be developed in the following section.

Figure 8.5.1-16 The electrophysiology of the gustatory “sweet” microvilli MOD UPPER RIGHT. This is the same histological and electrophysiology diagram as used for the olfactory sensory neurons. The shaded area on the left shows the electrical circuit configuration of the shaded histological area on the right. The C/D impulse response of this structure is shown at lower left (without considering any other circuit elements. See text.

[xxx the diagram assumes capacitive coupling instead of a dipole potential as source of drive to Activa. ] 104 Neurons & the Nervous System

The histological arrangement in the above figure is reminiscent of the simpler triune receptor concept of Hucho172, which was presented to, but has not gained traction in the pharmacology community. It differs from his type–I receptors involving a single protein bridging the lemma and his type–II receptors which involve three proteins, an ‘R’ on the external surface of the lemma, an ‘E’ on the inner surface and a ‘T’ connecting the two and defined as a coupling or G-protein. In this case, his concept is redefined as no proteins are involved. 8.5.1.3.2 The electrophysiology of the “sweet” gustatory sensory neuron

The physical similarity of all of the sensory neurons is impressive. The major morphological difference is whether they have extended length axons (like the visual and olfactory sensory neurons) or whether the pedicle of the sensory neurons is on the surface of the soma (like the auditory and gustatory sensory neurons). Based on these cytological similarities, Figure 8.5.1-17 shows the proposed electrophysiology of the gustatory sensory neuron. It is virtually identical to the auditory and olfaction sensory neurons and varies only in the basic transduction area from the visual sensory neurons.

172Hucho, F. (1993) Transmitter receptors–general principles and nomenclature In Hucho, F. ed. Neurotransmitter Receptors. NY: Elsevier page 7 Signal Generation & Processing 8- 105

Figure 8.5.1-17 Candidate cytology & electrophysiology schematics of the gustatory sensory neuron DUMMY ADD. The similarity to the other sensory neurons should be clear.

- - - - 106 Neurons & the Nervous System

. 8.5.1 ??? Renewal of the gustatory sensory neurons FIX COUNTER

Van der Heijden has noted, “The taste cells are renewed continuously; the renewal cycle occurs in 10 days to 2 weeks173.” This is the same renewal cycle as found in the visual and auditory sensory neurons. 8.5.1.1xxx How are taste sensations defined?

The chemical-sensing oriented portion of the psychophysical community has long speculated on the functional organization of the gustatory and olfactory systems. A major problem has been defining what is sensed by the gustatory and olfactory modalities. Initially, only the perceived responses of humans, as computed in stage 5 and expressed using stage 6 of the neural system, could be documented. More recently, electronic techniques have allowed recording sensations, in stages 1 through 4 within the neural system. However, no rational description of the ordering of gustatory or olfactory responses has been defined. Nor has the number of “dimensions” associated with that ordering been established. Squire et al. (pages 642-645) have provided a discussion of this situation. Pfaffmann et al. have discussed the delineation of the gustatory responses to different stimuli into four classes described as;

S group–sweet or sugary N group– salty, and specifically salty based on the presence of Na+ ion. Q group– bitter, letter derived from the relevance of quinine H group– acidic. U group–umami, a taste apparently unique to monosodium glutamate in some investigator’s eyes.

Johnson (page 753) has provided a discussion of these properties. Such properties are always discussed in narrative form and no two authors agree at the detail level.

Some investigators have attempted to define a unique channel, the umami channel, associated with mono-sodium glutamate. However, this compound appears to be a typical sodium-based member of the N group. An explicit unique definition of the perception of umami has not been found in the literature.

Squire et al. note (2003, page 613), “Most taste stimuli are hydrophilic molecules, including Na+ salt (salty), divalent salts, and KCl (salty and bitter), acids (sour), sugars (sweet), amino acids (sweet, bitter, and umami), and proteins (sweet and bitter). Some taste stimuli are lipophilic, including the bitter-tasting alkaloids and many synthetic sweeteners.”

Smith & Davis have provided a different behavioral perspective174, “These qualities and the behavior associated with them provide the means by which an animal makes ingestive decisions. Through these qualities, taste help to ensure the animals’ energy supply (sweet), maintain the proper electrolyte balance (salt), and avoid the ingestion of toxic substances (sour, bitter)”

A major area of study has been limited to studying the ionic portion of the chemical spectrum. Another major area has been focused on the study of organic chemicals, primarily but not exclusively the simple sugars. While Spector and Travers have conceptually recognized two broad classes of receptors, these two areas of study have yet to converge on a comprehensive functional model at a detailed level. They noted in 2005 (page 177), “For all of the behavioral and electrophysiological work

173Van der Heijden, A. (1993) Sweet and bitter tastes In Acree, T. & Teranishi, R. eds. Flavor Science. Washington, DC: American Chemical Society Chapter 3

174Smith, D. & Davis, B. (2000) Neural representation of taste In Finger, T. Silver, W. & Restrepo, D. eds. (2000) The Neurobiology of taste and smell, 2nd Ed.. NY: Wiley-Liss Chapter 14 Signal Generation & Processing 8- 107 that has been conducted to date, it is revealing that definitive evidence distinguishing various modes of neural coding of taste quality remains to be seen.” Many anecdotal remarks also appear in the literature but these have not generally been incorporated into a comprehensive functional model either. Hudspeth & Tanaka provided a brief review that only touched on the chemical senses, taste and olfaction175. They note the classical concept suggests the taste sensations of sweetness, sourness, saltyness and bitterness. They quickly note the sensitivity of the human system to fat as well. Many alternates to this brief listing have been proposed. Nearly a century ago, Ikeda suggested an additional axis to the taste map involving glutamate (or more specifically monosodium glutamate)176. He named the sensation associated with glutamate umami177. Sugimoto & Ninomiya have provided a review of more recent work related to umami. Axel & Buck suggested a very large number of independent olfactory sensor types. They inferred several relationships between the olfactory system and over 1000 genes. xxx add reference and brief comments re: sensing organic chemicals xxx

8.5.2.2 What are the structural constraints on tasting sweet?

Shallenberger & Acree presented a brief theory of sweetness in 1967 based on the presence of a glycol group in the stimulus178. It stated the initial interaction “is neither a proton transfer nor an electrostatic interaction, but probably involves London dispersion, the principle element of hydrogen bonds.” However, it is important to note the in vitro experiment involved neutral chemicals dispersed in water and not attached to any neural system. A much more comprehensive paper appeared in 1971179. After the appearance of a paper by Kier, Shallenberger & Lindley discussed a number of secondary structural features that affect the intensity of the sensation of sweetness180. The paper considered many alternatives and is very well structured. It also provided some information on the other basic tastes. Shallenberger presented a very comprehensive discussion of the structural aspects of sweetness in 1982 that appears to support the electrostatic premise181. In 1996, he provided a synopsis of his work based on an electrostatic assumption, i.e., that no reaction between the stimulus and the receptor occurred182. In 2000, Eggers, Acree & Shallenberger provided a review of their work over a 30 year span183. The critical nature of the specific conformation of a odorant was highlighted by Shallenberger. The Anti- anisaldehyde oxime is very sweet, yet its fraternal twin, Syn-anisaldehyde oxime, is tasteless.

175Hudspeth, A. & Tanaka, K. (2000) Sensory systems: editorial overview Curr Opin Neurobiol vol xxx, pp 443-446

176Ikeda, K. (2002) New Seasonings Chem Senses vol 27, pp 847-849 A reprint of original

177Sugimoto, K. & Ninomiya, Y. (2005) Introductory remarks on umami research: candidate receptors and signal transduction mechanisms on umami Chem Senses vol 30(suppl 1) pp i21-i22

178Shallenberger, R. & Acree, T. (1967) Molecular theory of sweet taste Nature vol 216, pp 480-482

179Shallenberger, R. & Acree, T. (1971) Chemical structure of compounds and their sweet and bitter taste In Beidler, L. ed. Taste: Handbook of Sensory Physiology, Vol IV, Part 2, Chap 12

180Shallenberger, R. & Lindley, M. (1977) A lipophilic-hydrophobic attribute and component in the stereochemistry of sweetness Food Chem vol 77(2), pp 145-153

181Shallenberger, R. (1982) Advanced Sugar Chemistry. Westport, CT: AVI Publishing Chapter 10

182Shallenberger, R. (1996) The AH,B glycophore and general taste chemistry Food Chem vol 56(3), pp 209-214

183Eggers, S. Acree, T. & Shallenberger, R. (2000) Sweetness chemoreception theory and sweetness transduction Food Chem vol 68(1), pp 45-49 108 Neurons & the Nervous System

Most recently, Erickson has written on the core ideas of taste184 and has drawn many comments. The paper contained no diagrams and provided little new information on the physiology of the gustatory modality. 8.5.2.3 The gustatory response versus molarity of stimuli

The output as a function of input intensity has proven very important in understanding the operation of the visual modality. Zotterman has provided data on the gustatory response of humans based on recordings of taste nerves185 with most of the data attributed to Borg et al. (1967). They confirmed their integrated responses from the chorda tympani relative to the molarity of citric acid exhibited the same slope on log-log paper as psychophysical responses, 0.5. They then showed the slope of N-best channels exhibited a slope of 1.0 while the slope of H-best channels showed a slope of nominally 0.67 psychophysically for 14 students. In parallel experiments, the S-best channels showed a slope near 1.0 and higher than that of the H-best channel. This data is very similar to that provided by Sato for the rat186. Sato more carefully recorded the saturation affects encountered at molarities near and above 0.1 M. Several of the figures in Sato and in Borg et al. show significant saturation that cannot be interpreted using a straight line as an overlay. No clear threshold levels were documented. The size of the database suggests more experiments are needed and saturation must be accounted for in reducing the data.

8.5.2 Analysis of perceived gustatory sensations–Multidimensional scaling applied to gustation

Three tools have emerged as the principle means of understanding the taste performance of the gustatory modality. As noted by many, taste is a perceived sensation that may not be due purely to gustatory sensory neurons. In many cases, the olfactory sensory neurons appear to contribute a major portion of the overall taste experience. The somatosensory neurons of the oral cavity appear to also play a significant role.

In common with other sensory modalities, ethical and cultural restrictions have limited the collection of data from the human species related to gustation, other than that from psychophysical experiments. As a result, the major data bases are based on behavioral data from the hamster, rat and occasional monkey.

The first goal of any gustatory analysis is to determine the minimum number of distinct sensory channel types required for a species to achieve its desired performance. This requires the collection of data using a variety of stimulus types. To interpret the available data, dendrograms and both two and three dimensional histograms have proven the most useful. 8.5.2.1 Dendrographic representation

Figure 8.5.2-1 shows a typical dendrogram from Smith et al. The figure is more complete than most and includes the designations on the right applicable to individual neurons in the set. However, it is less complete than some in only showing the clustering for three situations, S or sweet-best, N or NaCl-best and H or HCl-best. Note the perceived best response of the individual neurons listed on the right does not always correspond to the clustering on the left. This is a

184Erickson, R. (2008) A study of the science of taste: On the origins and influence of the core ideas vol 31, pp 59–105

185Zotterman, Y. (1971) The recording of the electrical response from human taste nerves In Beidler, L. ed. Handbook of Sensory Physiology, Vol 4/1: Olfactory Sensing. Chap 6

186Sato, M. (1971) Neural coding in taste as seen from recordings from peripheral receptors and nerves In Beidler, L. ed. Handbook of Sensory Physiology, Vol 4/1: Olfactory Sensing. Chap 7 Signal Generation & Processing 8- 109 problem in experimental protocol development and may also be confounded by the lack of a sufficiently broad range of stimuli to uncover the true best response of specific neurons. Unless a very large set of neurons is used, the statistical independence of the individual clusters is not assured as the right side of the figure is approached. In discussing this and similar figures (page , Smith & Davis employ three best conditions in this figure, four best conditions in immediately adjacent figures (adding quinone or Q-best) and at least provide marginal support for a fifth best condition (adding U or umami-best). They also note, citing several references, “Individual gustatory neurons, both peripheral and central, typically respond to stimuli representing several different taste qualities.” The abscissa is scaled using values from a fundamentally abstract analytical routine, which makes it difficult to compare, and account for the significant differences in, the work of different investigators. The cluster diagram does not provide any left terminus. The midpoint of the left-most vertical line can be assumed to represent the sum of the entire ensemble.

A similar, but more recent, dendrogram for a primate from Scott & Plata-Salaman omits the labeling of the best performance of the individual neurons but provides a different orthogonal scale from Smith. The scale remains abstract with both positive and negative values. It includes, S-best, N-best, Q-best and H-best categories. They have also added heavy lines attempting to show the level at which the different clusters achieve statistical independence. Their analysis shows that ~73% of the primate neural responses are to sensations described as sweet or salty. Those tuned to quinine constituted ~22% and only ~5% were oriented toward the detection of acids.

Brining et al187. examined the sensitivity of hamsters and describe the distance between their clusters in more detail than Smith et al. did.

8.5.2.2 ROC analysis

Lemon & Smith have recently consolidated their database and applied the “receiver operating characteristic (ROC) analysis technique of Green & Swets (1966) to action Figure 8.5.2-1 Cluster dendrogram of 31 hamster potential recordings from 162 (stge 3) PbN neurons based on similarities in their neural neurons at undefined locations on/within the response profiles across 18 gustatory stimuli. The NST of rats188. Their results included neuron numbers and their best-stimulus measured and standard deviation values. designations (S, H, or N) are shown on the right. Their reported spike rates were from a few The distance between neurons or cluster of neurons to less than 75 pps. They used an AC joined at each step is shown along the abscissa. S, coupled oscilloscope that degraded the form H, & N indicate the three major clusters. From of their action potentials significantly. They Smith et al., 1983. applied their stimuli to the apical tongue at

187Brining, S. Belecky, T. & Smith, D. (1991) Taste Reactivity in the Hamster Physiol Behav vol 49, pp. 1265-1272

188Lemon, C. & Smith, D. (2006) Influence of response variability on the coding performance of central gustatory neurons J Neurophysiol vol. 26(28), pp 7433-7443 110 Neurons & the Nervous System concentrations chosen to provide half-maximum response in the chorda tympani nerve. In applying the ROC procedure, Lemon & Smith did not report any neurons where the action pulses exhibited a high spontaneous discharge rate or where the action pulse rate decreased with stimulation of the sensory neurons. These facts suggest the neurons probed were either at the entrance to the NST or were at the exit and did not display any differential encoding. Their discussion suggests they did encounter variation in sensory neuron sensitivity or application protocol inconsistency that led them to assert, “Spike rate was found to be an unreliable predictor of stimulus quality for each neuron tested.” They also concluded, “Results revealed poor classification performance in some cases attributable to wide variations in the sensitivities of of neurons that compose a cell type.” These findings are not expected based on the very great adaptation capability of typical sensory neurons and the poor repeatability of stimulation of a specific neuron within a taste bud. In their discussion, they considered a number of stage 4 computational strategies without presenting any schematic of the appropriate sections of the neural system (or even a semantic model).

MacMillan & Creelman189 have recently published “Detection theory: A User’s Guide.” for those interested in exploring the ROC technique in detail.

189MacMillan, N. & Creelman, C. (1991) Detection Theory: A User’s Guide. NY: Cambridge Univ Press Signal Generation & Processing 8- 111

8.5.2.2 Two dimensional representation applied to gustation

A two-dimensional presentation can be obtained based on multidimensional analysis. This form provides the clearest separation of the perceived responses into distinct groups. Figure 8.5.2-2 shows such a presentation from Brining. The precise definition of position on this presentation is complex. The presentation shows there are two distinct perpendicular “dimensions” to the data. However, there is no defined orientation of the axes in this mode of analysis. The axes shown are arranged arbitrarily. The range of the data is scaled by the mathematical tools with the circle shown with a unit radius. It is interesting to note the location of the chlorine radical appears to be independent of these two dimensions. The sugars form a tight group isolated from the other less tightly grouped stimuli. The hydrogen ion present in the quinine hydrochloride sample appears to play no functional role in that stimulus. Smith & Scott (Doty, page 733) have noted the stimuli would be shown on the unit circle if their characteristics were determined exclusively by the two “dimensions.” Clearly, the N-best representations include an additional dimension while the other selected stimuli provide responses dominated by the two dimensions shown. This is a serious limitation of a two dimensional representation of data containing additional dimensions. This limitation clearly indicates the data needs to be replotted.

8.5.2.3 Multidimensional scaling applied to gustation

An adequate multidimensional scaling analysis of the taste system can provide strong evidence for resolving the question of the number of independent sensory Figure 8.5.2-2 Two-dimensional histogram of channels. A problem involves the is extreme hamster taste preferences for 12 different stimuli. sensitivity of the scaling procedure to the The data needs to be replotted in three dimensions. scope of the stimuli set. In 1963, Gesteland See text. From Brining et al., 1991. et al. distinguished eight types of neuroreceptors based on one set of criteria190. Holley et al in 1974 seven types of very selective cells and nine “categories” of less selective ones191. The multidimensional scaling technique has matured in recent years. Revial et al. used the technique to describe their data set obtained from the frog192. This set included the camphors and the benzenes and at least one thiophene (a class of sulfur compounds). “Data analysis revealed five factors that described 71% of the total variance, with one factor markedly prevailing over the other four.” “In other words, the olfactory space is multi-dimensional but, in the case of this particular set of compounds, one dimension is dominating.” [xxx this paragraph needs integration to previously numbered section ] More recently still, Smith et al. have provided a broad investigation into the gustatory modality in

190Gesteland, R. Lettvin, J. Pitts, W. & Rojas, A. (1963) Odour specificities of the frog’s olfactory receptors in Zotterman, ed. Olfaction and Taste I. London: Pergamon

191Holley, A. Duchamp, A. Revial, M. Juge, A. & Macleod, P. (1974) Qualitative and quantitative discrimination in the from olfactory receptors An N. Y. Acad Sci vol 237, pp 102-114

192Revial, M. Duchamp, A. Holley, A. & Cacleod, P. (1978) Odour discrimination by from olfactory receptors; a second study Chem Sens Flav vol 3, pp 7-22 & 23-33 112 Neurons & the Nervous System the hamster193. The more limited scope of the taste sensation was characterized using only three dimensions, except for one outlier representing quinine hydrochloride. By stretching the QHCl criteria, they placed it in the acid (H) response class. Using this criteria, multidimensional scaling suggested the taste sensations formed into three groups when observed in elements of the brainstem, sugar sensitive S-neurons, salt sensitive N-neurons and acid sensitive H-neurons that could be displayed in a two-dimensional space. These results support the conclusion that the answers investigators obtain in chemoreceptor research generally depend on what sample set they used. Figure 8.5.2-3 shows the result of such a multidimensional scaling analysis of glossopharyngeal nerve (NG) fiber of the rhesus monkey data by Hellekant, et al194. This analysis asserts there are three “dimensions” to the stimulus sensing mechanism in the mammalian gustatory system. Each of these dimensions constitutes a value continuum that is orthogonal to the other two dimensions. The challenge is to define these continua using sufficiently precise terminology. The figure also highlights the fact that these dimensions are continua in character and not binary. Similar recordings from many others195,196,197 have used a three-dimensional space to describe the perceived chemoreceptor space, at least for taste. However, both groups omitted several significant groups of stimulants. Using these additional groups may require additional dimensions to provide an appropriate framework for describing this wider set of stimulants.

Hellekant has written extensively on monkeys beginning in 1975.

193Smith, D. & Davis, B. (2000) Neural representation of taste In Finger, T. Silver, W. & Restrepo, D. eds. (2000) The Neurobiology of taste and smell, 2nd Ed.. NY: Wiley-Liss Chapter 14

194Hellekant, G. Danilova, V. & Ninomiya, Y. (1997) Primate sense of taste: behavioral and single chorda tympani and glossopharyngeal nerve fiber recordings in the Rhesus Monkey, Macaca mulatta. J Neurophysiol vol 77, pp 978-993

195Squire, L. et al. (2003) Op. Cit. pg 644

196Smith, D. Van Buskirk, R. Travers, J. & Bieber, S. (1983a) Gustatory neuron types in hamster brain stem J Neurophysiol vol 50(2), pp 522-540

197Smith, D. Van Buskirk, R. Travers, J. & Bieber, S. (1983b) Coding of taste stimuli by hamster brain stem neurons J Neurophysiol vol 50(2), pp 541-558 Signal Generation & Processing 8- 113

Figure 8.5.2-3 Multidimensional scaling of gustatory stimulants in monkey ADD. From figure 11 of Hellekant et al., 1997.

An important feature of the multidimensional scaling procedure is to achieve optimal alignment of the coordinate axes of the presentation. The procedure provides great flexibility in this area. For a review of the technique and references, see Shepard, et. al198. A figure of merit is used in these analyses to estimate the broadness of sensitivity of a specific sensory neuron channel. The figure used is an entropy given by the equation, H = .K(n)AΣpiAlogpi where the coefficient K(n) depends on the

198Shepard, R. Romney, A. & Nerlove, S. (1972) Multidimensional scaling; theory and applications in the behavioral sciences. NY: Seminar Press 114 Neurons & the Nervous System number, n, of stimulants in the set. Smith & Scott have given a brief discussion of this figure of merit199. The value of K(n) is adjusted to insure a maximum value for this function of 1.0.

A problem not addressed in the presentations based on multidimensional scaling relates to the underlying functions generating the three axes. The three-dimensional character of the multidimensional scaling is strikingly similar to that of the visual system200. The optimized dimensions in the visual case relate to the luminance channel and the two major chrominance difference channels of human vision201. These neurophysical channels describe the perceived scene as processed by stages 1 & 2 within the retina. In the case of vision, the two chrominance difference channels result in two of the three dimensions exhibiting zero’s or nulls along their axes. As a result, it is clear the chrominance outputs of the stage 2 signal processing are electrically bipolar signals. Because of the commonality of the spectral channels participation in the differencing, it is even possible to specify the relative polarity of the regions of these signals. If the signal processing in stage 2 of the chemoreceptor channels mimic that found in most other sensory channels, the knobs atop the vertical stems would point both up and down in [Figure 8.5- 1], the presentation would exhibit bipolar data sets. However the CNS does not provide bipolar perceptions. It assigns names, e. g., salty, sugary etc., to perceptions relating to the individual relative peaks in its perceptual space. Whether they were perceive based on bipolar data presented in the saliency map of the parietal lobe or not is irrelevant.

Hellekant et al. made only a brief attempt to define the value continua in this figure. They described dimension 1 as extending from a group of sweeteners in the background of the left side and a group of bitter compound in the foreground. They described dimension 2 as isolating a group consisting of citric and aspartic acid, NaCl, sodium cyclamate and MSG but described no counter to that group. No discussion of the third dimension was offered.

Smith & Scott have provided a similar three-dimensional figure (from Giza & Scott, 1991) showing the effect of amiloride on taste perceptions or rat for a variety of stimulants202.

8.5.2.4 Changes in multidimensional presentation

Giza & Scott have extended the multidimensional gustatory presentation to account for differences in protocol for the same materials. Figure 8.5.2-4 shows the effect of introducing amiloride into the protocol as a pre-wash. [??? xxx. ] Amiloride is a nitrogen and amine heavy cyclic hydrocarbon containing no alkali metal. In its pharmaceutical form, it is combined with HCl and water, C6H8ClN7O•HCl•2H2O, to form a potassium-conserving antikaliuretic-diuretic agent, Whether its activity in gustation is also distinct with respect to potassium is unclear.

This figure was un-dimensioned. The “dimensions” shown have been added for discussion. They approximate those in the above figure with dimension 1 being a Quinine-sugar axis and dimension 2 a sodium-sugar axis. The third dimension has been labeled the quinine-polycose axis for discussion. “Acid” stimuli appear under represented in the figure.

199Smith, D. & Scott, T. (2003) Gustatory neural coding In Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker Chapter 35

200Romney, K. & Indow. T. (2002) A model for the simultaneous analysis of reflectance spectra and basis factors of Munsell color samples under D65. . . Proc Natl Acad Sci USA vol 99, no 17, pp 11543-11546

201Fulton, J. (2005) Processes in Biological Vision. www.sightresearch.net Chapter 17 Part 1b

202Smith, D. & Scott, T. (xxx) Gustatory neural coding In Doty, R. ed. Handbook of Olfaction and Gustation. NY: Marcel Dekker, Chap 35 page 752 Signal Generation & Processing 8- 115 Smith & Scott have reviewed this figure in Doty (page 751). They assert, “taste offers no known analogy to the pathology of color blindness.” Actually, it appears to offer a significant analogy to conventional color vision (when interpreted as in this work (See Section xxx). Color vision can be explained fully using a three-dimensional color space. More approximately, color vision can be explained using an incomplete two-dimensional color space known as the Munsell Color Space. This is similar to the situation in the previous figure. One Smith & Scott comment may need modification. They note, “Blocking the NaCl response of the N-best neurons with amiloride resulted in the inability of the remaining cells to discriminate between sodium and non-sodium salts, . . .” Giza & Smith were more specific, “Responses to all 7 stimuli that contained Na+ or Li+ were suppressed by amiloride.” Even this statement in their summary appears subject to modification as monosodium glutamate was unaffected by the amiloride treatment. In the original paper, Giza & Scott noted, “The most striking findings of this study of NTS taste cells are (1) the effects of amiloride on responsiveness to NaCl and LIC1 are either profound or minimal, depending on the cell, . . . .” A larger statistical sample is needed to resolve the effect on the potassium (another alkali metal) salt data point. It is important to note the work of Ossebaard & Smith regarding amiloride in humans203. They suggest several subtleties indicating a lower effectiveness in humans than in some other species.

203Ossebaard, C. & Smith, D. (1995) Effect of amiloride on the taste of NaCl, Na-gluconate and KCl in humans: implications for Na+ receptor mechanisms Chem Senses vol 20, pp 37-46 116 Neurons & the Nervous System

Figure 8.5.2-4 Changes in three-dimensional taste spaces of one rat caused by amiloride as recorded at the NST. 15 stimuli were used in a before (A) and after (B) experiment where the tongue and palate were washed with amiloride. After amiloride application, all the Na+-bearing stimuli, except MSG, and the lithium salt migrate to the positions of the one organic acid, the non-sodium salts, and quinine. N1-N4, 0.01-0.3 M NaCl; Li, LiCl;.S; sucrose; Sa, Sodium saccharin; G, glucose; H, HCl; Ci, citric acid; Q, quinine hydrocholoride; K, KCl; Ca, CaCl2; M, MSG; P, polycose. From Giza & Scott, 1991.

8.5.2.5 Conclusions from analysis of the data

The collected papers of Schiffman provide the most taste data in one place, even though it has not all been collected and presented in a single multi-dimensional space. Some of the discussion accompanying this data may not be relevant in 2010. Figure 2 of her 2000 paper exhibits some strange artifacts of the graphic arts (example, the leading edge of the E/D response for NaCl leans to the left as it rises). The introduction of that paper also includes several descriptions that may be overly limiting. The individual data sets do not represent a portion of the overall taste Signal Generation & Processing 8- 117 sensation space. They are the result of individual MDS analyses on incomplete datasets. Lemon & Smith have provided data recorded at the NST of 45 adult male Sprague Dawley rats for a limited group of primarily bitter stimuli204. Their protocol did not employ quinine HCl as a stimulus and they grouped acid/bitter in their dendrogram and 3D histogram. Lacking a quinine HCl-like stimulus, their 3D representation is probably distorted based on applying MDS techniques to an incomplete dataset. The conclusion of Giza & Scott appears useful but incomplete in the absence of a group of organic acid stimulants and a larger subject sample size. “The results imply that specific receptors are responsible for the recognition and transduction of sodium salts and that this specificity in the peripheral taste nerves to be manifested in the NTS. The methods of multi-dimensional analysis are quite sophisticated and do not lend themselves to total computerization using a cook-book program. Beginning with Coombs in 1950, great progress has been made in ordering psychological test data using methods that uncover the underlying “dimensionality” of the data. The work of Shepard in the 1960's led to a technique labeled multidimensional analysis205. It could determine the number of relevant underlying dimensions and the character of the monotonic function relating the data points. A feature of Shepards 1962 paper was his analysis of when the technique would fail (p. 240). A more sophisticated technique labeled single value decomposition (SVD) was developing in parallel with Shepard’s version of multidimensional analysis. The SVD method was described in detail, with examples, in Weller & Romney206. A broader review of these and other techniques can be found in Shepard, Romney & Nerlove207.

Shepard asserted in 1962 that these techniques were not practical before the emergence of the high capacity digital computer because of the intense mathematical manipulations required (he used an IBM 7090 which was new in his time period). He could not have known that the fundamental requirements underlying these techniques would be frequently overlooked when it only required pressing a single key to invoke these powerful statistical techniques on a desktop computer.

The histograms plotted using olfactory and gustatory data have used several, but not all of the basis functions resulting from computerized analyses of the above types. In the case of vision, a complete SVD analysis provides four basis functions; a brightness or R–function versus spectral wavelength, and three basis functions (O–, P– and Q–functions) describing three chromatic difference functions versus spectral wavelength. It has been common in the past to ignore the O–function (the ultraviolet component) in vision resulting in an imprecise Q–function. In gustation, it can be expected that omission of the acids as a group in a test protocol will distort the resultant basis functions used to create the above histograms.

Figure 8.5.2-5 shows the underlying theory of the complete three-dimensional Munsell Color Space208. In practice, the lens of the eye truncates the effective spectral range at 400 nm and the two-dimensional Munsell Color Space results. Note carefully that the white point is not along the spectral locus forming the axes of the figure. Similarly, yellow at a nominal wavelength of 572 nm

204Lemon, C. & Smith, D. (2005) Neural representation of bitter taste in the nucleus of the solitary tract J Neurophysiol vol 94, pp 3719–3729,

205Shepard, R. (1962) The analysis of proximities: multidimensional scaling with an unknown distance function Psychometrika vol27(2), pp 125-139 & vol 27(3), pp 219-246

206Weller, S. & Romney, A. (1990) Metric Scaling: Correspondence Analysis. Series #07-075 Newbury Park, Ca: Sage Publications

207Shepard, R. Romney, A. & Nerlove, S. (1972) Multidimensional scaling; theory and applications in the behavioral sciences. NY: Seminar Press

208Fulton, J. (2005) Processes in Biological Vision. http://neuronresearch.net/vision/pdf/17Performance1a.pdf Section 17.3.3.1 118 Neurons & the Nervous System is not a node of the color space; although yellow is frequently perceived as a dominant color at higher cortical levels by combining brightness and chrominance information. A similar taste space is sought from the experimental data base using SVD.

Figure 8.5.2-5 The foundation for the chromaticity diagram of tetrachromatic vision. W marks the location of tetrachromatic “white” in the diagram. W’ shows the location of “white” for a long wavelength trichromat. W” shows the location of “white” for a theoretical short wavelength trichromat. Signal Generation & Processing 8- 119

Without a complete statistically relevant SVD analysis of the taste space in hand, a complete taste space can only be suggested. Figure 8.5.2-6 shows this suggested taste space. This space shows a primary locus of sensory neurons for acids at the zero coordinate point. It shows a second primary locus of sensory neurons for sugars along one axis of the three-dimensional space, another primary locus of sensory neurons for alkali ions along a second orthogonal axis and a final locus of primary sensory neurons for quinines along the third orthogonal axis. From a behavioral perspective, the acid sugar axis might be considered primary, indicating a likely food at its most positive extent and an undesirable acidic condition at its negative extension. Multidimensional analysis of a complete data set shows that Henning’s tetrahedron (1916) is inappropriate. Henning described the four tastes as located at corners of a equilateral tetrahedron, much as Young had done earlier for the perception of color. While conceptually simple, such a representation does not meet the requirements of orthogonality to avoid cross contamination. The use of selected corners of a cube does provide such independence in both areas of perception, taste and color vision. A right tetrahedron (all face angles at one vertex are 90 degrees) can represent the taste space more appropriately.

Note a right tetrahedron drawn within the orthogonal space shown (with its 90 degree vertices at H-best) occupies less than one third of the available taste space proposed here. Other basic tastes could occupy other vertices of a three dimensional cube (See analysis of Schiffman’s paper below). Figure 8.5.2-6 A proposed taste sensation space The proposed coordinate system has been based on an incomplete experimental database. overlaid with the data points from Smith et With a larger sample set, the values shown near al. (1983b, figure 8). The Q-best/N-best each “Best node” would converge on, and define plane has been rotated by 10 degrees from more precisely, each node. The dashed lines define Smith et al. This is a normal procedure and the total 3-D taste sensation space and a right totally permissible in multidimensional tetrahedron satisfying Henning’s description. See analysis. It is assumed that with a larger text. Data points from Smith et al., 1983. and more statistically defendable data set, the data points clustered around each axis would converge on a single value for each class of stimulant thereby defining precisely the locus of the “Best” point for each class of stimulants. The resultant best points would define the peak sensitivity of the underlying sensory neuron types.

[xxx careful with MDS versus SVD ] Smith et al.(1983a) used a very small stimulus data set in their multi-dimensional scaling (MDS) process, including only one stimulant clearly associated with the bitter channel. they used six inorganic compounds that are not relevant to this study. Their response data set only included 31 neurons divided into three groups. It is important to note the data in figure 1 of this paper. The data is from the parabrachial nucleus and not from the actual sensory neurons as might be acquired from nerves VII, IX or X. The distribution of sensitivity among their S–neurons and H–neurons suggest these neurons exhibit the result of a summation process and are not typical distributions of sensory neurons. The power of MDS developed clusters along the expected primary axes, figure 8 of Smith et al. (1983b), but the groupings may be less tight than would be expected of sensory neurons sensing only the organic compounds plus the sodium compounds. As a side note, it is of interest that sodium saccharine did not appear to ionize in solution and thereby influence the N-neurons. 120 Neurons & the Nervous System

The dimensionality of this figure is arbitrary. However, a convention is preferred. The axes shown are those commonly found in three-dimensional presentations following the right-hand rule. The scales of the individual basis functions have been linearly [xxx vectorially ] modified so that the mean of H-best occurs at zero and all other values are positive in all of these functions. This approach surfaces a critical thesis concerning gustation. The precise location and the degree of tuning associated with each descriptor, N-best, Q-best etc. are properties of the sensory neurons. In a statistically adequate group of stimuli. and target systems, these properties are independent of the stimuli employed. While the precise chemistry and the neurological circuitry associated with these scales is unknown, it can be assumed the neurological circuitry at the level of the NST forms differences between the signals from pairs of these nodes (probably limited to pairs along the individual axes). These pairs can be labeled much as they are in the neurological circuitry of color vision space. In the gustatory case, the axes can be associated with; Dimension 1 = N – H (x-axis) Dimension 2 = S – H (y-axis) Dimension 3 = Q – H (z-axis)

In this figure, the mean of the H-best locus is taken as the common junction defined by the basis functions of a new SVD analysis based on a larger, statistically adequate, data set. The salts of the alkali earth metals, Calcium, and Magnesium, generally appear along the axis between the H- best and N-best loci.

This analysis suggests, or supports the hypothesis that, there are four primary types of sensory neurons in gustation; S-best, H-best, N-best and Q-best. All gustatory stimulants excite one or more of these types. It may be useful to note that quinine hydrochloride contains the complicated organic molecule, quinine, which is considered an alkaloid (alkali-like) in many pharmaceutical applications. It does not appear to act as an alkaloid in gustation. Urea is a much simpler carbamide H2NCONH2 and is weakly basic. The data base is not large enough to address the question of a distinct umami sensitive sensory neuron. To expand the theory to include a fifth primary sensory neuron type would require; C development of a statistically relevant data base C a database in which all of the potential sensory neuron types were stimulated and C a SVD analysis to develop all of the relevant basis functions. 8.5.2.6 The basis functions of gustation

The basis functions, included as a step in the preparation of a histogram resulting from a multidimensional analysis or more complex single value decomposition (SVD), can provide a numerical scale for describing the tuning of individual sensory neurons. The SVD method is described in detail, with examples, in Weller & Romney209. A broader review of these and other techniques can be found in Shepard, Romney & Nerlove210. The work of Shepard in the 1960's led to a technique labeled multidimensional analysis211. It could determine the number of relevant underlying dimensions and the character of the monotonic function relating the data points. A feature of Shepards 1962 paper was his analysis of when the technique would fail (p. 240).

While many three-dimensional histograms of gustation have appeared in the literature, the

209Weller, S. & Romney, A. (1990) Metric Scaling: Correspondence Analysis. Series #07-075 Newbury Park, Ca: Sage Publications

210Shepard, R. Romney, A. & Nerlove, S. (1972) Multidimensional scaling; theory and applications in the behavioral sciences. NY: Seminar Press

211Shepard, R. (1962) The analysis of proximites: multidimensional scaling with an unknown distance function Psychometrika vol27(2), pp 125-139 & vol 27(3), pp 219-246 Signal Generation & Processing 8- 121 authors have not generally presented the basis functions used to define their orthogonal axes. In the case of vision, where it is easy to provide a constant amplitude stimulus across the visual spectrum, the basis functions are found to describe the mean location and tuning of the visual sensory neurons precisely (Section 17.3.5.2.4 of “Processes in Biological Vision”). Scott & Giza have collected their data and organized it to suggest sensory profiles in their figure 4. However, the relationship to the basis functions is tenuous. This paper also includes a broad discussion highlighting some of the problems in defining the neural coding of gustation. MDS is a very powerful analytical tool in the hands of an expert212. It involves considerable routine calculation that is amenable to and essentially requires computer support. Unfortunately, the use of a canned computer program in the hands of the naive can lead to beautiful but erroneous conclusions. The figures on pages 347 & 349 of Getchell et al. can be used as an example. The data is plotted in two dimensions, but as the captions note, 5 to 6% of the data is unaccounted for using only two dimensions. The phenomena being analyzed involves a three- dimensional space. By eliminating one of the degrees of freedom in the computer routine, suitable presentations are obtained but they force the data points into a two dimensional space. The rotation of MDS axes is an additional experimental tool in MDS. The axes selected by the computer routine may not be the primal axes of the data. The choice is determined by factors unrelated to the fundamental axes of the data set. This is particularly true if the statistical sample size and experimental error associated with individual odorophores is inadequate.

An argument can be made that the primal axes of the figure on page 347 (credited to Scott & Mark213) should pass through NaCl with one axis extending to Fructose/Sucrose and the other perpendicular to that axis as shown in Figure 8.5.2-7. Using the proposed axes, several important points can be made. C Sucrose is not a suitable sample for these experiments, since it is broken down into glucose and fructose by hydrolyzation in the process of salivation. C Both fructose and sucrose contain multiple odorophores based on their ring structure and their CH2OH groups. Thus, these materials are shown farther along the axis than appropriate due to their higher odorophore concentration than expected by the investigators. C The group of chemicals clustered along the axes sloping to the upper right can be separated into the organic acids in the plane of the paper and the picro-receptor stimulants such as quinine, brucine and strychnine out of the plane of the paper.. C The constituents shown on the lower one-dimensional scale can be replotted relative to the new axes and separated into in-plane and out-of-plane components. C The role of the inorganic molecules in gustation (except those containing sodium) have not been addressed in this work. See Section 8.6.3.4.4 for hydrogen sulphide’s role in olfaction.

212Weller, S. & Romney, A. (1990) Metric Scaling: correspondence analysis. Newbury Park, California: Sage University Press

213Scott, T. & Mark, P. (1987). The taste system encodes stimulus toxicity. Brain Res vol 414(1), pp 197-203. 122 Neurons & the Nervous System

Schiffman has presented a recent paper that provides data on a wider range of stimulants than usual in an attempt to demonstrate more than four basic tastes. She also attempts to show the equilateral tetrahedron of Henning is inadequate. It is interesting that Schiffman attempts to draw small equilateral tetrahedrons within the boundaries of her orthogonal three- dimensional taste space resulting from a conventional MDS analysis. It is argued here the orthogonal three-dimensional taste space represents a more modern theoretical right tetrahedron concept than did Henning’s equilateral tetrahedron. Schiffman presents a series of three-dimensional spaces derived from MDS for various limited ranges of stimulants. These spaces are necessarily incomplete.

Schiffman & Dackis presented an earlier paper that clearly recognized the multidimensional character of MDS analyses214. It contains very useful information on a range of stimuli. They used an early MDS analysis program by Guttman215. It appeared in the same journal Figure 8.5.2-7 A two-dimensional taste space with as the previous paper by Shephard alternate axes applied. The responses are described mentioned above. They chose to use a three- as taste qualities as determined by activity profiles dimensional space after considering up to across neurons. Dimension 1 accounts for 91% of five dimensions. In that paper examining the data variance in rats. Dimension 2 accounts for the role of amino acids, vitamins and fatty 4% of the variance. Five percent of the variance is acids in taste, they noted, “Since the four- unaccounted for by these two dimensions. Modified and five-dimensional solutions do not from Scott & Mark, 1987. appreciably decrease the error and since they do not reveal new relations among the stimuli, . . . a three-dimensional solution was considered appropriate for the data.” They gave the errors as a function of dimension number in the paper; 1D-36%, 2D-21%, 3D-14%, 4D-10% and 5D- 7%. Stopping at 3D with a 14% error is considerably poorer than in vision where the 3D error was below 5% in the Indow & Romney dataset.

It appears to be Schiffman”s challenge to show that her additional stimulants fall outside of the three-dimensional space of the four basic tastes. If they do, it is important to incorporate all of the taste sensations, including her metallic tastes in a new multi-dimensional analysis. That analysis should show the existence of more than three significant orthogonal basis functions (beside that for intensity alone). The results would be expected to define a four dimensional histogram which may be difficult to draw on paper, but nevertheless would exist. Schiffman’s interpretation of Henning’s and probably all other tetrahedrons as hollow with all tastes represented on its surface is probably inappropriate. A more likely interpretation is that the tetrahedron is a right tetrahedron where all tastes fall within its volume. Alternately, the three dimensional taste space has several corners (nodes) not presently occupied by any “basic

214Schiffman, S. & Dackis, C. (1975) Taste of nutrients: amino acids, vitamins, and fatty acids Percept Psychophys vol 17(2), pp 140-146

215Guttman, L. (1968) A general nonmetric technique for finding the smallest coordinate space for a configuration of points Psychometricka, vol 33, pp 469-506 Signal Generation & Processing 8- 123 taste.” By abandoning the tetrahedron concept altogether and allow the taste space to expand to a cubic volume, additional possibilities appear. Until proven otherwise, it can be assumed her metallic taste occupies one of the corners (Xmax, Ymax, 0 or Xmax, 0, Zmax or Xmax, Ymax, Zmax) in the taste space proposed here and shown above. [ xxx check it is still above ] It is appropriate to consider the proposal of the earlier Henning in 1916 as archaic and the use of MDS in the 1970's as primitive, not well understood and investigational. 8.5.2.7 The proposed human sensory space of gustation

Comparison between the multidimensional space of gustation and vision suggest a functional analogy. This analogy can be exploited based on the Electrolytic Theory of the Neuron. The analogy to be presented here is based on a number of hypotheses concerning gustation; C MDS analysis of a sufficiently large set of stimuli and large set of species specific subjects. C To exhibit effective gustaphores, the stimulant must be soluble in saliva. C The sensory region of the microvilli lemma exhibits a negative potential due to both the polarization of the sensory receptor molecules and the type 4 lemma of the dendroplasm. C The negative field in the region of the type 4 lemma is attractive to positively polarized ligands of stimuli molecules. C Transduction involves a coordination chemistry process (a narrow type of stereochemical process). C Transduction involves the change in the base potential of the Activa formed within the type 4 lemma of the microvilli, and the resulting change in current flow into the dendroplasm of the sensory neuron. C Transduction probability is the product of a sensory receptor capture cross-section for the gustaphore of interest times the correlation process reactivity coefficient for the sensory receptor type of interest. C The capture cross-section of the sensory receptor is the product of the number of sensory molecules within the area of the type 4 lemma times the number of reaction sites per sensory molecule. C The reactivity coefficient of a stimulus toward a specific sensory receptor is the product of the molarity of the stimulus in solution times the number of reactivity sites per molecule of the stimulus.

Figure 8.5.2-8 shows the proposed taste sensation space for gustation involving four primary taste sensations. Like in vision, signals from the individual sensory channels of gustation are treated as independent within the CNS. Thus, the primary taste receptors can be shown as orthogonal and located at the corners of a three-dimensional space. They are shown here with a color scale drawn from The Electrolytic Theory of Color Vision for orientation and comparison). The scale intervals are all equal in this figure, and equal to 0.10 Angstrom.

The figure shows the location of the four recognized sensory channels of the gustatory modality; the H-Best or acido-receptor at 1.4 Angstrom, the S-Best or gluco-receptor at 2.6 Angstrom, the N- Best of natro-receptor at 3.3 Angstrom and the Q-Best or picro-receptor at 4.2. Angstrom. The taste modality sensory neuron receptors have a narrow stereochemical capture range of about ±0.10 Angstrom, narrow band receptors compared to the wide band receptors of vision. Thus, an individual gustaphore must exhibit a d-value very similar to that of the receptor to stimulate the system. Many stimulants consist of multiple gustaphores. As a result, an effective d-value for the stimulant may lie anywhere along the d-value axis based on the relative effectiveness of the individual gustaphores. As a result of this functional configuration for the gustatory modality, a specific stimulus is defined by the relative ability of its gustaphores to be selected by a receptor and the ability of that gustaphore to create an analog potential in the sensory neuron proportional to its dipole moment. This protocol will be discussed in greater detail in Section xxx. An assertion by Smith & Davis (page362) can be restated in the context of this work. A universal characteristic of mammalian gustatory neurons is the simultaneous response of distinct sensory channels to a single stimuli (consisting of multiple gustaphores). 124 Neurons & the Nervous System

The potential for a stimulus to be perceived and described as exhibiting all four primary tastes is finite. Under this proposal, a great many active stimuli can be described as exhibiting multiple primary tastes simultaneously. By its name, it should be obvious that mono-sodium glutamate in solution is capable of stimulating both the gluco-receptor and the natro-receptor channels. The nomenclature of the chemical demonstrates why a separate channel for a umami gustaphore is not needed. The adaptation mechanism associated with each sensory neuron can result in the perceived taste of any stimulus to vary as a function of time, both short term and long term. The importance of this is the complex procedure used in formal wine-tasting.

8.5.2.8 The family of Neural Figure 8.5.2-8 Proposed taste sensation space for a mammalian species exhibiting four primary taste Response Functions sensations. The sensations are generated by the acido-receptors, the gluco-receptors, the natro- The concept of a neural response function receptors and the picro-receptors. Less specific (NRF) has appeared a number of times in identifiers are shown in parentheses. Based on the the literature. It is simple to define a neural d-value parameter of the Electrolytic Theory of response function as the response of a Taste & Smell. See text. specific region of the oral cavity to a range of selected stimuli (using only an arbitrary sequence of the stimuli rather than a calibrated horizontal axis). However, a more detailed definition of such a function, or functions, has been missing. Multidimensional analysis clearly shows the basic taste space is three dimensional and each “best” sensory channel exhibits a sombrero hat shaped neural response function in this space. These hats are the appropriate descriptions of the individual neural response functions.

Woolston & Erickson have developed their concept of an NRF from a totally philosophical and psychophysical perspective216. Their data set includes a variety of stimuli, including the stimuli normally associated with the four best channels. However, they chose to limit their analysis to a two-dimensional taste space. Their conceptual NRF’s are derived from their two-dimensional taste sensation space determined from the NST of 26 Sprague-Hawley rats. They used the Guttman-Lingoes MDS program and show the percent error as a function of the number of independent dimensions used using two different criteria. Frame A of their figure 8 is unlikely for several reasons. First, it does not represent a section through the taste sensation space as described above. Second, it appears the three absorption probabilities shown appear to have been individually normalized to a common height. The actual situation for a given subject and a real stimuli is not properly represented by their caricature. Based on the taste sensation space hypothesized here, a number of more specific NRF’s can be

216Woolston, D. & Erickson, R. (1979) Concept of Neuron Types in Gustation in the Rat J Neurophysiol vol 42(5), pp 1390-1406 Signal Generation & Processing 8- 125 detailed. The goal is to have one or more two-dimensional graphical NRF’s with one axis representing a calibrated variable and the second axis representing the response function. The problem is the three-dimensional character of the taste sensation space. It is possible to define a variety of two-dimensional NRF’s within this space. MDS provides an arbitrary calibration for each axis of the taste sensation space. These calibrations are stable as long as the stimuli set and subject set are broad enough to encompass the entire taste sensation space. While the calibration is arbitrary, the scale is linear and the distance between any pair of “best” loci is therefore linear. A percentage scale can be calculated

Figure 8.5.2-9 Proposed 2-D neural response function of X, NRFX. The individual responses are not normalized to the same level. The peak amplitude of each depends on the precise definition of NRF used. The relative neural responses for stimulus B are equal at a relative scale other than 50% because of the unequal heights of the individual responses. In practice, the individual sensory receptors are represented by the dashed lines. Many stimulants incorporate multiple gustaphores in order to excite separate sensory receptor channels. See text. using this distance between pairs of “best”loci as the denominator. Figure 8.5.2-10 shows a nominal NRF between H-best and N-best along the X-axis. If all of the scales of the taste sensation space have been adjusted to form spherical shells in the previous figure, the plane of this presentation is rotationally symmetrical about the X-axis.

The individual “best” response functions can represent several different combinations of factors. They can represent the relative density of sensory receptor molecules on the surface of the denrolemma for a given “best” locus. They can represent the product of the relative density and the number of potential binding sites per molecule. They can represent the unbound sites remaining after previous exposure of the sites to stimuli. Since it is also possible for the stimuli to reflect different reactivity to the different “best” sites, it is possible to plot the graph to show the unbound sites for a given “best” locus times the reactivity of a given stimuli to that locus. As shown below, the typical gustatory or olfactory receptor)shows a very narrow response function (dashed lines in the figure and there is little overlap between the individual response functions. This situation is compatible with the “quantized” character of the gustaphores and odorophores of chemical sensing. Because of the available molecular geometries, only a limited set of d-values are available to stimulate the chemical sensing system. On the other hand, many stimulants incorporate multiple gustaphores or odorophores targeting multiple sensory receptor channels. When fully annotated according to the above possibilities, the figure should prove more useful than the conceptual figure 8(A) of Woolston & Erickson and result in more useful scatter plots than the ones they conceptualize.. 126 Neurons & the Nervous System

In the context of gustation, the molar concentration of a stimulus does not necessarily indicate its total reactivity. That parameter is indicated by the molarity of the stimulus times the number of reaction sites associated with each molecule. [xxx edit ] Since a majority of the stimuli do not appear along one of the axes of the taste sensation space, NRFX, NRFY and NRFZ are of limited utility. An alternate approach is to calculate the likelihood of an individual stimulus’ interaction with each of the “best” sensory channels based on the above choice of parameters. The stimulus can then be described in terms of its relative participation with each of the best channels as [NaGlutamate]x,y,z or NaGlutamate:x,y,z where x,y,z are relative values indicating the salty, bitter and sweet sensations relative to the sour sensation respectively. Using H-best as the 0,0,0 point of the taste sensation space suppresses the role of H-best in the analysis. Since the space is linear, the zero point can be moved to anywhere in the space without introducing distortion. By moving the zero point to correspond to the zero point defined by the underlying MDS dimensions, the x,y,z values become more symmetrical about zero and the four “best” loci are treated more equally. Experience will demonstrate the best method of describing these stimulus values in a practical manner. Signal Generation & Processing 8- 127

8.5.3 Hypothesis of gustatory transduction

The literature of taste based on the chemical theory of the neuron contains a large number of almost completely conceptual descriptions of gustatory transduction. One of the most recent is presented by Gilbertson & Margolskee in Doty217. They describe all of their gustatory transduction mechanisms as dependent on a rise in intracellular Ca++ followed by the release of a chemical neurotransmitter at the pedicle of the sensory neuron. While many labeled materials and presumed operations are shown in their block diagram, no schematic or explanation of the steps in their operations are provided. They show the sensing of amino acids as entirely independent from the sensing of the sugars. Both are shown as requiring undefined pores through the microvilli. Their concepts do not account for the quiescent or dynamic potential at the pedicle of the sensory neuron, the adaptation characteristic of the sensory response, or the neural response function for any of the sensory channels. [xxx use a different term for dendroplasm and dendroplasm in the following sections, cilia or microvilli lemma and plasma? See page 48 of Cagan & Kare first. they use microvilli lemma ] [xxx may need pages 213 to 246 in Cagan & Kare ] After reviewing the literature of gustation from the perspective of the Electrolytic Theory of the Neuron, what conclusions can be drawn about the transduction mechanism of gustation? C The rapid replacement of the complete sensory neuron (~200 hrs) suggests transduction involves a substantive change in the structure of each sensory neuron. C The similarity of the gustatory pulse response to that of the visual, auditory and olfaction modalities suggests a quantum-mechanical mechanism is involved, probably involving a chemical reaction involving the outer lemma of the neurons. C The only detailed hypothesis relating to gustatory transduction in the literature relies upon the Law of Mass Action to describe a process described using a second order differential equation. C All previously discussed transduction mechanisms in this and related works have involved a change in state of one of the reactants/participants, thus ruling out the Law of Mass Action as an underlying premise. C The great variety of lipids found in the lemma of neurons suggest a sufficient variation in lipid reaction with stimuli to define a set of independent variables in gustation and explain the groupings of stimulus effects in many multidimensional analyses of stimuli.

Breslin’s work has suggested the phosphatidyl fatty acid of Q-best transduction. A globoside appears a candidate for the phosphoglyceride of S-best transduction. C The complex grouping of stimuli in various multidimensional analyses based on the stimuli suggest the chemical structures of the stimuli are not an independent variable in gustation. C As demonstrated by Kashiwagura et al., preadaptation with NaCl affects the transduction of CaCl, a compound devoid of sodium. The following proposals will follow the fundamental chemistry found to be operative in the discussion of olfaction, coordination chemistry between very complex molecular ligands associated with specialized (type 4) regions of the lemma of microvilli emanating from individual dendrites. The challenge is to determine the structure of these complex ligands and their electronic state prior to coordination with a variety of potential stimuli.

217 Gilbertson, T. & Margolskee, R. (2003) Molecular physiology of gustatory transduction In Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker Chap 34 128 Neurons & the Nervous System

The stimuli themselves range from the nonpolar, non-ionizing sugars and sugar derivatives to the highly ionic H+ and Na+ which may be present in solution in more complex coordinate forms. - - - - As a general introduction, the sensory neurons of the gustatory modality employ specialized (type 4) lemma on the microvilli emanating from their dendritic terminal. The specialized lemma consist of four different phospholipids known collectively as globosides, in the outer leaf of their bilayer structure. Mixed collections of these sensory neurons are grouped in taste buds embedded behind pores primarily in the epithelium of the tongue and oral cavity surfaces. In vivo, the globosides exhibit a negatively polarized molecular surface to the fluid environment of the oral cavity that offers a coordination chemistry association to molecules exhibiting a positive hydrogen ion or a positive sodium ion on their surface, and two more complex molecular arrangements, developed further below, commonly found in the sugars and in quinine hydrochloride. The possibility of additional sensory neuron types sensitive to the monosodium glutamate and various “metallic” salts cannot be dismissed at this time.. Negative ions, such as the chlorine ion, are not attracted to the globosides. The coordination chemistry involved appears to generate a stable state that is long lasting. This change of chemical state may contribute to the need to replace the sensory neurons on a 200 hour cycle time.

A conducting form of PtdIns is also a candidate for inclusion in the globosides used as receptors of the gustatory modality. Inositol is considered a sugar derivative although it lacks an oxygen within the ring structure of this cyclohexane. Inositol occurs as nine isomers. The inositols, particularly the epi- and myo- isomers, are known to participate in a variety of coordinate relationships with the alkali and alkali-earth metal ions218. PtdIns is a known phospholipid of the cell lemma. The inositol group of the phospholipid could participate in the necessary coordinate chemistry and are prime candidates for the N-best sensory neuron receptor. Additional background on the inositols is available219.

Lehninger fortuitously identified the four major globosides of gustation in 1970 based on their presence in neural tissue220. He also described their biological formation.

- - - -

Many multidimensional analyses and cluster analyses have shown multiple inconsistencies when interpreted in terms of a reversible chemical reaction leading to transduction. As in olfaction, the conflicts are sufficient to spread doubt that the underlying mechanism involves reversible chemistry. In all other excitation/de-excitation mechanisms studied, a quantum-mechanical mechanism was involved that was not reversible. Thus, it is useful to consider, non reversible mechanisms for gustation, particularly with the close association of gustation and olfaction.

A leading candidate for explaining transduction in gustation is the proliferation of different lipids found in specialized areas of the bilayer lemma of the sensory neurons. These lipids have polar terminals that are sugars, alcohols, amines, cholines, and many other complex chemistries. It is possible that the major stimulants attach stereographically or attack these lipids, causing a reaction and generating a free electron that is transferred through the nonpolar lipid tail to the plasma inside the neuron. The reaction also changes the chemical constituency of the lipid involved. As a result of the reaction, the particular lipid molecule is no longer viable. With time, the viability of the entire neuron is degraded in this way and it must be replaced in an orderly process to maintain nominal gustatory sensitivity. Failure to perform this continual replacement, due to age or otherwise, leads to a loss of gustatory sensitivity.

218Williams, R. & Atalla, R. (1981) Interactions of group II cations and borate ions with nonionic saccharides In Brant, D. ed. Solution Properties of Polysaccharides Washington, DC: American Chemical Soc. Chapter 22

219Cerny, M. Kocourek, J. & Pacak, J. (1963) The Monosaccharides. NY: Academic Press Chapter 35

220Lehninger, A. (1970) Biochemistry NY: Worth Publishing pp 196-200 & 522-526 Signal Generation & Processing 8- 129 Based on this hypothesis, the challenge is to identify the most likely lipid species able to react with the appropriate group of stimuli. The many multidimensional analyses in the gustation literature invariable display a two dimensional plane with the stimulants grouped in not more than four major locations. The various cluster analyses also show a few major branches with the various minor branches splitting from these. See also Wright & Michels for the similar situation in olfaction. It appears reasonable to hypothesize the gustatory sensory neurons operate in a mode very similar to the olfactory transducers and employ specialized regions of lemma encasing the protruding hair (villi) consisting of a few different types of lipid molecules. Under this hypothesis, the transduction function is primarily dependent on the type of lipid present and not on the precise nature of the stimulant. Thus, one type of lipid reacts to produce a signal that is interpreted as sweet, a second type as sour, a third type as xxx etc. irrespective of the chemical formula of the stimulant. A single stimulant could affect more than one type of receptor. The signals from these sensory neurons could be differenced higher up the neural pathway to provide finer bipolar signals describing the individual stimulants. Following the proposed hypothesis, the typical multidimensional analysis can be modified slightly in its support. Figure 8.5.3-1 shows a diagram from xxx with the added scales.

Figure 8.5.3-1 XXX modified multidimensional analysis based on the polar head of lipids EMPTY. 130 Neurons & the Nervous System

8.5.3.1 Previous theories of gustatory transduction

All previous theories of gustatory transduction have been based on the chemical theory of the neuron and have been necessarily largely conceptual. Scott & Mark described the state of understanding of the transduction mechanism in the gustatory modality in 1987, “Attempts to define the organization of the taste system in terms of the physical characteristics of stimuli have been largely unsuccessful.” Interestingly, after annotating reviews of gustatory transduction up through 1991, Getchell et al. note on page 153, “To date, there have been no examples of 2nd messenger modulating ion channels directly in taste cells.” They also note that in the case of at least one bitter compound, denatonium, no membrane conductance change is thought to occur at all; . . .” They conclude, “Since the final pathway converges and most taste cells respond to more than one taste modality, it is not yet clear how taste qualities can be discriminated.” This work will propose an entirely different theory of transduction based on the Electrolytic Theory of the Neuron. The theory eliminates the ambiguities encountered by earlier theories and demonstrates that individual receptors respond to only one specific stereochemical structure and measure the dipole potential of the gustaphore. It is this measurement that is passed to the CNS along a conventional sensory neural circuit. 8.5.3.2 The AH,B & AH,B,X coordination chemistry of the gustatory channel

The conditions required in coordination chemstry are quite complex. Shallemberger has provided a glossary addressing these conditions and their definition (pages 297-301)

The globoside receptors of the desirable/”sweet” gustatory channel are subject to attack by a variety of chemicals. Shallenberger has explored this possibility extensively221. Jakinovich has also offered ideas in this area222. Figure 8.5.3-2 shows the basic coordination chemistry he proposes as the mechanism resulting in excitation of the S-best sensory neuron. At the minimum, AH represents a hydroxyl proton and B represents a neighboring hydroxyl oxygen atom. While the configuration of these two units need not be identical on the two ligands, the situation strongly suggests a stereochemical relationship, at least in the local area of each ligand. The AH moiety may be OH, NH, NH2 or even CH in halogenated compounds. The B moiety may be O, N, an unsaturated center, or even the π-bonding cloud of the benzene ring.

Shallenberger & Acree noted a second stereochemical requirement can be used to describe the difference in sweetness between D-leucine and L-leucine. The latter is not sweet to the taste.

Figure 8.5.3-2 Proposed coordination chemistry of the S-best sensory neurons clarifying the condition described by Shallenberger. In the simplest case, all A’s & B’s are hydroxyl oxygen and H’s are hydroxyl hydrogen. His original text did not differentiate between the distance between AH and B. His later writings referred to the H,B distance as 3 Angstrom. Both numerics are ±7%. Modified from Shallenberger, 1971.

221Shallenberger, R. (1982) Op Cit. Chap 10, Sweetness: A stereochemical attribute.

222Jakinovich, W. (1981) Comparative study of sweet taste specificity In Cagan, R. & Kare eds Op. Cit. page 132 Signal Generation & Processing 8- 131 When originally investigating in 1967, Shallenberger & Acree asserted, the initial interaction “is neither a proton transfer nor an electrostatic interaction, but probably involves London dispersion, the principle element of hydrogen bonds.” This comment may be subject to modification where more is known of the environment involved. The biochemistry field has generally adopted the term Van der Waals forces as a synonym for the London forces of “London dispersion.” Wikipedia offers a good overview in this area. In 1970, Birch et al. initiated a study to determine if there was any reaction chemistry involved in the AH,B relationship223,224. They concluded that, “there is a chemical reaction basis (stoichiometric basis) for the initial chemistry of the sweetness phenomenon.” Shallenberger & Acree (page 263) also established the distance between the AH and the B moiety had to be 3 Angstrom (~2.86–3.2 Angstrom or ±7%). This parameter allowed them to define the specific members of various sugars and other molecules that could participate in the AH,B relationship. In the case of glucose, they showed that only OH-4 and OH-3 were the logical choice for a primary AH,B relationship. Using a galactose, they were able to establish that OH-4 was AH and the only remaining possibility for B is O-3 (Shallenberger, 1982, pp 265-275). After discussing some very sophisticated principles of advanced sugar chemistry, Shallenberger continues on page 275-276 to show that the receptor site structure is disymmetric, but not asymmetric. “This planar structure is capable of resolving the chiral recognition problem presented by the fact that the enantiomeric amino acids possess different taste, but the enantiomeric sugars do not.”

Based on the overall analysis, Shallenberger & Acree are able to account for virtually all of the variation in sweetness among the simple sugars, amino acids and other natural chemicals exhibiting the appropriate AH,B relationship. They have also described a mechanism for various hydrolyzed alkali and alkali earth ions to cause a perception of sweetness, particularly at low molar concentrations (page 269). After initially abandoning the tetrahedron concept of taste space in favor of an orthogonal space, Shiffman et al. have provided similar data on amino acids versus their acetylized relatives225.

Glutamic acid and aspartic acid, being the only two negatively charged amino acids, would not be expected to taste sweet. Shallenberger & Acree confirmed this observation for glutamic acid (page 246). They also noted that γ-aminobutyric acid (GABA) is not sweet because of the distance between the amine and carboxyl group is too large. This is due to the loss of the closer COO– group, for material within the neural system, by electrostenolysis.

- - - -

Working in the same time period as Shalllenberger, Kier has extended the idea of the AH,B coordination bond to include a tripartite or “three-point” coordination model226. This model supports additional discrimination between similar stimuli and also explains the “super sugar” qualities of many man-made sugar substitutes. Kier labeled the third point, X, while Shallenberger chose to use the label γ for “greasy.”

Shallenberger & Lindley (1977) re-examined the work of these two groups as reported by Shallenberger (1982, page 269) and surfaced the fact they were measuring distances from different references, Figure 8.5.3-3. Quoting Shallenberger, “In the former (Kier), the A to B distance is

223Birch, G. Cowell, N. & Eyton, D. (1970A) A quantitative investigation of Shallenberger’s sweetness hypothesis. J Food Tech vol 5, pp 277-280

224Birch, G. Lee, C. & Rolfe, E. (1970B) Organoleptic effect in sugar structures J Sci Food Agric vol 21, pp 650-653

225Schiffman, S. Moroch, K. & Dunbar, J. (1975) Taste of acetylated amino acids Chem Sens Flav vol 1, pp 387-401.

226Kier, L. (1972) A molecular theory of sweet taste J Pharm Sci vol 61(9), pp 1394-1397 132 Neurons & the Nervous System estimated to be 2.6 Angstrom. In the latter (Shallenberger et al.), the AH proton to B orbital distance is estimated to be about 3 Angstrom.” This difference appears to be significant and the orientation of the H relative to the triangle is also important, especially when differentiating between the stereographic structure of the receptor and potential stimuli. In the analysis to follow, the distance of primary interest will be taken as the distance between the two orbitals, A & B, as described by Kier as 2.6 Angstrom. The distance of secondary interest will be that to the location of X. The energy required to bring the hydrogen into the proper position for coordinate bonding will be considered a factor in determining the ultimate dipole potential of the receptor/stimuli union (its effectiveness in changing the base potential of the first Activa..

The coordination chemistry relationships developed by Shallenberger et al. and by Kier, and the Electrolytic Theory of the Neuron provides a comprehensive description of the sweet sensory neuron that does not involve any GPCR proteins or any movement of sodium ions through the wall of the cell. The described performance of these sensory neurons is much more complete and detailed than that of any other hypothesis.

- - - - Figure 8.5.3-3 Comparison of AH,B,X geometries. The primary structure of a stimulant that is Left, geometry reported by Kier. Right geometry perceived as sweet can be defined as its reported by Shallenberger group. See text. glycophore. Shallenberger ((1982, page 265) defined the glycophore of the majority of sugars and many sugar derivatives as consisting of the hydroxyls at position 3 and 4. OH-4 acts as AH and O-3 acts as B. In other chemicals, the glycophore is represented differently but still exhibits the nominal 3 Angstrom spacing between AH and B.

- - - -

Figure 8.5.3-4 shows several examples of chemicals considered to taste sweet but do not contain the structure of a sugar. Signal Generation & Processing 8- 133

Figure 8.5.3-4 Sweet tasting non-sugars and the their AH,B relationships, with fructose as a reference. From Shallenberger & Acree, 1971.

Kier extended the stereochemical requirement on an effective sweetener based on study of the amino acids and the growing number of man-made sweeteners (typically including amino acid ligands)227. [xxx edit ] Kier developed a three point union involving the AH,B coordination relationship (with a nominal spacing of 2.5 Angstrom) plus an additional bonding of undefined type about 3.5 Angstrom from the A entity and 5.5 Angstrom from the B entity. It is not clear if the difference between the nominal value of 2.5 Angstrom of Kier and the 3.0 Angstrom of Shallenberger & Acree is significant. The value of 2.5 Angstrom reported by Kier appears to involve more complex AH,B

227Kier, L. (1972) A molecular theory of sweet taste J Pharm Sci vol 61(9), pp 1394-1397 134 Neurons & the Nervous System configurations than the simple carboxylic arrangement found in most Shallenberger & Acree formulas. This may suggest, the 2.5 Angstrom value is off-peak compared to the 3.0 Angstrom nominal. Kier conjectured the third union involved “an electron-rich position capable of undergoing electrophilic attack, engaging in localized charge transfer, or capable of participating in some type of bonding involving the electron component such as a dispersion interaction.” Chemicals able to satisfy this three point union exhibit sweetnesses 10's to xxx,000 times greater than fructose, suggesting a significantly larger change in the dipole potential of the receptor per mass unit of.sweetener. Eggers, Acree & Shallenberger (2000) have discussed and developed the three-point hypothesis farther using the symbol γ to replace Kier’s X.

- - - - Signal Generation & Processing 8- 135

This sensitivity to attack and the resultant transduction signal intensity can be portrayed more graphically as seen in Figure 8.5.3-5.

8.5.3.3 Proposed transduction mechanism for “desireable/sweet”

It is important to have an understanding of the numbering system used in sugar chemistry and the possibility of sugars with the hydroxyl groups pointing in different directions. Figure 8.5.3-6 shows these relationships. Current convention calls for numbering to be clockwise beginning with the first carbon to the right of the oxygen in all hexose and pentose sugars. Note in the pentoses that the first carbon is taken as that in the methyl alcohol. Rotation of the axis of the moieties associated with a given carbon can affect the taste sensation significantly. For instance, α-D-mannose is sweet, but β-D-mannose, which is very similar in structure, is distinctly bitter according to Shallenberger & Acree (1971, pg 222) Figure 8.5.3-5 Proposed sensitivity of the desirable/”sweet” sensory channel ADD using the multidimensional graphic of xxx in overlay as an example. 136 Neurons & the Nervous System

[xxx rewrite the following to separate gangliosides from globosides and address third view of simpler receptor ] Using the description of the gustatory sensory neuron above, the question is what is the structure of the type xxx neurolemma covering the outer surface of the sensory hair(s). The exterior bilayer of the lemma of non-neural cells is known to be populated primarily by phosphatidyl choline (lecithin). However, in neural tissue, the exterior bilayer is known to consist of 20-25% of ganglioside, a very large family of phosphoglycerides228. Figure 8.5.3-7 shows the structure of two gangliosides. Only a few of the available configurations have been investigated in the laboratory229,230. The top frame shows a well studied ganglioside known as ganglioside GM2. Ferrier & Collins have provided an alternate Haworth

Figure 8.5.3-6 The numbering system of the simple sugars. Even the pentoses have six carbons. The axial elements associated with each carbon can invert structurally resulting in significantly different chemical properties.

228Christie, W. (2010) Gangliosides: structure, occurrence, biology and analysis http://lipidlibrary.aocs.org/Lipids/gang/index.htm

229Ledeen, R. ed. (1984) Ganglioside structure, function, and biomedical potential. NY: Plenum Press

230Porcellati, G. Ceccarelli, B. & Tettamanti, G. (1975) Ganglioside function: biochemical and pharmacological implications. NY: Plenum Press, c1976 Signal Generation & Processing 8- 137 Diagram of this ganglioside231. They describe one of the R moieties as ceramide, a saturated lipid. Similar forms of gangliosides are commonly found in the myelin sheaths of stage 3 neurons. The center of the frame shows a ganglioside with the complicated series of amino sugars replaced with a chain of D-galactose moieties bridged in the α(1–>3) arrangement. This arrangement provides three separate locations where a coordinate AH,B bond can occur between a stimulus and this molecule. Only the potential AH,B bond at O-4 and O-6 of galactose(I) is shown explicitly. One of the R moieties is shown fully saturated in order to provide conductivity along the length of the molecule. This modification may not be necessary as Hauser, and Sherer & Seelig have shown operation of the molecule as a molecular electrometer is possible without it (Section 8.xxx). This moiety is a polyethine, a member of the terpene family. Many variations are available within the polar head and the lipid tail of the gangliosides. Lehninger has shown a ganglioside with two

231Ferrier, R. & Collins, P. (1972) Monosaccharide Chemistry. NY: Penguin Books page 255 138 Neurons & the Nervous System

Figure 8.5.3-7 Two gangliosides associated with the neural system. Top; the ganglioside GM2 isolated from a Tay-Sach brain. Center; proposed ganglioside supporting the S-best channel of gustation. The ganglioside contains three galactose ligands chained at the α(1–>3) sites and a polyethine (fully conjugated) lipid for one of the R moieties. This structure provides multiple glucophore binding sites using O-4 & O-6. An optional sialic acid moiety is shown as A at the lower left. Bottom; a simpler ganglioside utilizing only one galactose moiety if AH,B coordinate bonding is only possible using O-3 & O-4. galactose moieties that could support two AH,B bonds232. The number of chained sugars can reach ten in some oligosaccharides. The first of three possible AH,B bonds is shown between the hydroxyl hydrogen at C-4 and a hydroxyl oxygen of the stimulus, and a hydroxyl oxygen at C-6 and a hydroxyl hydrogen of the stimulus separated by 2.6 Angstrom. The lower frame shows a simpler potential receptor with only one galactose moiety if the only acceptable AH,B coordinate bonding must use O-3 and O-4 as suggested by Shallenberger & Acree.

232Lehninger, A. (1970) Biochemistry NY: Worth Publishing page 201 Signal Generation & Processing 8- 139 Gennis has described a digalactosyl diglyceride using a α(1–>6) linkage arrangement that can be extended indefinitely while preserving the capability of O-3 & O-4 to support coordinate bond pairing233. The chain of galactose sugars (potentially labeled a galtan) can be extended indefinitely but the effect on the conductivity of the resulting structure is unknown. This family of phosphoglycerides is known to exhibit a negative polar head. In the case of the gustatory sensory neurons, this polarity is enhanced by the negative polarity of the interstitial liquid crystalline water of the type 4 microvilli bilayer. Thus, the in-vivo sensory ganglioside is highly attractive to positive ions introduced into the saliva in significant concentration. Shallenberger has described a series of n-galactose configurations employing different chaining arrangements (pages 224-230). Only two of the six arrangements provide the maximum opportunities for AH,B bonding.

- - - - Concatenation of the galactose moieties can cause an increased capture area for the sugars but potential interference with the L-configuraton sugars. The result would be an increased sensitivity of the channel to the D-configurations at the expense of the L-configurations.

- - - -

As noted in Chapter xxx, a bilayer of sphingomyelin (of the same super family of glycosphingolipids as ganglioside) is known to form an electrical diode, i.e. the bilayer is able to conduct electrical charges asymmetrically from one polar head to the other polar head, after surface treatment of one of the electrolytes with 10– 7 g/ml of alamethicin by Mueller & Rudin234. Alamethicin is a cyclopeptide antibiotic containing a variety of amino-acids and at least one carboxyl group. The precise role of alamethicin remains unknown.

The presence of a semiconducting region of type 2 lemma employing a ganglioside in its outer layer introduces the immense possibilities of polysaccharide (sugar) chemistry to the gustatory transduction function. Each of the four saccharide units of the ganglioside exhibit slightly different arrangements and the sugar moiety is subject to attack in a variety of ways. Such polysaccharides are known to be subject to decomposition by dilute bases and dilute acids. They are heteropolysaccharides. They contain both α(1–>4) and α(1–>6) linkages. The presence of a simple saccharide as a stimulus may result in the chaining of the simple saccharide to the heteropolysaccharides of the type 4 membrane. Any addition, decomposition or rearrangement that causes a significant change in current flow into the plasma of the neuron, via the first Activa of the sensory neuron, will constitute the fundamental step in transduction.

The typical lemma does not have two bilayers of ganglioside. It is more likely to have an outer layer of ganglioside and an inner layer of phosphatidyl ethanolamine (PtdEtn). The important feature is that the bilayer form a electrolytic diode that can transport an electron into the dendroplasm of the sensory neuron when such an electron is released from the polar head of the ganglioside. With the presence of a diode accounted for, any significant rearrangement, fragmentation or polymerization of the polar head could inject the negative charge associated with that head into the plasma. of the sensory neuron. In the laboratory, the sphingolipids as a family are known to be easily hydrolyzed (Lehninger, page 199).

233Gennis, R. (1989) Biomembranes. NY: Springer-Verlag pg 29

234Mueller, P. & Rudin, D. (1968) Action potentials induced in biomolecular lipid membranes Nature vol. 217, pp 713-719 140 Neurons & the Nervous System

[xxx edit ] The proposed transduction mechanism of the gustatory modality involves the disruption of the chemical structure of the ganglioside bilayer of the type xxx portion of the microvilli lemma and the injection of a negative charge (an electron) into the dendroplasm of the sensory neuron. The presence of the electron is sensed by the same electrolytic structure as employed in the olfactory modality. Most of the electrolytic structure is also shared with the visual and auditory modalities, however, the free electron is generated by different means. - - - - 8.4.4.6xxx Sugars as potential SSC’s in gustation

The significant presence of ganglioside or globoside attached to the surface of the dendrolemma of gustatory microvilli introduce the potential for these phosphoglyceride -sugars to provide the sensory substrate for gustation. Drefus et al. have defined the gangliosides as “a complex group of glycosphingolipids which contain one or more molecules of sialic acid235.” However, as Schauer et al. noted, “Sialic acids are derivatives of neuraminic acid with either an acetyl or glycolyl residue at the amino function and frequently one or more O-acetyl group(s) at C-4, C-7, C-8 and preferably at C-9236.” Thus the gangliosides are a very large family that has been only superficially explored in the literature. Merrill & Sandhoff, as an example, only discuss the saturated sphingolipids as a matter of expediency237. In the same volume, Cook & McMaster provide a set of nomenclature for the broader varieties of saturated and unsaturated fatty acids in phospholipids238. The gangliosides are widely associated with neuron tissue. The distribution of the gangliosides appears focused in the gray matter, the unmyelinated portions of neurons, but this remains controversial. Yamakawa writing in the same volume, noted the high concentration of at least a close relative, galactosyl ceramide, in the white matter of the brain239. Cook & McMaster discuss the wide spectrum of fatty acids needed within a mammalian species and note (page 187), “Unsaturated fatty acids also must be synthesized in cells, supplemented by essential fatty acids in the diet.” They also assert that trans-unsaturated fatty acids are not produced by mammalian enzymes. . .” Their statements about cis-unsaturated fatty acids may be too narrow to include the very small relative numbers of these acids needed in the body.

Galactose, which plays a major role in the following discussion was known as cerebrose in earlier times.

Benjamins et al. have discussed the cerebrosides in the context of their occurrence within the

235Dreyfus, H Urban, P. Harth, S. et al. (1976) Ritinal gangliosides: composition, evolution with age In Porcellati, G. ed. Ganglioside Function. NY: Plenum Press

236Schauer, R. Schroder, C. & Shukla, A. (1984) New techniques for the investigation of structure and metabolism of sialic acids In Ledeen, R. et al. eds. ganglioside structure, function, and biomedical potential NY: Plenum Press

237Merrill, A. & Sandhoff, K. (2002) Sphingolipids: metabolism and cell signaling In Vance, D. & Vance, J. eds. Biochemistry of Lipids, Lipoproteins and Membranes. 4th Ed. NY: Elsevier Chapter 14

238Cook, H. & McMaster, C. (2002) Fatty acid desaturation and chain elongation in eukaryotes In Vance, D. & Vance, J. eds. Biochemistry of Lipids, Lipoproteins and Membranes. 4th Ed. NY: Elsevier Chapter 7

239Yamakawa, T. (1984) Wonders in glycolipids-a historical review In Ledeen, R. et al. eds. ganglioside structure, function, and biomedical potential NY: Plenum Press Signal Generation & Processing 8- 141 brain240. They distinguish between sphingolipids containing sialic acid (gangliosides) and those that do not (globosides). They hypothesize the creation of these chemicals within the chemical milieu of the brain. They also provide additional information about the long chain fatty acids of the sphingolipids. The material does not address the operation of the sensory neurons or their receptors. They do describe galactocerebroside (CerGal) as associated with their description of the long chain fatty acid, sphingosine. Crescenzi et al. have described the interaction of the Na+ ion with iso-carrageenan in water at 25°C241. Pass & Hales have explored the changes in enthalpy and entropy when the alkali metal ions interact with polysaccharides242. `Williams & Atalla have discussed the interaction of hexose sugars complexing with group II cations such as calcium and magnesium243. They noted that either 3 or 4 coordinate bonds are formed between the cation and one or two sugar residues. Going beyond the simplest examples is difficult because of the immense number of isomers associated with a specific formula polysaccharide. They note that inositol, the polar head of a candidate phosphoglyceride, forms nine isomers. A three ring polysaccharide, as found in ganglioside, can exhibit up to 56 isomers244. However, the stereo preferences of these sugars may limit the useful number of these configurations. Williams & Atalla noted the exceptional effectiveness of the calcium ion in coordinating with these sugar alcohols in aqueous solution. They speculate this may be due to the specific parameters of the orbital electrons in this species. Sodium is also known to complex with inositol.

8.5.4 Operation of the gustatory sensory receptors

The overall operation of the gustatory sensory neurons are analogous to the operation of all other sensory neurons studied. For more details of its operation, see Section xxx. Each sensory channel employs a different sensory receptor based on the phospholipid forming the type 4 region of its villi.

This section will develop the stimulant/receptor relationship used by each of the sensory channels of the gustatory modality. Based on the data in the previous section, there are only four distinct channels, a considerably simpler situation than in olfaction.

- - - [xxx edit this down ] Shallenberger (1996) has summarized his thesis on the operation of each of the chemoreceptor types. The terminology is that of the advanced chemist and will not be reported here. In essence, there are at least four types of gustatory sensory neurons capable of sensing each historical class of gustatory sensations. The sensory neurons all rely upon coordination chemistry concepts for transduction and no reaction chemistry result is involved in the transduction process. The features of molecular symmetry/dissymmetry and chirality/pseudo-chirality play a major role in

240Benjamins, J. Hajra, A. & Agranoff, B. (2006) In Siegel, G. et al. eds. Basic Neurochemistry, 7th Ed. NY: Elsevier Chapter 3

241Crescenzi, V. Dentini, M. & Rizzo, R. (1981) Polyelectrolytic behavior of ionic polysaccharides In Brant, D. ed. Solution Properties of Polysaccharides. Washington, DC: American Chemical Society Chapter 23

242Pass, G. & Hales, P. (1981) Interaction between metal cations and anionic polysaccharides In Brant, D. ed. Solution Properties of Polysaccharides. Washington, DC: American Chemical Society Chapter 24

243Williams, R. & Atalla, R. (1981) Interactions of Group II cations and borate anions with nonionic saccharides In Brant, D. ed. Solution Properties of Polysaccharides. Washington, DC: American Chemical Society Chapter 22

244Hough, L. (1977) Selective substitution of hydroxyl groups in sucrose In Hickson, J. ed. Sucrochemistry. Washington, DC: American Chemical Society 142 Neurons & the Nervous System distinguishing between the various stimuli. As a general rule, the transduction process involves the generation of an electrical signal as the result of a pseudo-acid/base titration phenomenon. Shallenberger’s thesis relegates the topology of the chemical structures to a secondary role in transduction. His abstract summarizes his position; “When considered jointly, all tastes (sweet, salt, bitter, sour) are variations on a common electrostatic mechanism, and the primary distinction among them can be traced to the symmetrical nature of the interaction between the substance and the taste receptor. Sourness is a dissymmetric interaction between the hydronium ion (an acidophore) and the taste receptor, whereas saltiness is a concerted symmetrical electrostatic interaction between the Na+ and Cl– ions (the halophore) and the receptor. Sweetness is elicited through a bilaterally symmetrical and concerted dipolar interaction between a glycophore and the receptor, while bitterness can be traced to either dissymmetric ionic or dipolar interactions between a picrophore and the receptor. As no products are ever formed, taste phenomena are collectively grouped as being due to electrostatic recognition interactions.” Shallenberger expresses each of these relationships using an equation based on the notation of Belitz et al. (1981). In all cases, transduction occurs via a nucleophilic/electrophilic (n/e) taste receptor on the surface of the microvilli emanating from the dendrolemma of the gustatory sensory neuron.

Shallenberger concludes, “In taste chemistry, the substrate is not transformed, nor is there a direct physiological effect.” This statement is too simple. There is clearly a physiological effect in that the organism perceives a different state. More specifically, there is no physiological effect (that is physical transformation) recognized by the chemical theory of the neuron. There is a profound physiological effect associated with the electrophysiology of the sensory neuron that is reported to the CNS. The Electrolytic Theory of the Neuron recognizes this effect. A change in the dipole moment of the phospholipid receptor at bilayer interface results in a change in the potential at the base of the first Activa within the sensory neuron. This potential changes by a few millivolts, and results in a change in the electrical potential at the pedicle of the sensory neuron.

Beitinger et al. have reported the dipole characteristics of a variety of gangliosides, which may also be applicable to the globosides245. Their measurements were made using an upper air interface and a lower water interface by floating their material on a water pool. They note,

“Gangliosides are characteristic glycosphingolipids containing different numbers of negatively charged sialic acids. These molecules being particularly: abundant in nerve cell membranes of vertebrates are components of the outer bilayer leaflet and might be intimately involved in various cellular biological events.” “More than 2000 articles on gangliosides were published during the past decade, a lot of them with special regard to the large hydrophilic sugar moiety and its great potential for hydrogen-bonding. These reports give evidence that the polar headgroup determines the physicochemical properties of these molecules. These reports give evidence that the polar headgroup determines the physicochemical properties of these molecules. Several studies (t3C-NMR, ESR, X-ray diffraction) on the orientation of the sugar headgroup indicate that the polar headgroup is relatively rigid, and in mixed phospholipid/ganglioside bilayers fully extended and approximately perpendicular to the interface.”

“The formation of a monolayer can be measured as a change in surface pressure and surface potential. The surface potential is proportional to the change of the normal component of the dipole density with regard to the pure water surface. Numerous reports exist of measurements with respect to the total surface potential and the overall dipole moment, respectively, of simple giycosphingolipids and gangliosides.”

245Beitinger, H. Vogel, V. Mobius, D. & Rahmann, H. (1989) Surface potentials and electric dipole moments of ganglioside and phospholipid monolayers: contribution of the polar headgroup at the water/lipid interface Biochim Biophys Acta, vol 984, pp 293-300 Signal Generation & Processing 8- 143 “The phospholipids used for the experiments have been obtained with a degree of purity higher than 99%. The probes showed in each case a single spot in HPTLC and were used without further purification.” “In summary, we have given the sign and the magnitude of the potential drop across the Water/lipid interface for different ganglioside, phospholipid and sulfatide monolayers. The apparent dipole moment per ganglioside headgroup differs from other lipids. The most remarkable result is that among gangliosides the size and the number of charges do not cause large changes in the potential drop. Our potential data imply that the main purpose of nature to vary in biomembranes the number of charges per ganglioside headgroup, e.g., from one 10 three, is not 10 change the apparent potential drop across the headgroup region.” “The headgroup potential of the pure ganglioside monolayers reaches minus several hundred millivoits which can influence the electrostatic properties of neuronal bilayer surfaces strongly.”

Shallenberger briefly mentions a “miracle fruit extract,” from the berry of the tree known as Synsepalum dulcificum. Mixed with a sour stimulus, this extract is known to turn the perception of many sour stimuli remarkably sweet. 8.5.4.1 Operation of the “sweet” gustatory sensory neuron

Birch has provided an overview of the sugar sensing situation based on the SAR approach246. The focus is on the degree of sweetness of several families of chemicals and the effect of substitutions within these chemicals. He does assert the removal of the anomeric hydroxyl group in sugars does not affect their degree of sweetness. He also reports on the removal of one oxygen at a time at each of the five possible positions in pyranose structures. His findings suggest some alternate alignments between a given sugar and a receptor may be possible or multiple sugar receptors are present that operate according to different rules

Van der Heijden has tabulated the relative sweetness of a large variety of sweeteners. He has also provided chemical diagrams for each of them showing multiple sites of AH,B interaction with the sensory receptors in many cases. His discussion is the most recent advocating a third feature associated with each stimulant. He describes the AH,B distance as ranging from 0.314 nm to 0.333 nm in these materials.

The attack upon the globoside embedded in the microvilli lemma of the neuron causes a change in the potential of the base electrode of the Activa buried in the type 2 lemma of the microvilli. The globoside is no longer functional and it is replaced as part of the 200 hour renewal cycle of these sensory neurons. The injected electron is stored on the distributed capacitance of the plasma and results in a change in the base to emitter potential of the sensory Activa. This causes a modulation of the current through the collector-emitter circuit of the sensory Activa. capacitance. The sensory Activa and the distribution Activa are wired in a common emitter circuit. The change in the current through the first Activa causes a mirror change in current through the emitter-base circuit of the distribution Activa. This change results in a significant change in the collector potential (axon potential) of the distribution Activa of the neuron. The base-emitter circuit of the sensory Activa is normally biased just above cutoff and a quiescent current is typically present. This current is due to the presence of agents in the saliva that continually attack the globosides of the outer lemma, releasing a small continual electron current. As in the case of the visual modality, the rate of free electron generation within the base of the sensory Activa and the impedance of the collector current supply source are critical in the overall response of the sensory neuron. They control both the short term transient performance, the excitation/de-excitation (E/D) characteristic, and the long term adaptation properties of the

246Birch, G. (1976) Structure-activity relationships in the taste of sugar molecules In Benz, G. ed. Structure- Activity Relationships in Chemoreception London: Information Retrieval Limited pp 111-118 144 Neurons & the Nervous System neuron. Shallenberger & Acree (page 244) have reproduced a graph from Cameron, 1947, showing the sweetness as a function of concentration on a log-log graph. The slope is very nearly 1.0 for the simple sugars at concentrations up to 2.0%. Shallenberger has addressed the Lemieux Effect as additional confirmation of the AH,B theory of S-best channel sensory operation (page 267). Van der Heijden (page 108) has readdressed the potential for multiple sensory neuron receptors to account for the large range of chemical structures contributing to the sensation. Lindley (page 131), in the following article relating to sweetness antagonists reaches the opposite conclusion, “Currently available evidence is consistent with the conclusion that these sweetness inhibitors are competitive antagonists of sweet taste acting at a single receptor structure.” The limited set of stimulants used in prior multi-dimensional analyses cannot totally rule out this option. However, it appears unlikely after exploring the architecture of the neural system. Lindley (page 131), in an article following that of Van der Heijden and relating to sweetness antagonists reaches the opposite conclusion, “Currently available evidence is consistent with the conclusion that these sweetness inhibitors are competitive antagonists of sweet taste acting at a single receptor structure.”

The analysis of the models of taste cells available to Van der Heijden led him to draw much the same conclusions as in this work. [xxx tabulate ] 8.5.4.2 Operation of the “super sweet” sensory neuron & AH,B,X

Beginning in the 1970's, several sweeteners became prominent that offered a sensation of sweetness far exceeding the previous natural sweeteners. Kier (1972), followed by Shallenberger & Lindley (1977) explored the potential chemical structures that could account for this circumstance. Both groups defined slightly different mechanisms that involved a third element of a tripartite arrangement based on the previous bipartite, AH,B, arrangement. The 1977 paper discusses distances with reference to the two orbitals without considering the location of the hydrogen atom. They described this feature as hydrophobic in nature and capable of participating in a “dispersion” bond (a form of van der Waal bond) with an appropriate hydrophobic bond on the sensory receptor. Figure 8.5.4-1 shows the arrangement of the glucophores of two common sugars based on this tripartite concept. Signal Generation & Processing 8- 145

Figure 8.5.4-1 The “super sweet” tripartite glucophores of two sugars. The glucophores of the stimulants are descibed by the shaded areas and the subscripts C. Left; the C-6 methyl carbon acts as the hydrophobic feature. Right; the C-6 methylene carbon acts as the hydrophobic feature. The term γ of Shallenberger & Lindley is interchangeable with the term X of Kier. From Shallenberger & Lindley, 1977

Shallenberger & Lindley also described the glucophores of β-D-glucose and β-L-glucose using the Haworth notation to show how they interacted with the same undefined sensory receptor in a tripartite bond. These sugars show the same degree of sweetness.

The Kier paper discusses several more exotic sweeteners, involving nitrogen and sulfur chemical elements.

[xxx need to reference or consolidate parallel molecule structure in Figure 8.5.-51 ]

- - - -

Sugars have many polar hydroxyl groups (–OH) and are overall highly polar. One of the -OH groups is usually found in a –CH2OH group. Calculation of the overall dipole potential of the sugars is complicated. If a significantly polar group of another molecule is brought close to one of the hydroxyl groups, the electrical charge distribution near the hydroxyl, and therefore the dipole potential of the sugar, can be changed significantly. This appears to be phenomenon brought into play by the “super sweeteners.” The optimum sugar moiety for use in the receptor will have its various hydroxyl groups in optimum location relative to the AH,B group. Determining this degree of optimization requires careful examination of a 3-D representation of the sugar. If located appropriately, the moiety can be optimally esterified with phosphatidic acid to provide the desired optimum receptor. [xxx cite or consolidate with the figure “Concept: a stacked situation. . . “ The most likely sweetness receptor appears to be phosphatidyl galactose based on its known presence in many situations involving lemma. Figure 8.5.4-2 shows galactose projected on a plane including the axis between the presumed AH,B participants, O-3 and O-4. It shows two triangles that are not in this plane. There dimensions are therefore distorted in the figure. To be effective in the AH,B,X relationship, the three points of each triangle must be accessible by a potential stimulant (no significant other structure interfering in the plane of the triangle). It appears galactose can accommodate two different AH,B,X relationships within the present 146 Neurons & the Nervous System tolerances on the required dimensions of the triangles. Both the ring oxygen and O-2 are susceptible to an applied electronic force from the stimulant, thereby changing the dipole potential of the overall phosphatidyl galactose molecule. This makes galactose a preferred candidate for the super sweet (as well as the sweet) receptor.

8.5.4.3 Operation of the “acidic” gustatory sensory neuron

[ dipole moment of carboxylic acids Narasimhan57 in pdf ] While an acid sensing channel has been associated with the gustatory system since historic times, reports on the related transduction process are virtually nonexistent in the scientific literature. It is easy to assume any acid transduction would involve a hydrogen ion, this may be an oversimplification. Referring to both recent history and his limited range of subject species, Boudreau has noted247, “The system was labeled ‘acid’ because the most stimulating compounds were Brknsted acids and the least stimulating were Brknsted bases. The most excitatory compounds were carboxylic acids for all species. Also stimulating, but at a variable rate, were phosphoric acids and a small number of nitrogen compounds functioning as Brknsted acids.”

Boudreau’s comments suggest the subtleties Figure 8.5.4-2 Galactose in the plane of O-3 & O-4. between the classic acids, Lewis acids and Two potential AH,B,X triangles are shown. O-1 is Brknsted acids need to be reviewed before the normal site for esterification to the phosphatidic attempting to understand the acidic sensory acid. See text. receptors. The classic acid ionizes to form a simple proton that may or may not be + hydrated, H[H20]n . Lehninger (page 46) has noted, “A Lewis acid is defined as a potential electron-pair acceptor, and a Lewis base is a potential electron-pair donor. A Brknsted acid is a proton donor and a base is a proton acceptor. An acid-base reaction always involves a conjugate acid-base pair, made up of a proton donor and the correspond proton acceptor.” From a practical perspective, Lehninger has also noted, “the formalism introduced by J. N. Brknsted and T. M. Lowry is more useful than that of Lewis in describing acid-base reactions in dilute aqueous systems.” In transduction, no acid-base reaction occurs but dilute aqueous systems are generally involved. According to Morrison & Boyd (page 29), the Lewis acid concept is the most fundamental of the acid concepts. They also note, “To be acidic in the Lowry-Brknsted sense, a molecule must, of course, contain hydrogen.” However, there may be additional requirements on the stimulus besides presenting an available hydrogen ion. It is useful here to consider the inorganic acids as largely outside the realm of natural foods and the sharing of pairs of covalent bonds as the typical situation in gustatory transduction involving + organic acids. This formalism allows the ammonium radical, NH4 to be treated as an organic acid. It is also compatible with treating the carboxyl group as the primary component of most of

247Boudreau, J. (1989) Neurophysiology and stimulus chemistry of mammalian taste systems In Teranishi, R. Buttery, R. & Shahidi, F. eds. Flavor Chemistry: Trends and Developments. Washington, DC: American Chemical Society Chapter 10 Signal Generation & Processing 8- 147 the organic acids. In acid channel transduction, the use of the Lewis concept appears to offer compatibility with the other coordination chemistry concepts applicable to the other gustatory channels. This is particularly appropriate where oxygen is involved and it contributes a pair of electrons in a coordinate covalent bond (as in the AH,B coordinate arrangement). It is useful to note that in biochemistry, an apparent disassociation constant, K’, is used based on the analytical conditions of the investigator rather than the more precise disassociation constant, K, of thermodynamics. - - - - - Pitzer248 has noted the hydration of the hydrogen ion in water at biological temperatures and + pressures is H[H20)1 . [xxx rewrite this whole section based on the “browned flavor” sensation below. Ohloff went a different way ] Ohloff has presented a theory of H-best sensory receptor operation involving a specific configuration of sensory receptor capable of hydrogen bonding. Basically, the sensory receptor is capable of closing a ring by employing a hydrogen bond contributed by the stimulus. The essence of the method requires the sensory receptor to possess two oxygen atoms at adjacent locations on a larger chemical structure. One of the oxygen atoms must be divalently bonded to the larger structure as shown in Figure 8.5.4-3.

The sphingomyelin and phosphatidyl 3'-O- alanyl glycerol forms of phospolipids are known to be present in the outer bilayer of neural cell lemma, to possess a significant dipolar moment, and to possess multiple oxygen moieties in various configurations. Specific molecules from these families, called plasmalogens, with one of the lipid chains fully unsaturated, are good candidates for the sensory receptor role in the H-best sensory channel. [xxx need to consider sub- scripting the phosphatidyls of interest to Figure 8.5.4-3 Potential sensing of the hydrogen ion indicate their unsaturated nature ] in the H-best sensory receptor ADD. From Ohloff, 1981. - - - --

Based on this discussion and those of the previous sections, the sensory receptor of the acid-best (h-best ?) sensory channel is likely to be a structure that can form a coordinate structure with the carboxylic acids. A likely candidate would be PtdSer, a phospholipid known to be present in the lemma of neurons and to contain a carboxylic acid group as shown in Figure 8.5.4-4.

Figure 8.5.4-4 Phosphatidylserine shown in polar form ADD or REPLACE..

248Pitzer, K. (1982) Self-ionization of water at high temperature and the thermodynamic properties of the ions J Phys Chem vol 86, pp 4704-4708 148 Neurons & the Nervous System

In another depiction, the serine ligand can be described as in Figure 8.5.4-5. This ligand offers a variety of interpretations supporting an AH,B type coordinating situation. The potential role of the oxygen of the hydroxyl group acting as AH and the amine acting as B is obvious. Only the distance between them remains undefined in this figure. Alternately, the carboxyl group can provide an oxygen and a hydroxyl group corresponding to B and AH respectively.

Discussions of carboxylic acid chemistry frequently begin with a dimer as shown in Figure 8.5.4-5 Serine as the potential sensory Figure 8.5.4-6. While typically shown with receptor ligand. The oxygen at upper left forms the R and R’ identical in initial discussions, this ester with the phospholipid. The carboxyl group at is not a criteria. The structure shown is right forms the sensory receptor site of the acid planar. R and R’ may be quite different channel sensory neuron. without causing major differences in the coordinate bond relationship. Where the differences introduce strains in the bond configuration, the transduction process may be less effective and the sensation of“acidity” reduced for the stimulus. Almenningen et al. have provided detailed dimensions for the monomer and the dimer of formic acid249. They provide statistical limits on their parameters after reviewing the earlier work of others. Interestingly, the so-called hydrogen bond is measured from the centers of the two oxygen atoms.

It is proposed that this formation of a dimer between the serine of PtdSer and a carboxylic acid stimulus constitutes the major transduction process in the (organic) acid-best channel of the gustatory modality. The transduction requirement is that the stimulus form a coordinate bond with the carboxyl group Figure 8.5.4-6 A carboxylic acid dimer. The dimensions are shown of the serine, thereby for the carboxyl group of the serine portion of phosphatidyl serine changing the dipole potential acting as the (organic) acid receptor of the gustatory modality.

249Almenningen, A. Bastiansen, O. & Motzfeldt, T. (1969) A reinvestigation of the structure of monomer and dimer formic acid by gas electron diffraction technique Acta Chem Scand vol 23(8), pp 2848-2864 Signal Generation & Processing 8- 149 at the base of the first Activa formed by the asymmetrical bilayer lemma of the sensory neuron microvilli. Any stimulus capable of forming a coordinate bond with the serine ligand will elicit a sensation of acidity within the neural system. The hydrogen bond in each leg of such a dimer typically has an energy of 2-10 kCal/mole. This low energy illustrates the ease with which the bond can be broken following the transduction process. As suggested by the difference in bond lengths relating to the left carbon, resonance does not appear to play a role in this structure. However, more analysis may be warranted. Valla has described nine different coordinate bonding arrangements for the carboxylic acid group, three that involve two bonding paths250. Because of the carboxyl group in their intrinsic structure, all amino acids can coordinate with the proposed acid sensory receptor and stimulate the acid sensory channel to a degree. Boudreau (page 130) reviewed the relative intensity of these sensations in several species.

8.5.4.4 Operation of the “alkaline” gustatory sensory neuron

The alkali metals and the alkali earth metals are distinctly different and should not be lumped together when considering their theoretical taste performance. The alkali metals (Lithium, Sodium etc.) are singularly ionized while the alkali earth metals (Calcium, Magnesium etc.) are typically doubly ionized. Of greater importance is their different coordinate bonding potential.

It was found earlier that the alkali metal ions were perceived as sweet at low concentrations and this is believed to be due to their hydration resulting in an AH,B coordination capability with the S-channel receptors.

Avenet & Lindemann (1989) have noted, “Of the diversity of tastes induced by inorganic salts, that of NaC1 has found primary attention. For man, NaC1 is the only substance which has a pure salty taste, and the sodium ion appears to be more important for this taste than the anion.”

Quist & Marshall251,252 have described the hydration of NaCl and Na+ & Cl–. They note the nearly + constant hydration, Na[H2O]6 , regardless of temperature for concentrations above 1.2 moles/liter 0 and NaCl[H2O]2 below 1.2 moles/liter. Zumdahl has provided the hydration energy of the sodium ion, presumably for the fully hydrated case, as –402 kJ/mol253. He also provided the first ionization energy of sodium as +495 kJ/mol.

Marshall provided the ionic radii for Li, Na & K as 0.6, 0.98 & 1.33 Angstrom respecively254. He also provided the atom radii for these materials as 1.56, 1.86 & 2.23 Angstrom.

Shallenberger (1996) noted a general consensus that only NaCl elicited a true salt taste. His

250Valla, V. & Bakola-Christianopoulou, M. (2007) Chemical aspects of organotin derivatives of beta-diketones, quinonoids, steroids and some currently used drugs: A review of the literature with emphasis on the medicinal potential of organotins Synth React Inorganic, Metal-Organic, and Nano-Metal Chem vol 37, pp 507–525,

251Quist, A. & Marshall, W. (1968) The independence of isothermal equilibria in electrolyte solutions on changes in dielectric constant J Phys Chem vol 72(6), pp 1536-1544

252Quist, A. & Marshall, W. (1968) Electrical conductances of aqueous sodium chloride solutions from 0 to 800" and at pressures to 4000 bars J Phys Chem vol 72(2), pp 684-703

253Zumdahl, S. (1993) Chemistry, 3rd Ed. Lexington, MA: D. C. Heath page 329

254Marshall, S. (1956) Sodium: its manufacture, properties, and uses. NY: Reinhold 150 Neurons & the Nervous System words may be too casual. If the coordinate chemistry of the sodium ion is the principle contributor to the N-best channel’s response, it should be clear that it is the sodium ion (possibly at a specific level of hydration) that elicits the maximum (or “true”) N-best channel sensation. The presence of the chlorine ion is largely irrelevant. Figure 8.5.4-7 illustrates the two most common states of hydration of the sodium ion. In dilute + solution, it is believed to form Na(H20)2 . The distance between the oxygen atoms of this hydrate + is 4.7 Angstrom. In more concentrated solutions, it is believed to form Na(H20)6 with the water molecules arranged at the vertices of an octahedron. The distance between the water molecules, that can act as AH,B coordinate structures, are nominally 3.3 Angstrom

Figure 8.5.4-7 The sodium ion at hydration levels of 2 and 6. Only one of the three pairs of water molecules are shown on the right. See text.

The unique features of the sodium ion are two. First, the sodium ion is very small in diameter, nominally 0.95 Angstrom in diameter. Second, the ion is normally highly hydrated in aqueous solution. The hydration number is usually six (with the 2p shell fully involved), although it has been reported to be four, two or one in various special cases. With a hydration number of six, the ensemble exhibits a face centered cubic arrangement, with the water molecules located at th vertices of an octahedron. At a hydration number of six, the locations of the water molecules are at a spacing that is ideally arranged for coordination with a sensory receptor such as the oxygen rich PtdIns. This coordination may involve as many as three hydrogen bonds. Signal Generation & Processing 8- 151 The fully hydrated sodium ion exhibits three separate odorophores with a d-value of 3.3 Angstrom. This raises the probability that a given hydrate will be successful in coordinate bonding to a suitable sensory receptor. Figure 8.5.4-8 presents a potential hydrated sodium “dimer” similar to that shown for the carboxyl group. The dimensions are taken from Hille255 who credits them to Pauling. This figure shows the hydrogens of the water molecules are out of plane relative to the sodium and oxygen molecules. This suggests that one of the hydrogens can be moved into the plane of the molecules and form a hydrogen bond with a pair of electrons of the oxygen of the other hydrate. As in the case of the carboxyl dimer, this figure suggests the spacing between the AH,B group of the sodium sensitive sensory receptor should optimally have a spacing near 3.3 Angstrom. This small size is optimally matched to the coordination geometry of PtdIns. This makes the sensitivity of the N-best receptor maximally sensitive to the sodium ensemble. [xxx show inositol in lower part of figure ]

The chemistry of PtdIns is poorly documented. Parthasarathy & Eisenberg reviewed the subject in 1991, noting a major change in nomenclature in 1968. They also make confusing statements about the isomers of inositol. “Myo-inositol is the only stereoisomer to occur in phospholipids (pg 6)” Figure 8.5.4-8 A potential hydrated sodium “dimer and “This finding suggests that myo-inositol ADD.” Each of the units displayed consist of one is not the only stereoisomer found in inositol sodium ion fully hydrated with six water molecules, phospholipids (pg 14).” Their figure 6 only two of which are shown. [xxx where are requires careful study because the hydroxyl hydrogen bonds? ] groups are omitted from the chair conformation diagrams they present. A new set of rules was being prepared during preparation of their published article256.

[xxx probably condense to one sentence in favor of the following material based on Molecular Arts ] Inositol, or cyclohexane-1,2,3,4,5,6-hexol is a chemical compound with formula C6H12O6 or (-CHOH-)6, a sixfold alcohol (polyol) of cyclohexane. In its most stable conformational geometry, the myo-inositol isomer assumes the chair conformation, which puts the maximum number of hydroxyls to the equatorial position, where they are farthest apart from each other. In this conformation the natural myo isomer has a structure in which five of the six hydroxyls (the 1st, 3rd, 4th, 5th, and 6th are equatorial, whereas the 2nd hydroxyl group is axial. The counterclockwise notation is from the pseudonym Yikrazuul via Wickipedia.

Figure 8.5.4-9 shows this configuration combined with a partial view of hydrated sodium. The carbon-carbon bond lengths are 1.53 Angstrom. The carbon-oxygen bond lengths are 1.43 Angstrom. The net (out-of-plane) bond length between the OH-3 and OH-4 are estimated to be 3.3 about three Angstrom based on the spacing between the oxygen atoms of the hydrated sodium.

255Hille, B. (1971) The Hydration of sodium ions crossing the nerve membrane PNAS vol 68(2), pp 280-282

256NC-IUV (1988) Numbering of atoms in myo-inositol http://www.chem.qmul.ac.uk/iupac/cyclitol/myo.html 152 Neurons & the Nervous System

Figure 8.5.4-9 Myo-inositol, a sugar alcohol, and a fully hydrated sodium shown with coordinate bonds with a dimension, d, of nominally 3.3 Angstrom. Signal Generation & Processing 8- 153

Molecular Arts software package prepares an alternate representation of inositol. Figure 8.5.4-10 shows only one equatorial hydroxyl (1) with three above the plane and two below the plane.

The correct representation of inositol as it is esterfied to the phosphatidyl moiety is critically important to understanding the dimension, d, between various pairs of hydroxyls. The calculated distance between the oxygens alternating between above and below the molecule (3.24 Angstrom) is in good agreement with the distance between the oxygens of the hydrated sodium ion. Figure 8.5.4-10 Two perspective views of inositol Figure 8.5.4-11 shows the proposed bonding from the software package, Molecules 3D Pro. Only between the hydrated sodium ion and one hydroxyl is shown in equatorial position. See phospatidyl inositol. The figure can text. represent the natrophore bonding with the sensory receptor at O-3 and O-4 or O-4 and O-5. The out of plane length of the carbon bond is foreshortened in this figure.

8.5.4.4.1 The perception of sodium as sweet at low concentrations

Shallenberger & Acree have noted the perception of sweetness associated with salt in solution at low concentrations. The trait appears common among the low atomic weight alkali metal salts, Li, Na & K..

The literature related to the hydration of the alkali ions at low concentrations is extremely thin. The terminology is also contradictory due to age. An anhydrous compound, HNaO, Figure 8.5.4-11 The natrophore of hydrated sodium is described by the CAS Register as sodium and inositol in a coordinate bond. The length of the hydrate. In solution however, the sodium carbon-carbon bond is foreshortened in this ion is known to exhibit a variety of states of rendering. hydration. It appears sodium can support a coordinate number of 1, 2, 4 or 6 depending on the circumstances, and particularly the concentration of the ion in water.

Shallenberger & Acree have suggested that Na[4], with a tetrahedral form, can create a coordinate bond with the S-best sensory receptors because of the spacing between the pairs of hydrates, one acting as an electron acceptor and the other as an electron donor. Beginning in the same era, Marshall has indicated the ion is generally fully hydrated, Na[VI]+1, at low (gustatory) concentrations257. See Section 8.5.3XXX.4 [xxx resolve this paragraph and the reference. ]

- - - - [xxx check this ] The hydration of sodium at low concentrations explains the perception of a sweet taste to sodium salts. This would suggest that the sodium or N-best sensory channel is also a coordination chemistry based sensory channel that senses the sodium ion (without sensing the associated

257Marshall, W. (2008) Aqueous electrolyte ionization over extreme ranges as simple fundamental relation with density and believed universal; sodium chloride ionization from 0o to 1000oC and to 1000 mPa (10000 atm.) Nature Precedings : hdl:10101/npre.2008.2476.1 154 Neurons & the Nervous System negative ion). - - - - 8.5.4.5 Operation of the “bitter” gustatory sensory neuron

Bitter tastes are often associated with harmful substances like alkaloids and glucosides, but many other groups elicit a bitter sensation. The operation of the bitter sensory channel is not well documented. Belitz et al258. provided a theory of bitter taste related to the early work of Shallenberger. They suggest a topological requirement, involving a hydrophobic pocket, is involved in bitter or Q-best channel sensing. The concept appears to correlate well with experience involving a wide range of amides, amines and amino acids. However, Shallenberger & Acree noted in a larger context in 1971, “Only limited correlations between bitter taste and molecular structure can be found.” In 2000, Eggers, Acree & Shallenberger took a different position after reviewing the state of the art and noted, “These results suggest that both sweet and bitter reception share the same transduction components and that the non-sugar sweet receptor system is related to the bitter receptor system if it is not in fact the same.”

Kumazawa provided a discussion of the varied chemistry of bitter stimulants and potential bitter channel sensor operation but was unable to identify any receptor or provide any description of the pertinent sensory neuron259. The list of bitter tasting compounds is quite long and involves many chemical structures as shown by Spielman et al260. in Figure 8.5.4-12. Van der Heijden has given a similar list of the major bitter stimulants that includes their relative potency. He has also provided chemical structures for each of these materials. These diagrams illustrate the historic difficulty of finding a common structural feature to account for the bitter sensation.

258Belitz, H. Chen, W. Jugel, H. et al. (1981) Structural requirements for sweet and bitter taste In Schreier, P. ed. Flavour ‘81. Berlin: Walter de Gruyter pp 741-755

259Kumazawa, T. Nomura, T. & Kurihara, K. (1988) Liposomes as model for taste cells: receptor sites for bitter substances including n-c=s substances and mechanism of membrane potential changes Biochem vol 27, pp 1239

260Spielman, A. Huque, T. Whitney, G. & Brand, J. (1992) The diversity of bitter taste signal transduction mechanisms In Corey, D. & Roper, S. eds. Sensory Transduction. Woods Hole, MA: Marine Biological Laboratory Chapter 20 . Signal Generation & Processing 8- 155 Spielman et al., writing in a Society of General Physiology symposium record, speculate on whether a single sensory neuron could possibly accommodate the detection of this broad array of chemical structures, or whether multiple sensory neurons are required. They offer only working hypotheses on the character of the sensory neurons based conceptually on the chemical theory of the neuron. Their assumptions are; 1. there is no one mechanism that would apply to all bitter stimuli, 2. there must be several signal transduction processes attuned to the broader chemical categories of bitter compounds, 3. one group of bitter compound can be detected by several different mechanisms, and 4. there are still bitter compounds to be discovered or synthesized.

Van der Heijden reviewed the structural formulas for a Figure 8.5.4-12 The diversity of bitter compounds. See text. From variety of his bitter Spielman et al., 1991. stimulants (page 95-103) and concluded they did not meet the criteria for a suitable AH,B coordination bond because the structures did not meet his criteria for bitterness of 0.15 nm (1.5 Anstrom). His model of a sweet-bitter has not drawn favor in the taste research community. The 0.15 nm criteria applies to the H-best channel, not the Q-best channel. As shown in this work, the AH,B spacing criteria to coordinate with the bitter sensory receptor is 0.42 nm (4.2 Angstrom) as developed below.

The ability of the AH,B coordination structure to explain the operation of the S-best channel suggests a similar structure might be important in the Q-best channel. To date, only limited insight has been obtained into the operation of the Q-best receptor mechanism. Shallenberger & Acree have noted, “limited correlations between bitter taste and molecular structure exist because this taste sensation is not one that is enthusiastically evaluated.” More recently (2003), Drewnowski has noted261, “the biology of bitter taste perception is poorly understood.” and “The study of human taste genetics is largely the study of bitter taste.” The range of chemical structures stimulating the Q-best channel is wide. It includes many, but not all, alkaloids, tannins, methylxanthines, peptides, isoprenoids and many nonsodium salts. Care must be taken to use the complete chemical name of a bitter stimulant, the common names used in pharmacology and the food technologies are frequently less than specific.

261Drewnowski, A. (2003) Genetics of human taste perception In Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker Chap 40 156 Neurons & the Nervous System

Denatonium, Figure 8.5.4-13, usually available as denatonium benzoate and as denatonium saccharide, is the most bitter chemical compound known. Dilutions of as little as 10 ppm are unbearably bitter to most humans. Denatonium is a quaternary ammonium cation. It is a compound of a salt with an inert anion like benzoate or saccharide. The structure of denatonium, C28H34N2O3 is related to the local anesthetic lidocaine, differing only by the addition of a benzyl group to the amino nitrogen.

Figure 8.5.4-13 Denatonium benzoate, the most bitter compound known. Et; ethyl ligand. Mt; methyl ligand. PH; phenyl ligand. Signal Generation & Processing 8- 157 Figure 8.5.4-14 shows the chemical structure of quinine. Quinine hydrochloride is frequently used in the laboratory as the quintessential bitter stimulant. Its complex structure makes it difficult to analyze without a definitive set of requirements. However, based on the theory developed here, the bipartite AH,B coordinate bond with the Q-best sensory receptor is clear as illustrated. The spacing between the two oxygen atoms is estimated at 4.2 Angstrom after accounting for the two-dimensional projection of this three-dimensional molecule. If the actual spacing is not close to this value, an alternate AH,B coordinate bond will be required based on the presence of Nitrogen at several points or the presence of the ring structures.

While nitrogen plays a major role in many bitter stimulants, it is not a necessary component of a Q-best stimulus. The tannins (tannic acids) are a complex family of (a) so-called condensed tannins and (b) hydrolyzable tannins. The latter are sugar esters with one or more trihydroxybenzenecarboxylic acids as shown in Figure 8.5.4-15. The proliferation of hydroxyl radicals in these compounds make the potential for an AH,B coordinate bond of the required spacing quite likely. It is suggested the hydroxyls at the 4 positions of each ring satisfy the spacing requirement proposed by the theory presented here. Figure 8.5.4-14 Quinine, the preferred bitter taste in the laboratory.

Figure 8.5.4-15 Corilagin, a tannic acid & a complex sugar ester. 158 Neurons & the Nervous System

Figure 8.5.4-16 shows the chemical structure of caffeine, another bitter stimulus containing significant nitrogen but little oxygen, and its parent xanthine on the right. Determining the actual AH,B coordinate structure providing a spacing appropriate for the Q-best sensory receptor requires more careful mathematical analysis.

Quoting Glendinning et al., “Many different classes of compounds elicit bitter taste in humans and inhibit feeding in animals. These compounds include alkaloids, tannins, methyl- xanthines, peptides, isoprenoids, and many non-sodium salts. Because virtually all naturally occurring poisons taste bitter to humans, this taste quality Figure 8.5.4-16 Caffeine, 1,3,7-trimethylxanthine is thought to have evolved as a and its parent xanthine on the right. mechanism for avoiding toxic substances.”

[xxx rewrite next two paragraphs ] Breslin may have uncovered the key to the operation of the Q-best sensory neuron (Section 8.xxx). This may be an excellent clue to the operation of the Q-best sensory channel since phosphatidic acid is a potential transducer in the type 4 lemma of the Q-best sensory neuron microvilli. If the phosphatidic acid is reacting with the quinine hydrochloride with the precipitation of quinine, the phosphatidic acid may become chlorinated and become negatively charged. This action could mirror the nominal reaction between the phosphatidyl fatty acid of type 4 lemma and quinine hydrochloride, resulting in the precipitation of the quinine, permanent alteration of the phosphatidyl fatty acid and the injection of an electron into the plasma of the microvilli. A comparison of the state of polarization of phosphatidic acid and both PtdCho and PtdSer would appear to be useful. PtdSer is very similar structurally to phosphatidic acid and shows a positive polarization, just the opposite of the more common PtdCho forming a majority of the outer leaf of plasma lemma.

Breslin (page 440) has noted the genetics-based work of Kalmus262. Kalmus found a N–C=S bond in a set of anti-thyroid drugs that may be significant. Individual humans exhibit considerable difference in sensitivity to these two compounds. As many as 25% of the population may be taste blind to the bitterness of these materials, suggesting a “spectral blindness” similar to protanopia in vision. Since these taste-blind subjects are not blind to other quinine related stimuli, it has been suggested there may be more than one Q-best receptor types.

Shallenberger & Acree have provided some information concerning two possible structural arrangements important in the Q-best sensory channel.

Figure 8.5.4-17 shows two structural configurations that both elicit a bitter sensation but appear to have little in common at the 1971 level of understanding. The thio-carbamide was originally found in phenyl-thio-carbamide (PTC) by Fox (1932)

262Kalmus, H. (1971) The genetics of taste In Beidler, L. ed. Handbook of Sensory Physiology: Vol. IV, Chemical Senses NY: Springer pp 165-179 Signal Generation & Processing 8- 159

Shallenberger & Acree have also reported the work of Kubota & Kubo263 suggesting an AH,B relationship with a 1.5 Angstrom spacing might be important in bitter channel transduction. However, they also suggest three electronegative oxygen atoms lying in a single plane may be a characteristic of quinine class stimuli, such as picric acid with three NO2 ligands. Kubota & Kubo examined the diterpenes from a specific species of plants, Isodon, extensively. Using slightly different terminology than Shallenberger & Acree, possibly mis-translating their concept and relying on the Brknsted acid concept of acids and bases, they assert that a proton donor group (DH) and a proton acceptor group (A) must be within 1.5 Angstrom and form an intramolecular bond in order to form a “bitter unit.” They do not address the stimulus/receptor relationship.

The assertion of a distance between coordinate bonds of 1.5 Angstrom for bitter compounds has not appeared in the recent literature, except in its restatement by Kubo in 1994. A paper by Yamada et al264. in 1999 obtained detailed information on the Figure 8.5.4-17 Two structures believed to cause structure of several more diterpenes from the bitter taste sensation ADD. B; a thio-carbamide. Isodon and explored their bitterness relative From Shallenberger & Acree, 1971. to quinone. However, they did not explore the transduction process.

263Kubota, T. & Kubo, I. (1969) Bitterness and chemical structure Nature vol 223, pp 97-99

264Yamada, Y. Sako, N. Ando, E. et al. (1999) New bitter diterpenes: Rabdosianone I & II: Biosci Biotechnol Biochem vol 63(3), pp 524-529 160 Neurons & the Nervous System

- - - - [xxx move this up before other examples of stimuli ] A major problem for previous investigators has been their lack of any guidance on what to look for. With the exception of the comment about three electronegative oxygen atoms lying in a single plane by Shallenberger & Acree, the typical investigator has been looking at structures involving less distance between the specific atoms. The rectilinear sensation space of this work shows a quinine best receptor must involve A B spacings greater than 3.7 Angstrom to be independent of the other sensory channels. xxx add here The most probable phospholipid of the outer bilayer membrane of the microvilli capable of operating as the sensory receptor is a form of Ptd3'Og with one unsaturated fatty acid providing electrical conductivity along the length of the molecule. Figure 8.5.4-18 shows the structure of this candidate along with its predicted sensitivity in taste sensation space. As shown by the unspecified group, R, Ptd3'Og.is actually a large family. In their experiments, Silvius et al. found R to be mostly in the form of alanine265.

The active portion of the overall phosphatidyl molecule is shown at upper left beginning with the ester to phosphoric acid on the left. The pertinent feature is the distance of about 4.2 Angstrom between the oxygen atom, B, and the amine, AH. This broad spacing suggests this sensory receptor is positioned as shown in the lower frame of the figure, achieving a sensation space with minimum overlap with the other sensory channels. As noted earlier, the mean values of each distribution is calculable but the distributions are shown only conceptually.

The upper right of the figure shows a very complex molecule known for its bitterness, quassin (or Surinam quassia in commerce). It has a bitterness threshold of 1:60,000. Note the symmetry of this molecule suggesting it could form the AH,B bond in multiple ways. A similar graphic can be Figure 8.5.4-18 Candidate sensory receptor prepared showing coordinate bonding with performance for the “bitter” channel. Upper left, brucine, another important bitter stimulant. active ligand of bitter sensory receptor. Upper right; quassin shown oriented so as to form an AH,B coordinate bond with the sensory receptor. Bottom; rectilinear taste space showing the quinine channel centered at an A B spacing of 4.2 Angstrom. See text.

265Silvius, J. Mak, N. & McElhaney, R. (1980) Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii b Biochim Biophysica vol 597(2), pp 199-215 Signal Generation & Processing 8- 161

Figure 8.5.4-19 shows the proposed sensory receptor coordinate bonding to the common laboratory stimulant quinine. The nominal spacing between the coordinate bonding couple is 4.2 Angstrom. The receptor orbitals are oxygen and nitrogen while the stimulant orbitals are both oxygen. R is shown as carboxylic acid in the figure. The left-most oxygen forms the ester with the conductive phosphatidyl group. The potential for an enhanced bitter sensation resulting from a third site forming an AH,B,X bond like that of the sugars should not be dismissed. If the bitter sensory receptor is in fact 3'-O-aminoacyl glycerol, it exhibits a potential X site at either of the two oxygen atoms associated with the glycerol moiety and at either of the two oxygen atoms of the carboxyl group.

Figure 8.5.4-19 Candidate picric sensory receptor and quinine coordinate bonding RESCALE. SHOW left-most O as ester. 162 Neurons & the Nervous System

8.5.4.6 CaCl2 & MgCl2 as gustaphores

CaCl2 and MgCl2 are generally perceived as bitter as well as or rather than salty. The reasons for this will be developed below.

Both CaCl2 and MgCl2. are described as deliquescent, forming crystals including six molecules of water. The anhydrous calcium salt is deliquescent; it can accumulate enough water in its crystal lattice to form a solution. When calcium chloride is added to water (solubility–74.5 gr/100 ml), much heat is liberated. Magnesium chloride acts similarly. Its solubility at biological temperatures is 60 gr/100ml. While these materials completely ionize in water, it is the coordination chemistry of the hydrated cations that is significant in gustation. The anion plays no active role. Magnesium sulphate (milk of magnesia) is also perceived as both bitter and salty. When in solution, the cations of these chemicals form coordinate bonds with the water molecules. Calcium generally bonds to six or seven water molecules, occasionally eight or less than six. For six ligands, the structure is octahedral. For eight ligands, the structure is hexagonal bipyramidal. Other bonding levels typically employ one of these forms with one or more coordinate locations empty (although some sources report a pentagonal bipyramidal structure for this salt). At biological temperatures one calcium is predominantly bound to six water molecules. Calcium forms complexes with many molecules. The ligand distances vary between 2.1 and 2.6 Angstrom. When coordinate bonded to water, the ligand distance is normally 2.431 Angstrom.

For a coordination number of eight, the hexagonal bipyramidal structure exhibits angles of 60 degrees between the six oxygen ions in the horizontal plane and 90 degrees between the oxygen ions in that plane and the two oxygen ions at the points of the pyramids. Figure 8.5.4-20 shows the medial plane of the octahedral structure of the calcium ion fully coordinated with six water molecules after CaCl2 is dissolved in water. The ability of the oxygen atoms to participate in paired coordinate bonds with a sensory receptor is obvious. The nominal calcium to oxygen 266 +2 spacing is from Kim et al . As shown, the Ca(H2O)6 complex could exhibit gustaphores with d- values of 3.437, 4.210 and 4.862 Angstroms. +2 The smaller Mg(H2O)6 exhibits equivalent values of 2.9, 3.585 and 4.14 Angstrom Glusker et al., 1999). Bock et al. (1994) have noted that for MgCl2, the hydrogen atoms are at an angle of 120-127 degrees from the Mg–O bond. This angle would be 127.7 degrees if the pHOH was the nominal 104.5 degrees

Figure 8.5.4-20 Calcium cation fully coordinated with water when in solution. Coordinate bonds are shown dashed. The cation retains its conventional valence of +2. Dimensions in Angstrom. See text.

266Kim, S. Gregor, W. Peloquin, J. Brynda, M. & Britt, R. (2004) Investigation of the Calcium-Binding Site of the Oxygen Evolving Complex of Photosystem II Using 87Sr ESEEM Spectroscopy J Am Chem Soc vol 126, pp 7228-7237 Signal Generation & Processing 8- 163 Yang et al. discuss variations in the angles between the oxygen ions in stressed complexes267. Magnesium complexes in the same manner as calcium and with a similar variation in coordinate levels (with six being the predominant value 79% of the time268). The oxygen to oxygen spacing in the octahedron form is 2.9 Angstrom. The spacing between opposed oxygen ions is 4.14 Angstrom. [xxx duplicates above ] [xxx Is either form of calcium or magnesium good matches for the values of a picrophore, d=4.2 Angstrom, or Natrophore, d=3.3 ] The d-value of 4.14 Angstrom for the opposing oxygen ions of the hexagonal Magnesium complex makes this structure a potential picrophore if it is able to satisfy the stereo-chemical requirements and reach the appropriate sensory receptor site.

Figure 8.5.4-21 shows the relationship between the these “salts” and the receptive range of the gustatory sensory receptors. It clearly shows why these salts are perceived as bitter. Both have d- values matching the values of the bitter sensory channel. CaCl2 and MgCl2 are both picrophores. Both exhibit d-values that could qualify them as natrophores also, depending on the sensitivity range of the sensory receptors..

The d-values of these “salts” aid in determining the receptive range of the sensory receptors. In the case of the picrophores, they exhibit +2 calculated-values of 4.14 (Mg(H2O)6 ) to 4.22 (Quinine xxx). These values suggest a receptive range of at least 0.08 around a center value of 4.18. On the other hand, the gustaphores of these salts at 3.437 and 3.585 are less effective in stimulating the sodium sensitive channels and help define the senstivity range of the sodium sensitive channel. The sodium sensitive channel can be considered to be centered at d = 3.30 with a range at most only marginally higher than 3.437. These values suggest a center value of 3.30 with a range on the order of 0.28 for the sodium sensitive channel.

This analysis suggests the picrophores of Magnesium salts are formed by the oxygen atoms separated by 120 degrees about the cation while the picrophores of calcium salts are formed by the oxygen atoms on opposite sides of the cation. In both cases, these hydrated ion structures exhibit multiple picrophores associated with each cation, raising the probability that these structures will form dual coordinated bonds with the bitter sensory receptors. The nearest oxygen atom pairs of Figure 8.5.4-21 The potential gustaphores of CaCl these structures do not act as effective 2 and MgCl2 ADD. Both salts exhibit significant gustaphores. picrophores, at 4.14 and 4.21 Angstrom. The calcium salt may also exhibit a natrophore at 3.437 Angstrom, depending on the receptive range of the 8.5.4.7 The “water” gustatory sensory neuron receptor. sensory response

267Yang, W. Lee, H-W Hellinga, H & Yang, J. (2002) Structural analysis, identification, and design of calcium- binding sites in proteins Proteins struct funct genet vol 47, pp 344-356

268Glusker, J. Katz, A. & Bock, C. (1999) Metal ions in biological systems Rigaku J pp 8-16 164 Neurons & the Nervous System

Several investigators have reported a “water” response for their sensory neurons. With an understanding of the concept of pre-adaptation, this response should not be surprising. A water flush after stimulation with any stimulant or even normal saliva can be expected to constitute a change in the taste bud environment. This previous exposure to a stimulant or saliva can be considered a pre-adaptation step with the water wash a subsequent stimulus test interval. The sensory neurons can be expected to report this change. Pages 262- 268 in Cagan & Kare discuss their experiments.

8.5.4.8 The “browned flavors” sensory response

Hodge, followed by Ohloff269, have studied the characteristics of a large range of molecules associated with the sensations resulting from the browning of food products during cooking. Hodge analyzed the chemical structure of a wide variety of molecules he associated with the browned flavor sensation270. He described these sensations as; “They are essential for the recognition and acceptance by taste of many processed foods, especially cereal-derived foods. Browned flavors include caramelized sugar aromas (1); food aromas that have been described variously as toasted, baked, nutty, or roasted (2); corny and amine-like aromas from cooked grains and meals; and both the desirable and undesirable burnt aromas and bitterish tastes of roasted malt, nuts, coffee, chicory, cocoa, meats, fruits, and vegetables (2). The objectionable burnt and bitter flavors of overheated or long-stored dehydrated foods also fall within this class.”

Whereas Hodge noted the ability of many of these molecules to form an intramolecular hydrogen bond between a divalent oxygen and an adjacent hydroxyl moiety, this relationship did not suggest any arrangement between these molecules and a sensory receptor. Ohloff, in the same time period as Shallenberger & Acree were working on the sugars, proposed the “browned flavor” stimulants employed the same AH,B coordinate bonding to a sensory receptor as the sugars (with a spacing of less than 3 Angstrom) but with an additional relationship. He suggested this relationship involved the hydrophobic part of the molecule. He was unable to specify the precise relationship between this apolar portion of the molecule and the receptor as a substrate.

No multidimensional analyses involving these flavors could be located. It appears the “browned flavor” stimulants could support a dual channel sensation involving the S-best channel and one or more other best channels.

8.5.4.9 The role of amines & amino acids in the taste sensation TIE 8.6.2.5

[ rewrite and broaden to cover both categories ] Belitz et al. have provided a simple table showing the critical role of the carboxyl group and a ligand containing some form of nitrogen in giving the amino acids a sweet taste271. More recently, Van der Heijden (page 104) has described the relative sweetness and bitterness of the amino acids based on their steric form. Of those amino acids addressed, the D– forms are invariably sweet and the L– forms invariably bitter. Van der Heijden provides a brief list of properties associated with amino acids and peptides and the sensation of bitterness. [xxx list ] Kier272, has provided a

269Ohloff, G. (1981) Bifunctional unit concept in flavour chemistry In Schreier, P. ed. Flavour ‘81. Berlin: Walter de Gruyter pp 757-770

270Hodge, J. Mills, F. & Fisher, B. (1972) Compounds of browned flavor derived from sugar-amine reactions Wash, DC: Agricultural Research Service, U.S. Department of Agriculture.

271Belitz, H. Chen, W. Jugel, H. et al. (1979) In Bourdreau, J. ed. Food Taste Chemistry. Washington, DC: American Chemical Society Chapter 4

272Kier, L. (1972) A molecular theory of sweet taste J Pharm Sci vol 61(9), pp 1394-1397 Signal Generation & Processing 8- 165 description of the sensations elicited by the amino acids that differs significantly from that of Van der Heijden. Kato et al. have provided considerable background on the gustatory properties of the amino acids273. The discussion makes it very clear that it focuses on the requirements of the sensory receptors and not the obvious properties of the stimulants that determine the gustatory sensations. Because the amino acids exhibit such a variation between sweetness, non-sweetness and bitterness, they appear to offer a chance to develop a better understanding of the structural relationships required to elicit sweet and bitter tastes in general.

8.5.4.10 The putative “umami” sensory response

The Japanese, beginning in 1908, have suggested a fifth fundamental taste sensation called umami. The term appears to have been derived from their word, umai (delicious). The designation appears to have a strong representation in their culture. Whether this sensation is the result of a mixture of the other four sensations, an enhancement of the overall taste sensation or a fifth sensation is yet to be resolved. Yamaguch & Ninomiya have recently asserted, “Umami makes a variety of food palatable, although it is not palatable by itself274.” Yamaguchi has been the leading investigator of umami. He has prepared the most extensive study of this putative sensation, including extensive lists of chemicals believed to involve the umami sensation275. More recently, he has presented some results of multi-dimensional analyses and asserted that umami is represented by a different dimension than the other four historical sensations276. However, the printed record of this poster presentation does not include any graphical material and speaks of dimensions and vertices of the taste sensation space that may suffer in translation. It does reference, but not cite in the paper, a larger paper of ten years earlier277. This paper did not include a full multi-dimensional analysis. It specifically did not include the basis factors demonstrating a four dimensional taste space. It leaves the question of whether umami is a taste enhancer or is sensed by a distinct sensory channel unanswered. It is yet to be resolved whether their interpretation introduces a fourth dimension in taste space or merely occupies another vertex of the three dimensional space.

The principal stimulant associated with umami is mono-sodium glutamate, an unusual chemical that includes the primary N-best channel stimulant (sodium) and a well studied stimulant, glutamate. The glutamate ligand exhibits the ability to coordinate with the H-best sensory receptor via its carboxyl group(s), and to potentially coordinate with the S-best sensory receptor via its combination of an NH and O combination providing an AH,B union. The spacing of these atoms is probably not close to the nominal 2.6 Angstrom. The naturally occurring L-glutamate is reported to be tasteless while D-glutamate is sometimes reported to be sweet. The sensations elicited by mono-sodium glutamate are primarily those of salty, with a slightly acidic, and to some a slightly sweet taste. These conclusions are consistent with Belitz et al (1979).

273Kato, H. Rhue, M. & Nishimura, T. (1989) Role of free amino acids and peptides in food taste In Teranishi, R. Buttery, R. & Shahidi, F. eds. Flavor Chemistry: Trends and Developments. Washington, DC: American Chemical Society Chapter 13

274Yamaguchi, S. & Ninomiya, K. (1999) Umami and food palatability In Teranishi, R. Wick, E. & Hornstein, I. eds. Flavor Chemistry: Thirty Years of Progress. NY: Kluwer Academic/Plenum Chapter 36

275Yamaguchi, S. (1979) The umami taste In Boudreau, J. ed Food Taste Chemistry. Washington, DC: American Chemical Soc Chapter 2

276Yamaguchi, S. & Komata, Y. (1987) Independence and primacy of umami as compared with the four basic tastes Annals NY Acad Sci vol 510, pp 725-726

277Yamaguchi, S. (1979) The umami taste In Bourdreau, J. ed. Food Taste Chemistry. Washington, DC: American Chemical Society Chapter 2. 166 Neurons & the Nervous System

The glutamate anion also plays a critical role in powering the sensory neurons. In this role, it is frequently described as a neuro-facilitator. However, in excess, it is known to cause excessive neural performance. In this case, glutamate can increase the intensity of the taste sensation due to its increasing the static potential at the collector of the first Activa formed within the microvilli. The resulting increase in sensory neuron sensitivity could be reflected in the sweet channels and potentially in the acid and sodium channels as well. Kurihara & Kashiwayanagi addressed umami in 1998 and described the principle chemicals associated with it as mono-sodium glutamate, disodium inosinate and disodium guanylate278. Disodium guanylate incorporates both a sodium ion and a glucophore. Disodium inosinate is unique in that the sodium excites the N-best receptor, PtdIns and the inosinate is a derivative of the family forming the N-best receptor, PtdIns. There is little evidence these chemicals excite an independent sensory channel. In 1991, Yamaguchi reported a large scale test comparing the sensations of Orientals and Caucasians of European origin279. No significant differences were found. Figure 8.5.4-22 shows a selected list of the broad range of chemicals claimed to be associated with the umami sensation based on purely psychophysical tests280. Note the difficulty of determining any specific structural commonality between these chemicals. However, note the commonality of the carboxyl group in a majority of the chemicals. The presence of this group suggests at least one of the sensations elicited by this family is the acidic sensation due to coordination with the serine sensory receptor (representing the H–best channel). The pair of adjacent OH groups in the pentane ring may be separated sufficiently to be able to coordinate with the AH,B receptor site of serine. The hydrated sodium ion of MSG is obviously able to elicit the salty sensation simultaneously.

278Kurihara, K. & Kashiwayanagi, M. (1998) Introductory remarks on umami taste In Murphy, C. ed. Olfaction and Taste, XII. NY: New York Academy of Sciences pp 393-397

279Yamaguchi, S. (1991) Basic properties of umami and effects on humans Physiol Behav vol 49, pp. 833-841

280Schlichtherle-Cerny, Affolter, M. & Cerny, C. (2004) Taste-Active glycoconjugates of glutamate: new umami compounds In Hoffman, T. Ho, C-T & Pickenhagen, W. eds. Challenges in Taste Chemistry and Biology. Washington, DC: American Chemical Society Chapter 14 Signal Generation & Processing 8- 167

Figure 8.5.4-22 A selected group of stimulants represented as “umami” type in the literature. Note the presence of a carboxyl group in nearly every chemical (some shown more explicitly than others. See text. From Schlichtherle-Cerny et al., 2004

Thus, it is conceivable that mono-sodium glutamate represents a group of chemicals that stimulate more than one of the basic sensory channels. However, there are other chemicals in the group that do not contain either sodium or glutamate, including a long list of inosinates. The inosinates may be particularly liable to associate with the active portion of the sensory receptor of the N-best channel which is believed to be an inositol ligand. Two organic acids are said to be 5-30 times more effective than monosodium L-glutamate in eliciting the umami response. 8.5.4.11 Transduction of the hydrophilic sugars ?NEW HOME

To simplify the following discussion, only stimulants belonging to the sugars, polyhydroxyl aldehydes or ketones will be considered. Any polysaccharides (particularly sucrose) applied to the chemical sensing modalities will be assumed to be hydrolyzed into monosaccharides by the mucosa. The resulting monosaccharides will be considered non-electrolytes. Jakinovich has noted a requirement that the three hydroxyl groups of the monosaccharide must be in the equatorial positions for the sugar to be sensed by flies281. His graphics also suggest the critical steric feature of the monosaccharides is the presence of an oxygen in the ring at position 6 adjacent to a carbon at position 5 that is supporting a CH2OH group. All monosaccharides are reducing sugars. They will reduce Fehling’s, Benedict’s, Nylander’s or Tollens’ reagents. Fehling’s reagent involves a Cupric ion complexed with tartrate ions. Benedicts’s reagent involves a Cupric ion complexed with a citrate ion. Nylander’s reagent involves bismuth complexed with a tartrate. Tollen’s reagent contains the diamminesilver(I) ion, [Ag(NH3)2]+. These reactions produce products that are frequently highly colored. This coloration

281Jakinovich, W. (1981) Comparative study of sweet taste specificity In Cagan, R. & Kare, M. eds. Biochemistry of Taste and Olfaction. NY: Academic Press pg 119 168 Neurons & the Nervous System may play a part in explaining the coloration of the mucus associated with the olfactory sensory neurons. Wikipedia provides the following description of a reducing sugar. A reducing sugar is any sugar that, in basic solution, forms some aldehyde or ketone. This allows the sugar to act as a reducing agent, for example in Benedict's reaction. Reducing sugars include glucose, fructose, glyceraldehyde, lactose, arabinose and maltose. Significantly, sucrose and trehalose are not reducing sugars. Benedict's reagent is used to determine if a reducing sugar is present. If it is a reducing sugar, the mixture will turn brick red. Fehling's solution can also be used for the same purpose, as both contain copper (II) ions, which are reduced to a brick red precipitate of copper (I) oxide when the solution is heated. Copper (II) forms a distorted tetrahedral coordination geometry in organic molecules282 that is not directly related to its conventional inorganic structures based on its valence of Cu2+. Cu(I) and Cu(II) both exhibit a conventional valence of 2+ even though they coordinate with four other atoms or ligands (Spiro, pg 114). Electron parametric resonance (EPR) measurements clearly identify three types of Copper in these molecules, Cu(I), Cu(II) and Cu(III) that are illustrated by Spiro (page 122). The complexity of the coordination of copper in a protein is illustrated on pages 35 and 310 of Spiro.

A reducing sugar occurs when its anomeric carbon is in the free form. Since sugars occur in a chain as well as a ring structure, it is possible to have an equilibrium between these two forms. When the hemi-acetal or ketal hydroxyl group is free, i.e. it is not locked, not linked to another (sugar)molecule, the aldehyde (or keto-) form (i.e. the chain-form) is available for reducing copper (II) ions. When a sugar is oxidized, its carbonyl group (i.e. aldehyde or ketone group) is converted to a carboxyl group. [xxx This last sentence can only be interpreted for sugars described using a straight chain chemical representation. All monosaccharides present in solution are properly represented using a ring representation (Lehninger, pg 220)]

8.5.5 Defining the gustatory sensory neurons and receptors

Significant information is now available concerning the steric geometry required of the sensory receptors of gustation. The challenge is to ascertain the most likely organic molecules used within the microvilli of the sensory neurons that can provide a voltage change required by the Electrolytic Theory of the Neuron to elicit a gustatory sensation. The most specific information relates to the distance, d, between the two electron-pair sharing atoms found to be present in all known gustatory stimulants.

The analyses performed here suggest the simplest steric forms of the gustatory receptor are molecules containing ligands that are dimers of the ligands of the gustaphores, except probably in the case of the sodium sensory receptor. True dimers tend to be planar structures. However, the tripartite form of some glucophores suggest the interaction between the stimulant and the receptor is probably not planar but involves a parallelism between the rings of the stimulant and the receptor to support the juxtaposition of the two X features in the AH,B,X coordinate bonding arrangement. This possibility introduces the question of the stimulant and the receptor being enantiomers, either totally or at least with respect to the elements related to the AH,B,X coordinate bonding. Because of the wide range of stimulants known to elicit a response via a single receptor, it appears likely that only a degree of enantiomerism is required to satisfy the coordinate bonding requirement.

Ferrier has described the unit cell of β-D-glucose as consisting of four molecules in an orthorhombic configuration283. The molecules exhibited a complex orientation between each other with hydrogen

282Spiro, T. ed. (1981) Copper proteins. NY: John Wiley & Sons pg 6

283Ferrier, W. (1960) The crystal structure of β-D-glucose Acta Cryst vol 13, pp678 Signal Generation & Processing 8- 169 bonding involving the ring oxygen & O-6 bonded to the adjacent O-3 & O-2 respectively. The hydrogen atoms are not shown explicitly. This bonding was planar in character. The O-1 and O-4 oxygen play no role in coordinate bonding within the cell. The dimeric relationships assures an ability of the receptor to provide the necessary steric match to the major stimulants and provides a gradation in the sensation intensity as a function of the dimensional mismatch between the receptor and the less than ideal stimulant geometries. The phospholipids known to be associated with the sensory neurons, if not the actual receptor areas of those neurons, appear to provide this general capability. When present in the form containing one conductive fatty acid chain (probably conjugated), these phospholipids provide a generic receptor capability for each of the major taste sensations and an electrolytic path to the space between the bilayers of the respective lemmas. The literature of fatty acids originally assumed all fatty acid chains in lemma were fully saturated, then were present with a single conjugated link near the middle of the chain and more recently were present with a conjugated bond at every third carbon as in 4,7,10,13,16,19-docosahexenoic acid (Siegel, 1999, pg 49; 2006, pg 35). The structure on page 34 is alleged to be the isoprene subunit in natural polymerization. This material can be polymerized by two paths. One path employs addition polymerization that leads to the squalenes (double bonds at every third carbon). Cholesterol is the most common squalene. The second approach employs condensation polymerization and leads to the terpenes (double bonds at every other carbon) with the release of water. Vitamin A is the quintessential example of a fully conjugated terpene. Additional research is likely to uncover very small amounts of the fully conjugated fatty acids (a double bond at every other carbon according to the isoprene rule) in type 4 lemma dedicated to only the sensory receptor function. C Carboxylophore receptor– PtdSer provides a carboxyl ligand ideally located relative to the surface of the microvilli lemma. The two oxygen atoms of the nonresonant carboxyl group exhibit precisely the same dimension, d = 2.34 Angstrom, as do the acidophores associated with this sensory channel.

The ability of a single sensory receptor to coordinate with both organic acids and amino acids is illustrated in Figure 8.5.5-1.

The serine ligand of PtdSer is able to coordinate bond with other organic acids as well as with the amino acids.

C Natrophore receptor– PtdIns provides a ligand ideally located relative to the surface of the microvilli lemma and is known to associate with sodium in other processes. Its cyclic structure provides the opportunity to provide two electron-sharing atoms positioned to match the required dimension, d = 3.3 Angstrom, closely.

C Glucophore receptor– The variety of cyclic ligands capable of forming phospholipids and also coordinate bonding with the potential sweet and super sweet stimulants is very large. However, only a few of these ligands have been reported as present in sensory neuron tissue. [xxx edit this after extending above paragraph ] Phosphatidyl xxx provides a ring structure capable of coordinate bonding with the Figure 8.5.5-1 The serine ligand coordinate bonding principle tripartite glucophores of the with organic acids and amino acids. 170 Neurons & the Nervous System sweetness stimulants (and the bipartite glucophores of a much wider range of sweetness stimulants). - - - - - Based on its presence in globosides, it is likely the sensory receptor ligand is a form of galactose. Such a ligand could coordinate bond with a galactose molecule acting as a stimulant in a planar dimer (like found in the natural crystal of glucose) or it could coordinate with a galactose molecule in a more complex three dimensional configuration.

Figure 8.5.5-2 shows a dimer situation with α-D-galactose representing both the sensory receptor and the stimulant. O-3 and O-4 of each molecule are forming the AH,B coordinate bonds and the four atoms define a plane (like that found in Ferrier).

Figure 8.5.5-2 EDIT A dimer situation with galactose [spell?] as both sensory receptor and stimulant. The radii associated with the X element intersect at O-2 of the stimulant but do not intersect at any element of the receptor. Exchanging the centers of the rings results in an intersection near O-6 (or possibly C-6). See text. Signal Generation & Processing 8- 171

C Picrophore receptor– Ptd3'Og provides a long ligand terminated by oxygen and the amine of nitrogen that is ideally located parallel to the surface of the microvilli lemma to coordinate with the picrophores. The dimension, d = 4.2 Angstrom. Van der Heijden addressed the chemical structures related to bitterness (pages 94– 108). However, the treatment is briefer and some confusion appears between sourness and bitterness when discussing the AH,B dimension, d. He asserts the dimension is 0.15 nm for bitterness (compared to 0.14 nm for the acidophores) instead of the value of 0.42 nm (4.2 Angstrom) for the picrophores developed above. His table VI describes picric acid as the most potent of the picrophores. He summarizes his findings in 1993 as, “Although some progress has been made upon more precise characterization of bitter-receptor sites, the specifications among the various classes of bitter principles are lacking. Even the existence of a bitterness receptor is rather speculative. The relationship between the bitter taste and the shape of molecules is more complicated.” The last sentence indicates the criteria for a bitter structure remained unknown at that time. 8.5.5.1 Artificial and Anti-sweeteners

[ Methanol CH3OH, or CH4O, has a significant dipole moment of 1.71 Debyes, Methanal (formaldehyde, CH2O has a value of 2.33. CH3NO2 has a large value of 3.5 debyes ] Figure 8.5.5-3 shows a dimer situation with α-D-galactose representing both the sensory receptor and the stimulant. O-3 and O-4 of each molecule are forming the AH,B coordinate bonds and the four atoms define a plane (like that found in Ferrier). 172 Neurons & the Nervous System

Figure 8.5.5-3 ED A dimer situation with galactose as both sensory receptor and stimulant. The radii associated with the X element intersect at O-2 of the stimulant but do not intersect at any element of the receptor. Exchanging the centers of the rings results in an intersection near O-6 (or possibly C-6). For illustration only, the distances must be calculated in three-dimensional space. See text.

Circles are overlaid on the coordinate oxygen molecules of the stimulant to locate X with radii as estimated by Kier. Their intersection occurs at the nominal location of O-2 but do not intersect near any element associated with the receptor. The circles do not intersect at the methylene C-2 of the stimulant in this projection. By switching the centers of the circles, their intersection occurs near C-6 and O-6 of the stimulant. If O-2, or C-6 or O-6, are significant in the sensation of super sweetness, the coordination mechanism must bring one of these elements into some form of bond with the receptor, or an adjacent receptor molecule of the sensory receptor complex. Figure 8.5.5-4 conceptualizes an alternate coordinate bonding arrangement where the galactose receptor is bonding to an Signal Generation & Processing 8- 173 unspecified glucophore. In the case of the glucophore, the electron-pair orbitals O-2, O-3 and O-4 need not be oxygen-based. They can be any of the atoms or other species identified earlier in Section 8.5.3.1.

Figure 8.5.5-4 Concept: A stacked situation with the sensory receptor galactose interfacing with the glucophore of an undefined stimulant. A third (but strained) coordinate bond is shown between the O-2 atoms, completing a tripartite union (triangle). 174 Neurons & the Nervous System

8.5.5.1 The nominal gustatory receptor EDIT

Figure 8.5.5-5 places the galactose-based sensory receptor in context with the sensory neuron. The sensory receptor is esterfied to the electrolytically conductive fatty acid portion of the phospholipid. The molecule normally exhibits a dipole potential. This dipole potential is a function of the overall configuration of the combination of the sensory receptor and any coordinate bonded stimulus. The resulting dipole potential at the bilayer membrane interface can change by δV based on changes in the bond state of the sensory receptor. The smallest change in δV is associated with the bipartite bonding via O-3 and O-4. This bonding is associated with a majority of the natural sweeteners. A super-sweet sensation (per molar concentration of sweetener) is associated with either tripartite bond, O-2, O-3 & O-4 or O-3, O-4 & O-6. The galactose-based sensory receptor offers the possibility of a “super-super-sweet” sensation resulting from the coordinate bonding to all four of its identified coordinate sites (O-3, O-4, O-2 & O-6) at once. Van der Heijden has supported this assertion (1993, page 79).

Van der Heijden (pages 77–91) has attempted to move the understanding of enhanced sweeteners forward based on two papers

Figure 8.5.5-5 The galactose-based receptor in context with the sensory microvilli. A bipartite coordinate bond (d = 2.6 Angstrom) is the minimal requirement for eliciting a sweet sensation. Extension of the bonding to either tripartite configuration elicits a super- sweet sensation. A super-super-sweet sensation is possible using a quadripartite bond (O-2, O-3, O-4 & O-6). See text. Signal Generation & Processing 8- 175 published in 1985284, 285. He provided a series of figures describing potential sites he labels δ for enhanced sweetness relative to the nominal AH,B sites of sweeteners. He did note the importance of the distance A B rather than H B in describing the baseline feature associated with sweetness. His calculations, based on a modified computer program, STERIMOL, assume the broadest possible definition of the AH,B condition and all possible auxiliary sites within a given sweetener. As a result, many potential auxiliary sites are defined that may or may not contribute to perceived sweetness. He did not address the structural arrangements required to bond maximally with the potential sensory receptors. However, he did note the most effective sweeteners typically had an angle of 125 degrees at the A vertex.

8.5.5.1.1 The electrolytic properties of the receptors

The phosphatidyl moieties defined above, when present in liquid crystalline form constitute the receptor areas of the sensory neuron villi. Excellent, although limited data is available on the electrolytic (particularly the surface potentials and the electron density profiles) characteristics of the materials of interest here. Nearly all of the available data relates to the two insulating and electrically inert, type 1, phospholipids, PtdCho and PtdEtn. However, much of the orientation data related to these moieties should be useful. Hauser286 and Hitchcock et al287. have provided some very early but invaluable information about these two moieties. The 1981 Hauser et al. paper added considerable information about the packing of the phospholipids when in both crystalline and liquid crystalline form288. Hitchcock et al. examined phosphatidyl xxx. Many have built on their initial findings. Weiner & White provided more information on a bilayer of phosphatidyl choline, PtdCho (which they label DOPC) in 1992289. Flewelling & Hubbell describe the high internal potential of a typical lemma bilayer and tentatively describe the source of the potential as the dipole moments of the phosphatidyl moieties forming the bilayer290. Davis has provided data, contradicting Hitchcock et al. to a degree. Davis found the individual phosphatidyl xxx molecules rotate about their long axis when in the liquid crystalline state at biological temperatures. Seddon & Marsh have provided X-Ray data showing additional detail regarding the organization of liquid crystalline films of two PtdEtn’s of different chain lengths291. Sherer & Seelig have explored the orientation of phosphatidyl choline polar heads and their dipole moment when subjected to the

284Van der Heijden, A. van der Wei, H. & Peer, H. (1985) Structure-activity relationships in sweeteners. I. Chem Sens vol 10(1) pp 57-72

285Van der Heijden, A. van der Wei, H. & Peer, H. (1985) Structure-activity relationships in sweeteners. II. Chem Sens vol 10(1) pp.73-88,

286Hauser, H. (1976) Phospholipid model membranes: Demonstration of a structure-activity relationship In Benz, G. ed. Structure-Activity Relationships in Chemoreception London: Information Retrieval Limited pp 13-24

287Hitchcock, P. Mason, R. Thomas, K. & Shipley, G. 1974) Structural chemistry of 1,2 dilauroyl-DL-phosphatidylethanolamine: Molecular conformation and intermolecular packing of phospholipids Proc. Nat. Acad. Sci. USA vol 71(8), pp 3036-3040

288Hauser, H. Pascher, I. Pearson, R. & Sundell, S. (1981) Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine Bioehim Biophys Acta, vol 650, pp 21-51

289Wiener, M. & White, S. (1992) Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data: III. Complete structure Biophys J vol 61(2) pp 434-447

290Flewelling, R. & Hubbell, W. (1986) The membrane dipole potential in a total membrane potential model: Applications to hydrophobic ion interactions with membranes Biophys j vol 49, pp 541-552

291Seddon, J. Cevc, G. Kaye, R. & Marsh, D. (1984) X-ray diffraction study of the polymorphism of hydrated diacyl and dialkylphosphatidylethanolamines Biochem vol 23(12), pp 2634–2644 176 Neurons & the Nervous System presence of strong electrolytes292. Significantly, they note, “In spite of intensive research, the specific functional roles of these headgroups are still, by and large, unknown. Also of interest, they used the term molecular electrometer (a scalar device) to describe the quite accurate response of the phospholipid to the electric charge at the membrane surface. It is the scalar dipole potential that is the proposed result of initial transduction in this theory. Sherer & Seelig attempt to rationalize the nomenclature of phospholipid chemistry and explore both the crystalline and liquid crystalline form of these materials. White & Wimley have explored the presence of simple peptides with phosphatidyl choline293. They address five fundamental questions. They also note the dipeptides remain associated with the polar head. Marsh has reported on the water content of the interior of bilayer membranes of PtdCho under various conditions294. PtdCho and PtdEtn are particularly simple phospholipids that do not offer the requisite oxygen rich binding sites required of receptors. All of the candidate type 4, semiconducting, phospholipids exhibit significant oxygen atoms arranged to support coordinate bonding. Figure 8.5.5-6 to show the operation of the microtubules (villi) at the molecular level. Lowe & Gold have shown that odorant sensitivity and the odorant-evoked inward transduction current are uniformly distributed along the cilia295. Based on this premise, Gold has asserted, “Thus, all components of the transduction mechanism must be present in the cilia296.” Lowe & Gold also showed that the latency of the transduction current is independent of the region of the cilia that are stimulated: This implies that current is generated at the site of odorant binding.

The figure starts with the two bilayer membranes separated by only a thin layer of water crystallized into a hydronium liquid crystal by the confined quarters. The two bilayers are both present as liquid crystals. They are formed of insulating lipids except in the shaded areas. The insulating lipids are phosphoglycerides with fully saturated nonpolar tails (typically PtdEtn). In the shaded areas, at least one of the tails is not saturated and is conductive like the polar head. These phosphoglycerides are typically from the family of globosides. They are typically associated with neurons and exhibit more complex polar heads and unsaturated tails. It is proposed these globosides constitute semiconducting electrolytic materials. The complex heads provide the steric arrangement required to accommodate either the electrostenolytic process or the OR mechanism shown. The more detailed discussion of these materials appears in [Section 5.2.4.1 ]

The shaded area on the left constitutes a first Activa. The semiconducting lipid of the dendrolemma constitutes a region of P-type electronic material. The semiconducting lipid of the reticulum also constitutes a region of P-type material. The region of hydronium between them is an N-type electronic material. The sandwich forms an active electrolytic PNP transistor device, an Activa. The polar head of the dendrolemma facing the mucosa provides a steric site suitable for accommodating the electrostenolytic power supply providing –150 mV to the Activa collector as shown (See Section xxx). The polar head of the reticulum facing the dendroplasm passes the electron current directly into the dendroplasm as shown by the dashed arrow. The magnitude of this current is controlled by the bulk potential of the hydronium N-type material.

292Scherer, P. & Seelig, J. (1989) Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles Biochem vol 28(19), pp 7720–7728

293White, S. & Wimley, W. (1994) Peptides in lipid bilayers: structural and thermodynamic basis for partitioning and folding Cur Opin Struct Biol vol 4, pp 79-86

294Marsh, D. (2001) Polarity and permeation profiles in lipid membranes PNAS vol 98(14), pp 7777–7782

295Lowe, G, Gold, G. 1991. The spatial distributions of odorant sensitivity and odorant induced currents in salamander olfactory receptor cells. J Physiol vol 442, pp 147–68

296Gold, G. (1999) Controversial issues in vertebrate olfactory transduction Annu. Rev. Physiol vol 61, pp 857–71 Signal Generation & Processing 8- 177

Figure 8.5.5-6 The proposed molecular operation of the microtubules in ORN’s based on the Electrolytic Theory of the Neuron. A portion of the surface of a microtubule extending from the dendritic knob is shown. The portion is typically replicated many times. This proposal can be compared to the equivalent conceptual transduction mechanisms of the chemical theory of the neuron, such as Ache, 1994. See text.

The N-type material of the Activa is connected to an adjacent P-type region on the right, again formed of a semiconducting globoside. The junction of the P and N materials forms a PN diode junction and an electrical connection to the stimulus sensing complex, SSC. The diode is forward biased and acts like a “low” impedance electrical conductor. The polar head of the globoside forming the semiconducting dendrolemma is sterically selected to support a steric coupling with the SSC in liquid crystalline form. The polar head also constitutes one plate of a capacitor, CT, with the SSC as the dielectric. The other plate is formed by the interface between the SSC and the mucosa.

The SSC may or may not exhibit a dipole moment in the absence of a odor stimulant (horizontal bar inside left half of SSC). However, in the presence of a sterically appropriate odor stimulant, it is a zwitterion and will exhibit a dipole moment as shown by the inclined bar on the right. This second dipole moment is due to a quantum-mechanical change in the configuration of the SSC molecule(s). This change in dipole moment between the absence and presence of a stimulant causes a change in the dielectric properties and the resultant voltage on the capacitor, CT. The change in voltage is passed through the forward biased PN junction to the base of the Activa. This voltage controls the amplitude of the current passing through the Activa. The change in the dipole moment of the SSC acts to repel the stimulant from its steric coupling with the SSC. Thus, the presence of the stimulant in a steric relationship is only temporary. The resultant voltage change at the Activa is also temporary. The stimulant molecule is free to go without encountering any chemical change in character. The waveform at lower left shows the current waveform entering the dendroplasm resulting from a group of stimulant molecules coupling and uncoupling from the SSC within a time period short 178 Neurons & the Nervous System with respect to the time constant of the overall quantum-mechanical-electrolytic circuit. The mathematical form of this waveform, including its latency, Δ, are developed in Section xxx.

Hauser has described the stereographic structure of PtdCho (he uses the common name lecithin) and PtdEtn and demonstrated how the dipole moment of these two materials is quite different. The dipole moment of PtdCho is aligned with the long axis of the molecule and perpendicular to the surface of the membrane. The dipole moment of PtdEtn is aligned nearly perpendicular to the long axis of the molecule. This would suggest the surface potential of these materials when formed into a liquid crystalline monolayer would be quite different. However, when so aligned, it is not the vector quantity, the dipole moment that is important by the scalar dipole potential. Vogel et al. have demonstrated the conductivity of the typical phospholipid is sufficient to allow the measurement of a finite potential between its two ends when present as a monolayer film297. They have measured the potential across phosphatidyl choline and phosphatidylethanolamine when in the single layer liquid crystalline state. These potentials are found when the liquid crystalline film has reached an equivalent molecular cross-sectional area of 40 sq Angstrom for these materials. The potential is given as 555 mV for PtdEtn and 669 mV for PtdCho at 18-19C with the hydrophobic surface negative. These are large voltages relative to the input dynamic range of the first Activa of the sensory neuron (see the following sections) .

It is proposed that the net surface potential between the mucosa and the interior layer of the type 4 bilayer membrane consists of three scalar components, the intrinsic dipole potential of the phospholipid, the contribution to the total dipole potential due to the coordinate bond pairing with the odorophore (AH,B), and any additional change in dipole potential caused by the third-point association (X) between the odorophore and the receptor molecule. The intrinsic dipole can be considered the quiescent contribution of the phospholipid to the biasing of the first Activa of the sensory neuron with the sum of the contribution to the dipole potential from the AH,B pairing and the X association constituting the signal element in the total electrical potential. With the nominal gain of 200x for the Activa and a sensitivity of about 0.1 to 1.0 mV for the following circuitry, a change in the quiescent potential of the phosphatidyl moiety of the sensory receptor of less than five microvolts would constitute an adequate signal for eliciting a sensation within the CNS. Such a small change is not currently measurable in the laboratory, but the presence of such changes are indicated by the psychophysical data of Imamura et al. and of Randebrock for changes in the potential due to coordinate coupling of the receptors with alcohols and carboxylic acids of different chain lengths and internal bond arrangements (Section 8.6.2.3).

While Hauser has also suggested that the molecules of a monolayer membrane of PtdEtn are interlocked by hydrogen bonds between the nitrogen and the adjacent oxygen of the phosphorous oxide group, the more recent and comprehensive data of Davis suggest the individual molecules are rotating rapidly about their long axis at biological temperatures. Such rotation would appear to aid the contact between the receptor molecules and the stimulant molecules in the mucosa.

Many empiricists have attempted to graphically conceptualize the transduction mechanism of olfaction. See Ache298 reproduced in Schild & Restrepo (page 455), as an example. The chemical neuron approach is much more complex, usually employs sweeping arrows and symbolism, and has not shown itself amenable to verification or deterministic calculations.

297Vogel, V. & Mobius, D. (1988) Local surface potentials and electric dipole moments of lipid monolayers: contributions of the water/lipid and the lipid/air interfaces J Colloid Interface Sci vol 126(2), pp 408-420

298Ache, B. (1994) Towards a common strategy for transducing olfactory information Semin Cell Biol vol 5, pp 55-63 Signal Generation & Processing 8- 179 The above figure provides a closed form, verifiable and deterministic model of olfactory transduction. It employs no putative pores or channels passing complex ions under the control of undefined protein processes. ------The artificial sweeteners–Saccharin and Aspartame Aspartame has been found to play an important but controversial role in the sweetening of foods. Its perceived side effects have spawned a cottage industry of de-toxifying programs. The perceived problem is its breakdown into individual amino acids, and potentially methanol, following ingestion after performing its desired gustatory task. Aspartame is considered 150-200 times sweeter than sucrose. This is probably due to both its molecular structure and the change it causes in the dipole potential of the sweetness sensory receptor. Figure 8.5.5-7 shows its principle “supersweet” glucophore is probably formed by the two nitrogens, able to form a pair of AH,B coordinate bond with a distance between them of 2.6 Angstrom and the oxygen shown acting as X. The structure is nearly identical to the O-3, O-4 and O-2 arrangement of galactose in the sweet channel receptor.

Figure 8.5.5-8 shows the reason aspartame can taste bitter. It contains a picrophore that is nearly a perfect structural match to the sensory receptor, 3'-O’aminoacyl glycerol. The reason is both are based on the same ligand, aspartic amino acid. The chemical can also elicit a sour taste due to its carboxyl group. Figure 8.5.5-7 Aspartame showing the axis of its AH,B,X “supersweet” glucophore, the two nitrogen atoms, and the location of the oxygen at the X position. See text. 180 Neurons & the Nervous System

Aspartame incorporates three distinct gustophores. - - - -

Figure 8.5.5-8 Aspartame bonding to the picric The structure of saccharin is relatively sensory receptor ADD HYDROGEN BONDS and simple. However, its role as a sweetener spacing of 4.2 Angstrom.. probably introduces known but new chemistry to this discussion. Figure 8.5.5-9 shows the structure of saccharin. Deprotonation of the nitrogen leads to the normal anion of saccharin. Sulfur exhibits 5 shareable electron pairs with four involved in oxygen bonding in this example.

The bond lengths and angles relating to saccharin are well documented299,300. Shang et al. also describe the distortions of the molecule relative to common descriptive drawing practice. Like other chemicals, the individual bond lengths can vary by up to ten percent from standard bond lengths in a specific moiety. The spacing between the two oxygen atoms associated with sulfur is within the 7% tolerance (2.42-2.78 Angstrom) of the spacing originally derived from Shallenberger & Acree for sweet channel stimulation. The third oxygen appears too far from the initial pair to represent the X of the AH,B,X relationship. However, the phenol ring appears well placed to satisfy this requirement. The shared electrons of the π bonds are known to provide the necessary field potential. However, the elevation view of the molecule shows the symmetrical arrangement of the AH & B oxygen atoms. As a result the triangle formed between these atoms and the X location is isosceles.

299Zhang, Y. Wang, Y & Yu, H. (1995) Structural properties of o-sulphobenzoimide in complex crystals Cryst Res Technol vol 30(6), pp 831-835

300Matos, M. Miranda, M. Morais, V. & Liebman, J. (2005) Saccharin: a combined experimental and computational thermochemical investigation of a sweetener and sulfonamide Molec Phys vol 103(2–3), 221–228 Signal Generation & Processing 8- 181 The bitter after-taste associated with

Figure 8.5.5-9 The structure of saccharin using standard bond lengths (see text). The spacing between the two oxygen atoms is within the tolerance for a sweet odorophore based on standard bond distances. The triangle involving the phenol as X is also isosceles based on the elevation view of the molecule. 182 Neurons & the Nervous System

saccharin appears to be due to the odorophore formed between the isolated oxygen atom and the phenol with a d-value of about 4.2 Angstrom (Section 8.5.xxx).

- - - - 8.5.5.2 Blocking agents

Breslin has addressed the question of blockade of gustatory stimulants301. He also discusses enhancement, synergy, suppression and masking by multiple agents simultaneously. Although discussing, chlorhexidine, gymnema sylvestre, lactisole and a combination labeled PALG, the emphasis is on amiloride.

Breslin describes two known sweetness suppressants. He notes that gymnema applied to the oral cavity prior to application of a sweet tasting substance results in suppression of the perception of sweetness. He also notes lactisole is a fast-acting competitive antagonist to almost any compound tasting sweet. Lindley has written on the chemistry of anti-sweeteners302. He summarized as follows, “Currently available evidence is consistent with the conclusion that these sweetness inhibitors are competitive antagonists of sweet taste acting at a single receptor structure.”

Breslin asserts the sodium ion is the primary known blocker of the bitter taste sensation. He offers no description of the mechanism causing this action. Blocking in this manner could be related to a neural differencing in either the dimension 1 = Q – H or dimension 2 = N – H channels described in the multidimensional analyses above. The importance of the differencing might be less than that due to the summation of the various stimuli joining in forming the overall taste sensation.

He discusses PALG, a protein (especially lactoglobulin) bound to phosphatidic acid, as a good blocker of bitter tasting compounds, especially quinine which it precipitates. His terminology is that of a pharmacist or specialists in food product development. The designation phosphatidic acid describes a fatty acid lipid that is a precursor for many of the potential fatty acids in the sensory neuron lemma. Quinine in soluble form generally relates to quinine hydrochloride. This may be an excellent clue to the operation of the Q-best sensory channel since phosphatidic acid is a potential transducer in the type 4 lemma of the Q-best sensory neuron microvilli. If the phosphatidic acid is reacting with the quinine hydrochloride with the precipitation of quinine, the phosphatidic acid may become chlorinated and become negatively charged. This action could mirror the nominal reaction between the phosphatidyl fatty acid of type 4 lemma and quinine hydrochloride, resulting in the precipitation of the quinine, permanent alteration of the phosphatidyl fatty acid and a significant change in the flow of current into the plasma of the microvilli. A comparison of the state of polarization of phosphatidic acid and both PtdCho and PtdSer would appear to be useful.

As Breslin noted (page 437), “. . .a potent selective blocker of salty taste in humans remains to be discovered.” He also noted (page 438), “There are no known blocking agents for acid sourness or umami in humans at present (2000).”

8.5.5.3 Neuro responses to gustatory stimulants and blocking agents

[xxx review Breslin in Finger pgs436-437. Bring in amiloride information ]

301Breslin, P. (2000) Human gustation In Finger, T. Silver, W. & Restrepo, D. eds. The Neurobiology of taste and smell, 2nd Ed.. NY: Wiley-Liss Chapter 16

302Lindley, M. (1993) Sweetness antagonists In Acree, T. & Teranishi, R. eds. Flavor Science. Washington, DC: American Chemical Society Chapter 4 Signal Generation & Processing 8- 183 Kurihara et al. have provided comprehensive data on the neural responses of the gustatory system of several species303. Their responses are compatible with the excitation/de-excitation mechanism associated with other sensory modalities. Nakamura & Norgren have provided good information on the neural responses of the nucleus of the solitary tract using a broad range of stimuli304. Nishijo & Norgren have provided good information on the neural responses in the parabrachial tract of the awake rat305. Their expanded array of stimuli is identical to that used for the NST experiments of Nakamura and Norgren. They performed a number of cluster analyses that highlighted the difficulty of employing “standard” stimuli. “A hierarchical cluster analysis of the neurons substantially confirmed the best-stimulus categorization but separated the NaCl-best cells into those that responded more to Na+-containing salts and those that responded more to Cl–-containing salts. The cells that responded best to the Na+ moiety actually were somewhat more correlated with the sucrose-best cells than with those that responded to the Cl–-containing stimuli. Citric acid–best neurons and the lone quinine-best unit formed a single cluster of neurons that responded well to acids, as well as to NH4Cl and, to a lesser extent, NaNO3 .” These stimuli are clearly not the keys to understanding gustation. Figure 8.5.5-10 shows their dendrogram of 44 chemicals. This diagram has the flavor of Bayesian Statistics where you predetermine the possible outcomes and then calculate the statistics based on that predetermination. In this case, there appear to be an alternate set of fundamental parameters that are unrelated to the common chemicals selected by non-invasive psychophysical testing within a given culture.

303Kurihara, K. Miyake, M. & Yoshii, K. (1981) Molecular mechanisms of transduction in chemoreception In Cagan, R. & Kare, M. eds. Biochemistry of taste and olfaction. NY: Academic Press Chap 13

304Nakamura, K. & Norgren, R. (1993) Taste responses of neurons in the nucleus of the solitary tract of awake rats: an extended stimulus array. J Neurophysiol vol 70, pp 879–891

305Nishijo, H. Norgren, R. (1997) Parabrachial Neural Coding of Taste Stimuli in Awake Rats J Neurophysiol vol 78, pp 2254-2268 184 Neurons & the Nervous System

Figure 8.5.5-10 Dendrogram of 44 parabrachial neurons ADD. See text. From Nishiho & Norgren, 1997.

8.5.5 The electrophysiology of the gustatory sensory neuron 8.5.5.1 Circuit description of the gustatory sensory neuron EDIT

[ refocus on gustatory neuron ] With the exception of one simple two-terminal circuit applicable to chemoreception in general proposed by Kurihara et al., no circuit diagram of the olfactory sensory neuron could be found in the literature306. Their circuit diagram contains no capacitors or active elements and does not address the detailed operation of the olfactory sensory neuron. Figure 8.5.5-11 shows the proposed sensory neuron of olfaction based on the common functional circuit elements found in other sensory modalities. The circuit is amenable to direct quantum-mechanical stimulation of the base region of the first Activa (as in photoreception) or to the application of an electrical potential to the base electrode (as in phonoreception). Because of the expected low energy of stimulation, the figure adopts the configuration used in hearing.

306Kurihara, K. Miyake, M. & Yoshii, K. (1981) Molecular mechanisms of transduction in chemoreception In Cagan, R. & Kare, M. eds. Biochemistry of taste and olfaction. NY: Academic Press Chap 13 Signal Generation & Processing 8- 185

Figure 8.5.5-11 Candidate circuitry of the olfactory sensory neurons. All stage 1 and 2 elements operate in the analog domain. The Activa of the afferent synapse operates as an “active diode” with a bypass capacitor. The electrolytic power supply forming the battery associated with impedance (1) in the Activa collector circuit is shown in its hydraulic analog at the top of the figure. The stage 3 ganglion (functioning as an encoding neuron) cell is frequently labeled a mitral or tufted cell in olfaction.

In the case of phonoreception, the first Activa is biased into the operating range required by a conventional amplifier. The circuit is capable of accepting electrical signals in the tenths of a millivolt to ten millivolt range. Such a range appears compatible with the energy levels involved in the odor reception scheme suggested by Turin.

In hearing an AC electrical potential is created between the plates of a capacitor by piezo-electric action. In the case of olfaction, it is proposed a similar AC electrical potential is created between the plates of a capacitor by dielectric polarization due to an electronic rearrangement of the dielectric. the dielectric is formed by a stimulant sensing complex, SSC. This rearrangement is temporary and caused by the temporary presence of a stimulant in steric coupling with the SSC. The proposed configuration creates a quantum-mechanical shift in the electronic state of the SSC every time an appropriate stimulant couples with the SSC. This coupling at the molecular level would be a low probability event unless the SSC was present in a liquid crystalline form on the surface of the input structure of the sensory neuron. It is proposed that the SSC is present in such a form and is located on the surface of a specialized section of the dendroplasm of the sensory neuron. It is this specialized section that creates the Activa just as it does in other modalities. [xxx eliminate the capacitor part ] It is further proposed that the SSC is present in a liquid crystalline form where part of the crystalline configuration forms the dielectric of a capacitor between the electrolyte of the mucosa and the base region of the Activa within the sensory neuron. Indicator dyes are characterized by their multiple electronic configurations. Each of these configurations exhibits a moment. The change in configuration can be described by a transition dipole moment. A non-zero transition dipole moment change in a material forming the dielectric of 186 Neurons & the Nervous System a capacitor will result in a change in the potential between the electrodes of that capacitor. The concept is discussed briefly in Adamson307. Maron & Lando go into the subject of dipole moments, and the various spectra related to molecular structure308. They provide a pedagogical energy diagram. For molecules of more that three atoms, the complexity of th spectra becomes quite high. However, the change in polarization associated with the vibration-rotation spectra are of interest here. The energies of interest are in the millivolt range and the associated wavelengths are in the near infra-red range. Petersen & Cone have provided an excellent paper discussing many aspects of the dipole moment of proteins, particularly rhodopsin, the disk protein associated with vision309. It forms a good tutorial as well as providing valuable protocols and experimental data. They provide a footnote; “the dipole moment, μ, is in Debyes (D), where 1 D = 10–18 statcoulomb-cm. Thus an electron and a proton separated by 1 Angstrom produce a dipole moment of 4.8 D. In other words, 1 charge-D = 4.8 D.” They show that most small proteins separate into two classes based on their dipole moment and their molecular weight, those with values of μ/W of 5 x 10–3 D/Dalton and a second group with μ/W clustered about 20 x 10–3 D/Dalton. Rhodopsin with a dipole moment of 720 Debye and a molecular weight of 35,000 ± 2000 Daltons, has a μ/W value of 20 x 10–3. Gerber et al. note that the proteins with the larger dipole moments all tend to aggregate and suggest that the dipole moments aid in the aggregation process. This ability of the protein to aggregate is crucially important in the formation of the outer disks of visual sensory neurons. Aggregation may play a similar role in forming the SSC’s of olfaction. The change in the dipole moment of rhodopsin upon saturating illumination is only 25 D (5 charge-D) or about 3%. Petersen & Cone give precise data on the dielectric constant of rhodopsin solutions and its change with applied frequency in a standard test cell. In the in-vivo situation, a value for the aggregated material is believed to be necessary.

Many complex organo-metallic compounds exhibit a dipole moment in the 2-5 Debye range310. More comprehensive general lists are available but they tend to be old311. Following further isolation of the chemistry of interest, a more detailed search may uncover the desired values for their dipole moments. The lower molecular weight of these organo-metallic materials (the molecular weight of copper tartrate is only 329 Daltons, give them much higher μ/W values than those of proteins. Nominal static μ/W values for the organo-metallics are in the 2–6 x 10-3 range. The transition dipole moment may be much larger relatively because of the small size of the molecules.

Takashima has provided excellent background on the dipole moment312. An on-line calculator for calculating the dipole moment of arbitrary proteins is available313. However, the structure of the protein must be known in detail.

307Adamson, A. (1973) A Textbook of Physical Chemistry. NY: Academic Press pp 911-913

308Maron, S. & Lando, J. (1974) Fundamentals of Physical Chemistry. NY: Macmillan Chapter 5

309Petersen, D. & Cone, R. (1975) The electric dipole moment of rhodopsin solubilized in Triton x-100 Biophys J vol 15, pp 1181-1200

310CRC (1975) Handbook of Chemistry and Physics. Cleveland, OH: CRC Press pg E-66

311Smyth, C. (1941) Dipole moment and bond character in organometallic compounds J. Org. Chem vol 06(3), pp 421–426

312Takashima, S. (1999) Computation of the dipole moment of protein molecules using protein databases Colloids and Surfaces A vol 148, pp 95-106

313Felder, C. Prilusky, J. Silman, I. & Sussman, J. (2007) A server and database for dipole moments of proteins Nucleic Acids Research vol 35, special Web Servers Issue. Signal Generation & Processing 8- 187 Two technical challenges arise in attempting to define a steric mechanism of transduction. First, the capture cross-section of the transduction mechanism must be as large as possible. Second, the transition dipole moment must be as large as possible and it must be optimally sensed. Forming the SSC’s into a liquid crystalline structure is one method of increasing capture cross-section and potentially increasing the efficiency of sensing the net transition dipole moment. - - - - Not shown explicitly in these diagrams are the large dipole potentials introduced by the inner and - - - - Figure 8.5.5-12 shows the circuit along with its cytological equivalent. Frame A shows the cytological layout of the proposed typical olfactory sensory neuron. All plasmalemma are electrically insulating bilayer membranes, except for specialized sections that are semiconducting. These specialized sections are active participants in forming active semiconductor devices, Activa, or in supporting the electrostenolytic process electrically powering the cell. All of the sensory neuron except for its axon is located within the olfactory epithelium and its mucosa. The body of the sensory neuron peripheral to the soma is arbitrarily defined as the dendrite portion. That portion between the soma and the olfactory bulb is arbitrarily described as the axon. 188 Neurons & the Nervous System

Figure 8.5.5-12 Proposed cytological and electrolytic description of the olfactory sensory neuron ADD & MODIFY. MODIFY frame A to show stimulation. Signal Generation & Processing 8- 189

The bulbous extreme end of the dendrite is labeled the knob. Its internal structure is not reported in the literature. Multiple individual microtubules emanate from the knob. These function as dendritic spines with multiple sensitive areas typically associated with synapses in non sensory neurons. In the sensory neurons, these areas are modified to support transduction. Only the microtubules (cilia) lie in the mucosa. The number of microtubules emanating from the dendritic knob varies from species to species, from as few as four to as many as thirty. The typical length of the microtubules is about 50 microns in mammals314. The microtubules are splayed into a planar surface within the xxx thick mucosa. The three horizontal arrows in the dendroplasm indicate where the multilayer sandwich to their left actually extend up into the individual cilia of the neuron. The character of these layers changes from insulating to semiconducting at multiple points along each cilia just as they do in the microtubules of the visual sensory neurons. The two insets, A1 & A2, illustrate how the transduction mechanism may appear at one point along the length of the cilia. Inset A1 shows an Activa (black rectangle) supported by its electrostenolytic supply (E.S (1)) and the transduction element consisting of an SSC forming the dielectric of a capacitor, CT, with its outer conducting surface formed by the mucosa. Inset A2 shows an alternate configuration where an auxiliary electrostenolytic source (E.S. (0)) appears on the outer surface of the capacitor. This source provides an electrical bias to the capacitor that may provide a higher sensitivity to the overall circuit as discussed in Section xxx.

The dashed arrows associated with each electrostenolytic supply show the direction of electron charge flowing into or out of the respective plasmas. The “conventional charge” defined erroneously by Benjamin Franklin flows in the opposite direction.

Charges from each of the Activa in each cilia are introduced into the dendroplasm and generate a potential between the dendroplasm and the podaplasm. This causes the Activa shown between the dendroplasm and the axoplasm to transfer approximately 200 times as much charge to the axoplasm. This additional charge passes through impedance associated with E.S. (4) and generates the output voltage at the synapses shown.

The sensory neuron delivers a tonic generator waveform at the remote glomeruli by diffusion. Because of the length of the axon required to pass through the cribriform plate and the low rate of signal propagation by diffusion, the bandwidth of the olfactory sensory neurons is necessarily low.

Frame B shows the same circuit configuration as in Frame A. [xxx Expand ]

Frame C shows the same circuit configuration as in Frame B but redrawn to illustrate the commonness of the circuit. It is described as an PNP type asymmetric differential pair in conventional electronic engineering terms. The left (or first) Activa is base driven and the right (or distribution) Activa is emitter driven. The left Activa provides a high impedance input and a gain of nominally 200:1. The right Activa provides a low impedance output at 1:1 gain suitable for driving multiple synapses. [xxx Expand ]

Frame A1 has been expanded further and discussed in detail in Section 8.5.5.1. 8.5.5.2 Circuit parameters of the olfactory sensory neuron EDIT [ Refocus to gustatory modality ] Schild & Restrepo have included considerable parametric data (with citations) on the axonal portion of the olfactory sensory neurons. Statements like “whole cell capacitance” presume a two terminal structure for the neurons. Under the Electrolytic Theory of the Neuron, this label should be modified to the whole axoplasm capacitance as it was obtained by injecting charge into the axon and measuring the resulting voltage rise. Values in the 2-10 pF range are consistent with the sensory neurons of other modalities. They report a wide range of axon resting potentials,

314Menco, B. (1983) The ultrastructure of olfactory and nasal respiratory epithelium surfaces In Reznick, G. & Stinson, S. eds. Nasal Tumors in Animals and Man . . . Vol 1. Boca Raton, Fl: CRC Press pp 45-102 190 Neurons & the Nervous System undoubtedly because of the lack of control of the dendrite environment while investigators made these measurements. A value of –70 mV would be expected and falls near the middle of the range quoted. They report an average time constant of the axoplasm of ~ 60 ms, giving a first order RC filter corner frequency of ~16 Hz. While lower than in other modalities, these values are consistent with the extended length of the axon required to pass through the cribriform bone. Measured time constants in some species have been as long as 100 ms. They did note an important point, the delay displayed in their generator potentials of figure 4 are proportional to the amplitude of the stimulus (page 433). Unfortunately, they have continued to repeat the reports based on conclusions improperly drawn the work of Gesteland in 1971315. That assertion was that sensory neurons exhibit action potentials (beginning on page 434). Schild & Restrepo note, “These reports were discussed controversially, see Getchell316.” Gesteland was very clear that his measurements were extracellularly, and in fact of the crudest kind. His measurements were made between the two sides of the olfactory epithelium. Because of his technique (his figure 1), he actually measured generator waveforms of opposite polarity to that at the axon of a cell “and with action potentials spikes superimposed” (page 144). He was measuring the complement of the generator waveform at the collector of the first Activa instead of at the collector of the second Activa (the normal axon potential (See Section xxx). It is now known his current path in figure 1 requires changing to reflect the documented role of the poditic terminal.(Section xxx). Gesteland does provide several parameters supporting the neural model developed here. [xxx review gesteland 1971 ]

Additional data showing such action potentials has not appeared subsequently. It appears Gesteland relied upon data collected extracellulary (intercellularly). It exhibits low amplitude action potentials riding on top of generator waveforms. The combined waveform is easily explained as due to capacitive coupling under the Electrolytic Theory of the Neuron. Schild & Restrepo show generator waveforms on page 444 that are free of action potential features.

Schild & Restrepo reproduce a figure from Kurahashi317 that may represent the diode characteristic of the axon load, Figure 8.5.5-13. If so, the load diode had a reverse cutoff current (I0) of –25 pA. Unfortunately as noted in the original caption, the current traces shown on the left were arbitrarily shifted. The nominal horizontal axis of each waveform does not correspond to zero current. The holding voltage would have been accompanied by a holding current that is not shown explicitly, but can be seen on the right.

315Gesteland, R. (1971) Neural coding in olfactory receptor cells In Beidler, L. ed. Handbook of Sensory Physiology, Vol 4, Part 1: Chemical Senses. Berlin: Springer-Verlag pp 132-150

316Getchell, T. (1986) Functional properties of vertebrate olfactory receptor neurons Physiol Rev vol 66, pp 772- 818

317Kurahashi, T. (1989) Activation oby odorants of cation-selective conductance in the olfactory receptor cell isolated for the newt J Physiol (Lond.) Vol 419, pp 177-192 Signal Generation & Processing 8- 191

Figure 8.5.5-13 Odorant induced currents at various holding potentials from a newt, Cynops pyrrhogaster ADD. Current traces were arbitrarily shifted. See text. Modified from Kurahashi, 1989 192 Neurons & the Nervous System

8.5.5.3 Analog waveforms generated by xxx’s

[ Refocus on gustatory modality ] The literature provides many analog generator waveforms from olfactory receptor neurons (Schild & Restrepo, page 443-444). These waveforms exhibit a latency as a function of stimulus intensity that is not compatible with experiments involving the putative photoexcitation of cAMP. This latency as a function of intensity is characteristic of the excitation/de-excitation mechanism proposed in this work. Farbman has provided a very clear patch-clamp recording from the olfactory cilium of a frog (page 110). The change in amplitude of up to 20 mV suggests the recording was from the axoplasm of the sensory neuron. The variation in the amplitude of the signal over time suggests some logarithmic compression due to the current to voltage conversion but no sign of hard saturation or of action potential generation.

8.5.6 Potential stage 2 & 3 neural projection in gustation

There appears to be no stage 2 signal processing within the gustatory tract prior to the brain stem (except possibly simple summation of analog intensity signals from adjacent sensory neurons of the same channel at the taste bud). Stage 3 signal encoding appears to occur at the base of the taste bud.

Very little data is available to determine whether the NST and/or parabrachial tract operate as stage 2 or stage 4 elements. Their location within the brainstem would suggest stage 4 processing.

- - - - [xxx move to section on stage 2 processing within gustatory modality. ] The lack of any obvious differencing neurons related to stage 2 signal processing within the taste buds suggests the gustatory signaling is similar to that employed by the olfactory signaling system (See figure 8.6.3-13 & -14) [xxx showing diode matrices and glomeruli. ]. The gustatory nerves (VII from the anterior 2/3 of the tongue, IX & X from the posterior third and base of the tongue) travel to the nucleus solitarius (NST, sometimes referred to as the nucleus gustatorius). The nucleus solitarius may play a role similar to the glomeruli of the olfactory modality (defining it as the stage 2 signal processing element in gustation).

Nejad explored the action potential streams in the nerves leading to the NST and reported no example of a differencing channel signal based on over a dozen combinations of sugars and simple salts among 22 adult Sprague-Dawley rats318.

Travers et al319. reported 26 specific, 24 summation and only 3 difference channel action potential streams at the NST of Sprague-Dawley rats (their Table 1) based on a statistical analysis. The summations originated from different regions of the tongue. Note of the difference channel response was limited to a comment in a caption to the table. No mention or discussion occurred in the text. During the same time period, Sweazey & Smith reported no difference channels in the NST of hamsters320.

318Nejad, M. (1986) The neural activities of the greater superficial petrosal nerve of the rat in response to chemical stimulation of the palate Chem Senses Vol.11(3), pp 283-293

319Travers, S. Pfaffmann, C. & Norgren, R. (1986) Convergence of lingual and palatal gustatory neural activity in the nucleus of the solitary tract Brain Res vol 365, pp 305-320

320Sweazey, R. & Smith, D. (1987) Convergence onto hamster medullary taste neurons Brain Res vol 408, pp 173- 184 Signal Generation & Processing 8- 193 Subsequently, Travers & Norgren did not report any differencing signals as recorded at the NST of rats321. In 1991, Nakamura & Norgren made an important comment while investigating the rostral NST of Sprague-Dawley rats322, “The entropy measure introduced by Smith and Travers (1979) is useful because it permits criterion-free comparisons among neurons. It poses difficulties because it is sensitive to the overall rate of activity, which encourages factoring out spontaneous rate, but is unable to accept negative values, i.e., inhibitory responses. In the present sample, the inhibitory responses were relatively small and never reached significance, so the discussion focuses on the values calculated from the excitatory activity alone (Italics added).” They included an extensive dendrogram based on conventional stimuli. The figure of merit introduced by Smith and Travers is used in these analyses to estimate the broadness of sensitivity of a specific sensory neuron channel. The figure used is an entropy given by the equation, H = .K(n)AΣpiAlogpi where the coefficient K(n) depends on the number, n, of stimulants in the set. Smith & Scott have given a brief discussion of this figure of merit323. The value of K(n) is adjusted to insure a maximum value for this function of 1.0.

In 1995, Travers & Norgren expanded this class of investigations to separate the effect of somatosensory stimulation of the tongue from chemical stimulation as measured at the NST324. “Approximately half of the neurons could be activated by gustatory stimulation (G neurons), whereas the other half exhibited no chemosensitivity, but instead usually responded to mechanical stimulation, mainly of the oral mucosa (M neurons).” Their figure 8 shows a clear example of differencing due to mechanical stimulation of neurons delivering signals to the NST. They also noted, “As mentioned previously, three NST M neurons fired in synchrony with the rhythm of artificial respiration. Thus, in addition to oral mechanical input, these cells appeared to have an input from airway receptors.”

[xxx resolve differencing between the above and this paragraph ] While these investigations are not conclusive, they strongly suggest that differencing of gustatory neural signals does not occur either prior to or within the NST. Summation was found based on both the stimuli employed and on spatial grounds. The introduction of spurious mechanical stimulation, from a brush or even flowing water, is obviously a factor in this class of experiments.

It is worth noting, the recordings of action potentials by the above investigators invariably involve AC-coupled oscilloscope techniques that result in significant undershoot in the action potential representations. They appear virtually bi-symmetrical instead of monopolar.

- - - -

Doetsch & Erickson have made the argument that their electrical recordings are from the output of the NST based on the fact they were probing the soma of the neurons for signal that arose in a very small area of the NST325. Such claims are reasonable but their report does not describe the characteristics of their signals from a signal message or signal projection perspective. They

321Travers, S. & Norgren, R. (1991) Coding the sweet taste in the nucleus of the solitary tract: differential roles for anterior tongue and nasoincisor duct gustatory receptors in the rat J Neurophysiol vol 65(6), pp 1372-1380

322Nakamura, K. & Norgren, R. (1991) Gustatory Responses of Neurons in the Nucleus of the Solitary Tract of Behaving Rats J Neurophysiol vol 66(4), pp 1232-1248

323Smith, D. & Scott, T. (2003) Gustatory neural coding In Doty, R. ed. (2003) Handbook of Olfaction and Gustation, 2nd revised and expanded edition. NY: Marcel Dekker Chapter 35

324Travers, S. & Norgren, R. (1995) Organization of orosensory responses in the nucleus of the solitary tract of the rat vol 73(6), pp 2144-2162

325Doetsch, G. & Erickson, R. (1970) Synaptic Processing of Taste-Quality Information in the Nucleus Tractus Solitarius of the Rat J Neurophysiol vol 33, pp 490-507 194 Neurons & the Nervous System characterized their neurons as generating action potentials at up to about 40 pps, and little ability to follow artificial stimulation beyond 100 pps at normal body temperatures. The artificial stimulation was introduced electrically into the neurons of the chorda tympani. They suggest the average firing rate of NTS output neurons is 4.3x higher than for chorda tympani neurons. Their analysis involved mostly statistical data related to pulse rates. Where used, they employed a two- dimensional MDS. They were unable to treat sucrose effectively in their two-dimensional space. The higher pulse rates encountered at the output of the NST could be considered due to summation of the input signals, conceivably in either phasic or analog signaling space. No signals reflecting a diminution of pulse rates or subtraction of signals within the NST were reported. Grigaliunas et al. have provided Figure 8.5.5-14 showing idealized in-vitro patch clamp recordings for “geniculate ganglion neurons” of the embryonic rat they associated with gustation326. The stage 3 ganglion neurons were located in the tongue and delivered signals to the chorda tympani. The neurons were cultured in a series of complex pharmacological mediums, including neurotrophins, for multiple days before investigation. As a result, the waveforms cannot be assigned to an equivalent in-vivo specimen. However, the waveforms can be considered typical. The term “parametric” has been placed in the figure caption to indicate the cell was not operating in response to stimulus signals received via the neurites of the ganglion neuron. The charge shown was injected into, and the voltage waveforms shown were obtained from the same compartment (assumed to be the axoplasm) of the neuron. The data provides strong support for the Electrolytic Theory of the Neuron as applied to stage 3 ganglion neurons.

In this figure, the responses to currents of 100 pA and higher are not shown. They would have exhibited increasing pulse rates with increasing currents. For negative currents, the neuron acted as an analog amplifier instead of a pulse generator.

Grigaliunas et al. employed a patch clamp technique that established the potential of the plasma they contacted at a nominal –52 mV using a high impedance clamp source. As a result, when current was injected into or extracted from the same plasma, the voltage of the plasma was free to move higher or lower, subject to the impedances of the circuit under test and the impedance of the clamp source.

Grigaliunas et al. did not describe the reference potential that they used to determine the value of –52 mV shown in the Figure 8.5.5-14 “Parametric” In-vitro patch clamp figure. [xxx ?true ] They did subtract 10 mV recordings from embryonic rat. The threshold from their readings to compensate for the voltage, as represented in the axoplasm, was 18 mV assumed potential between the pipette filling more positive than the quiescent level. Clamp probe and the bath solutions. As a result, it is tip impedance, 6-8 megohms. Test temperature, 20- difficult to interpret the precise meaning of 22°C. See text. Modified from Grigaliunas et al., the voltage scale. During in-vivo operation, 2002. this type of neuron is normally powered from a –154 mV metabolic source present on the surface of the neurons plasma membrane. The axoplasm potential is normally near –140 mV before the first oscillation and is driven to about –20 mV at the peak of the single pulse oscillation. By patch clamping the plasma to –52 mV, the circuit is in an abnormal condition.

326Grigaliunas, A. Bradley, R. MacCallum, D. & Mistretta, C. (2002) Distinctive Neurophysiological Properties of Embryonic Trigeminal and Geniculate Neurons in Culture J Neurophysiol vol 88, pp: 2058-2074 pdf file Signal Generation & Processing 8- 195 Grigaliunas et al. described the current that resulted in hyperpolarization as positive. When the injected current is positive, the emitter-to-base potential is driven in the opposite direction to the threshold potential. The current causes a complex analog response reflecting the coupling between the axoplasm and other plasmas of the neuron. The response suggests it is dominated by a resistive impedance rather than a capacitive impedance. The axoplasm potential reflects the emitter-to-base potential as expressed in the collector circuit of the neuron. The circuit is operating as a class A amplifier in this regime with a gain of less than one. It will not result in the generation of any single pulse oscillation (except as described below).

For negative currents, the current is integrated by a capacitive, rather than a resistive, element resulting in the emitter-to-base potential rising linearly as reflected in the change in the axoplasm potential suggested by line A. When the emitter-to-base potential exceeded the threshold potential, as reported by the related change in axoplasm potential (nominally 10 mV in this case), the gain of the overall circuit becomes positive, the axoplasm potential departs from line A, and the circuit goes into single pulse oscillation. The decay portion of the single pulse oscillation results in a change in the emitter-to-base potential. This change in potential is coupled into the emitter-to-base potential and, in conjunction with the rate of potential change suggested by line A, results in line B. When the emitter-to-base potential again exceeds the threshold value, the overall gain of the circuit exceeds unity and a second (and subsequent) single pulse oscillation occurs.

Note the last pulses shown on the right (asterisk) overlay each other and are unrelated to the pulses on the left. The pulses on the right are due to a capacitive coupling that drove the emitter- to-base potential of the Activa above threshold during the relaxation transient following the end of the charge injection. The resulting pulses occurred after a time that varied slightly in proportional to the voltage of the patch clamp plasma at the end of the charge injection.

By introducing the coupling capacitances between the collector (axoplasm) and the base (podaplasm), it is possible to describe the transient waveforms more completely.

Grigaliunas et al. provided more information about their ensemble of geniculate ganglion cells and also trigeminal ganglion cells (leading to the trigeminal, or fifth crania, nerve). However, much of the data must be reinterpreted in light of the Electrolytic Theory of the Neuron. An example, the “input impedance” is actually the parametric collector to ground impedance of the axoplasm. It has nothing to do with the input impedance at the dendritic or poditic input terminals of the neuron. In addition, the impedance is sensitive to the protocol used (current injected or withdrawn from the axoplasm) and the instantaneous operating condition of the Activa. They confirmed they were measuring only the axoplasm parameters by injecting Lucifer yellow into their recording pipette. The fact their dye did not permeate the dendroplasm or the podaplasm provides good confirmation of the multi-chamber character of these neurons and support the fact the Activa provides a barrier between these chambers.

Grigaliunas et al. examined 150 “geniculate ganglion neurons” parametrically. While they showed relatively small standard deviations from the mean values for the full set, their ranges were large (frequently 30% of the mean value for a particular measurement). The collector-to-ground time constant for their geniculate ganglion cells was 32 ± 14 ms. The collector-to-ground impedances varied considerably during their tests, that they attributed primarily to growth within the cell. Their action potential heights averaged 102 ± 12 mV. The recovery transient following current injection was relatively constant at 88 ms total. They also reported parametric results for 254 “trigeminal ganglion neurons” found at the same location. These units always exhibited a single oscillatory pulse followed by an offset continuing for the duration of the stimulation. 35% of the geniculate ganglion neurons produced multiple pulses. The variability in pulse output performance can be explained as a function of the capacitive coupling between the collector (axoplasm), and the emitter (dendroplasm) and base (podaplasm). 8.5.6.1 Potential neural code for gustation

As Spector & travers noted, no description of the neural code appropriate to gustation had appeared by 2005. This problem is accentuated by the shortage of electrophysiological recordings of 196 Neurons & the Nervous System gustatory sensory responses. An aggravating aspect is that various authors use the term neural code differently. Are they talking about the code used across the neuron space of the stage 1 sensory neurons? Are they talking about the code used to convert stage 1 or 2 output signals to the phasic code of action potential propagation? Are they trying to find the code employed to transduce a wide range of stimuli interactions with a small range of sensory neuron types? As of recently, the community remains struggling to define a conceptual level, and hence poorly defined, code327. It is safe to say the stage 3 encoding of these channels is the same as that of other sensory modalities. It is the organization of the analog data in stage 2, signal processing, that remains undefined. However, the details need to be developed. Page 144 of their paper develops the current terminology and concepts on which to develop these details. They note they were not going to deal explicitly with intensity encoding. Page 145 asserts, “The ability to definitively test the validity of various models of the neural coding of taste quality has been hampered by a nearly complete reliance on correlative statistical procedures and a lack of available techniques that allow selective manipulation of putative classes of gustatory neurons.” Spector & Travers describe two forms of “spatial” analog encoding. The labeled-line hypothesis of encoding proposes there are only a few classes of sensory neurons and that the activity in a given class generates a specific perception (similar to the manner in which color vision is implemented). The across-neuron pattern theory postulates than any given qualitative perception arises from patterns of activity in large ensembles of neurons (similar to the manner in which information is extracted from both auditory and foveal vision signals in the perigeniculate nuclei). These concepts are not mutually exclusive. They are both used within the gustatory modality.

Current wisdom, as reviewed by Spector & Travers, is that there are specific gustatory sensory neurons sensitive to;

“Narrowly tuned gustatory sensory neurons”

N-fibers; sodium (and lithium) salts. This type is probably also responsive to potassium salts. S-fibers; perceived as sweet tasting. Q-fibers; sensitive to quinone.

“Broadly tuned gustatory sensory neurons” A-fibers; sensitive to ammonium chloride – do not respond to quinone. H-fibers; sensitive to acidic stimuli, presumably due to the presence of the hydrogen ion.

As quickly noted, these designated channels are generic and not directly related to specific chemical structures, with the possible exception of the H-fiber sensitivity to hydrogen ion.

Pages 152-155 of Spector & Travers provide a very large bibliography of research.

Hill & Bour have demonstrated the responses of the gustatory neurons of rats conform to the E/D equation of this work, Figure 8.5.5-15328. They recorded and integrated action potential pulses from an exposed chorda tympani nerve. The recordings show the typical initial peak followed by a decline to a steady state value followed by a exponential decay following cessation of the stimulus. This is the expected performance of a parasol ganglion neuron encoding intensity information. The duration of each stimulus was 20 seconds.

327Scott, T. & Giza, B. (2000) Issues of gustatory neural coding: Where they stand today Physiol Behavior vol 69, pp 65–76

328Hill, D. & Bour, T. (1985) Addition of functional amiloride-sensitive components to the receptor membrane: a possible mechanism for altered taste responses during development Brain Res vol 352(2), pp 310-313 Signal Generation & Processing 8- 197

The stage 3 electrophysiological results of Hill & Bour presented above demonstrate the response of the gustatory sensory neurons is the typical quantum-mechanical E/D response of all sensory neurons. The E/D response was demonstrated for the chlorides of sodium, potassium, lithium and ammonium. Based on their work, it appears there is a single sensory neuron class responding to some feature of the ions of the Group 1a members of the Periodic Table and ammonium. However, other work reviewed in the Spector & Travers paper (page 158) shows this may be an oversimplification. The results also show that amiloride does suppress the response to potassium just as it does that of sodium, only to a lesser degree. The responses of Hill & Bour also demonstrate the stage 3 channel shows minimal to zero action potential activity prior to stimulation and is therefore a summation (or intensity) channel encoded by a parasol ganglion neuron.

The literature suggests the sensory neurons Figure 8.5.5-15 Integrated chorda tympani adapt rapidly to changes in the local chemical responses to 0.5 M chlorides before and after environment. It speaks of adaptation application of amiloride in 12 -day postnatal rat and occurring within seconds. Based on the E/D in adult rat. Stimulant application for 20 seconds. equation, it is likely that adaptation occurs in Time constant of integrator, 0.5 sec. From Hill & milliseconds (a time much to short to be Bour, 1985. evaluated by typical topical application of fluids to the tongue).

No two-step adaptation measurements, as commonly used in visual experiments, were located in the gustatory literature. Adaptation is a stage 1 mechanism designed to provide an initial alarm signal to the CNS but subsequently maintain a proportional but not saturating response for purposes of analysis within stage 4.

Data demonstrating operation of a midget ganglion neuron encoding difference information within the gustatory system may be difficult to find since investigators naturally look for an on/off type response in the stage 3 neural channels. What is needed is data from a channel exhibiting a continuous stream of action potentials under quiescent conditions and an increasing and/or decreasing pulse rate under stimulation. Such data would demonstrate the presence of a differential stage 3 encoding channel associated with gustation.

8.5.6.2 Variation in stage 3 response with concentration MOVE

Performing experiment where the concentration of the stimulant (or gustaphore) varies is difficult because of the imprecision of application to the taste buds and the potential variation in the gustaphore strength in the solution. Particularly in inorganics, the concentration of a given gustaphore may change with total stimulant concentration. This is often due to changes in coordinate chemistry changes due to changes in the hydration of the inorganic ligand. Berg & xxx have provided data showing potential examples of variation in response with concentration even among organics. Figure 8.5.5-16 reproduces some of their data. 198 Neurons & the Nervous System

8.5.7 Potential stage 4 signal processing (information extraction) EMPTY

Very limited data is available concerning stage 4 signal processing. The nerves from the taste buds find their way to the nucleus solitarius of the brain stem and then find their way to the ventral posteromedial nucleus of the thalamus according to Noback (page 137). This area is distinct from the portion of the nucleus associated with somatic sensing. “The neural path then ascends through the posterior limb of the internal capsule to the neocortex in the lower end of Figure 8.5.5-16 Dependence of subjective intensity the postcentral gurus (opercular region of the of taste sensation with concentration ADD. From parietal lobe) and possibly in the insula and Berg et al. 1967. upper portion of the temporal lobe.”

Faurion et al. provided 3 Tesla fMRI images of the gustatory sites in the human brain in 1998329. They used only three stimulants, NaCl, Aspartame and Quinine HCl. Scott & Erickson have reported data recorded in the thalamus of the rat330, based on a study by Emmers et al., 1960, where, “the taste nerves projected to the medial half of . . . a morphologically distinct sub-nucleus in the most ventral and posterior part of n, ventralis.” The text suggests the neural path was direct from the NST to the thalamus. They found there anaesthetics played a critically important role in their obtaining data from the thalamus. They reported the relative importance of the major stimuli was drastically different in the thalamus than as recorded in the CT and NST of earlier studies. They also noted their action potential sequences did not exhibit the initial peak followed by a lower pulse rate plateau typical of the E/D equation and both CT and NST recordings. The pulse trains frequently varied in pulse interval, and frequently ended, during stimulation. They also encountered reductions, 163 out of 631 recordings, in the spontaneous pulse rate of at least 7.5% indicative of a subtractive process. They also reported a distinct “water response” following cessation of many of their stimuli, particularly those causing a reduction in spontaneous discharge rate.

Scott & Erickson made one important observation, “Perhaps the most interesting aspect of the results presented here, and most difficult to interpret, is that they imply different functions for the first (medulla) and second (thalamic) major relays in the taste system.” The difference is clearly associated with the difference in responsibilities between these stage 2 and stage 4 sites. The thalamus is responsible for signal manipulation (information extraction) while stage 2 is primarily responsible for signal processing related to optimizing the messages prior to stage 3 propagation. 8.5.6 Perceptual performance in gustation

Rodgers et al. have begun the task of analyzing the recently acquired olfactory performance database into a tree of knowledge focused on bitter molecules331. The analysis focuses on analyzing the structural fragments associated with a certain degree of quality. They perform several cluster

329Faurion, A. Cerf, B. LeBihan, D. Pilliasm A-M. (1998) fMRI study of taste cortical areas in humans In Murphy, C. ed. Olfaction and Taste, XII. NY: New York Academy of Sciences pp 535-545

330Scott, T. jr. & Erickson, R. (1971) Synaptic processing of taste-quality information in thalamus of the rat J Neurophysiol vol 34, pp 868-884

331Rodgers, S. Busch, J. Peters, H. & Crist-Hazelhof, E. (2005) Building a tree of knowledge: analysis of bitter molecules Chem Senses vol 30, pp 547-557 Signal Generation & Processing 8- 199 analyses and begin the construction of a cladogram (the diagrammatic technique underlying common phylogenic trees). They describe their cladogram as a “phylogenetic-like tree.” The work highlights the importance of oxygen in the molecules they describe as bitter tasting. They examined 31,179 molecules found in the Dictionary of Natural Products for 2004 and found 23,179 included at least one of the structural fragments they associated with gustation. 758 (3.2%) of these molecules were associated with their bitter database. They also identified seventy-five molecules that were known to elicit a bitter response but did not contain any of the identified structural fragments. They defined these as singletons that fall outside of their cladogram at this time. They reported 116 nodes in the “bitter” section of their cladogram. They did not provide a graphic of their complete cladogram because of its significant size. The representation of 25 of their nodes focuses on very complex molecules dominated by multiple-ring structures and the presence of oxygen. 8.5.4xxx Interpreting the rectilinear & 3-D taste spaces

The proposed theory supports a rectilinear taste space related to the distance between the A & B atoms associated with a stimulant in a AH,B coordinate bond. It defines certain specific distances as equal to the distance between the A & B atoms of the sensory receptor involved. By asserting that the central nervous system treats the signals received from the individual signaling channels associated with the individual types of sensory receptors, as independent, the theory proposes that the rectilinear taste space can be folded to present a three-dimensional taste space that is congruent with the three-dimensional taste space obtained from multi-dimensional analysis of laboratory taste tests. However, it has not addressed how and why stimulants vary in their ability to create an intense sensation equivalent to certain stimulants typically described as causing a maximum taste sensation for that class of stimulants.

It is proposed the ability of a stimulant to elicit an intense response in a specific taste channel is proportional to many factors. These factors include questions of solubility and concentration of the stimulant in the saliva, and a myriad of potential influences related to the internal stereographic arrangement within the ligand forming the AH,B or AH,B,X bonds with the sensory receptors. It is proposed that these factors can be combined into a single effective A B distance and this A B distance can be determined from multidimensional analysis. Such analyses provide these effective A B distances for all members of the set of stimuli relative to the positions of the sensory receptors routinely but on a scaled (as opposed to an absolute position) basis. . 8.5.6 A dichotomy: the labeled-line and across-neuron-pattern theories

How the different stimuli are initially sensed in the peripheral portion of the gustatory system has evolved along two separate paths, the labeled-line theory and the across-neuron-pattern theory. These two approaches are seldom precisely defined and may not be totally distinct. Several authors offer different interpretations of their meaning.

Christensen & White provided a comprehensive overview of this subject332 from its introduction by Dethier in 1976. They note, “In the ‘labeled-line’ model, the sensory axons carrying information to the CNS are ‘absolutely restricted’ with respect to selectivity, whereas in an ‘across-fiber’ code, ‘each stimulus would produce a different and characteristic total response profile’ across the entire population of sensory neurons. [xxx add and reference olfactory portion, Section 8.6.6 & 7 ] 8.5.7 The labeled-line and across-neuron-pattern theories resolved

Data is now available to resolve the labeled-line versus across-neuron-pattern dichotomy with respect to gustation.

332Christensen, T. & White, J. (2000) Representation of olfactory information in the brain In Finger, T. Silver, W. & Restrepo, D. eds. The Neurobiology of Taste and Smell, 2nd Ed. NY: Wiley-Liss 200 Neurons & the Nervous System

Data such as Figure 8.5.5-17 from Pfaffmann et al., and similar individual neurons make it very difficult to support the labeled-line concept in the gustatory modality organization333. They also highlight the analog character of the initial waveforms at the receptor neurons and within that modality. These individual neurons are responsive to a wide variety of stimuli and may contribute to an across-neural-pattern theory. Alternately, they may contribute to a simpler difference channel approach as used in vision.

333Pfaffmann, C. Frank, M. Bartoshuk, L. & Snell, T. (1976) Coding gustatory information in the squirrel monkey Chorda Tympani, In Sprague, J. & Epstein, A. eds. Progress in Psychobiology and Physiological Psychology. NY: Academic Press. vol 6, pp 1-27 Signal Generation & Processing 8- 201 202 Neurons & the Nervous System

Figure 8.5.5-17 Records of total chorda tympani responses to water and taste stimuli applied to the anterior tongue. The first two deflections of the marker at the lower edge signal the flow of distilled water, the third deflection signals duration of stimulus flow, the fourth indicates reinse with distilled water. I division = 5 seconds. From Pfaffmann et al., 1976 Signal Generation & Processing 8- 203

8.5.8 Correlating genes to gustatory receptors

Dahanukar et al. have made some important observations concerning the gustatory genes of Drosophila in an extensive paper334. They have associated Gr5a with the sensing of a disaccharide sugar, trehalose. Such disaccharides are typically broken down before sensing in gustation. They also noted, “Gr5a-labeled neurons are responsive not only to trehalose, but to sucrose and other sugars.” Subsequently, they find that, “Gr5a is required for detection of a small subset of sugars including trehalose. We generate deletion mutants lacking Gr64a and find that it is required for response to a complementary subset of sugars. Strikingly, flies lacking both Gr5a and Gr64a do not show electrophysiological or behavioral responses to any tested sugar. These results demonstrate that the sugars divide into two classes that are dependent either on Gr5a or on Gr64a for their responses.” They go on, “Classic physiological and biochemical studies led to the proposal of a ‘‘fructose’’ site in sugar-sensing neurons. Our studies provide a molecular and genetic identity to this site: fructose response is completely abolished by loss of Gr64a and is completely restored by the addition of a Gr64a transgene.” Whether humans involve two separate sweetness receptors is not yet clear. Either or both of these genes can be considered directly associated with the proposed human sweetness receptor, proposed here to be PtdGal, phosphatidyl galactose.

They have associated gene Gr66a with the bitter sensory channel. This gene can be directly associated with the proposed bitter receptor, Ptd3'Og, phosphatidyl 3'-O-aminoacyl glycerol.

8.4.4.9 Rationalization of the lipid versus protein versus sugar debate in gustation EMPTY

8.5.9 Man-made taste sensors

Julia Tsitron et al335.have recently reported on a Bayesian model of a man-made gustatory sensor focused on the reception of only four chemicals related to glucose. [See reference papers xxx. ]

334Dahanukar, A. Lei, Y-T. Kwon, J. & Carlson, J. (2007) Two Gr genes underlie sugar reception in drosophila Neuron vol 56, pp 503–516

335Tsitron, J. Ault, A. Broach, J. & Morozov, A. (2011) Decoding Complex Chemical Mixtures with a Physical Model of a Sensor Array PLoS Comput Biol vol 7(10): e1002224. doi:10.1371/journal.pcbi.1002224 204 Neurons & the Nervous System

Table of Contents 25 December 2011

8 Stage 1 & 2, Signal Generating & Processing Neurons...... 2 8.1 Introduction...... 2 2.1 Introduction...... 3 2.1 Introduction...... 5 8.1.1 Important background...... 7 8.1.1.1 Genetics of the sensory neurons...... 7 8.1.1.2 Genetics of the sensory neurons...... 8 8.1.1.2.1 Genetics of vision EMPTY...... 11 8.1.1.2.2 Genetics of hearing EMPTY...... 11 8.1.1.2.3 Genetics of chemoreception...... 11 8.1.1.2.4 Role of “Holes” in Genetics ...... 13 8.1.1.3 Transduction, neurotransmitters vs chemical signals ...... 13 8.1.1.4 Chemicals of major importance in the chemical theory of the neuron ...... 17 8.1.1.5 A redefinition of the “messengers” of the neural system...... 18 8.1.1.6 Ultimate detection thresholds for the sensory neurons ...... 19 8.1.2 Comparative anatomy and histology in neuroscience...... 20 8.1.2.1 Areas of specialized lemma in sensory neurons...... 20 8.1.2.2 The transduction mechanism of sensory neurons...... 21 8.1.2.3 Uniqueness of the transduction mechanism of sensory neurons . . 22 8.1.2.4 Sensory neuron regeneration ...... 23 8.1.3 Sensory modalities addressed in this chapter ...... 23 8.2 The photoreceptor neurons of vision EMPTY ...... 24 2.2.3.2 The two-terminal biological transistor of the photoreceptor cell MOVE ...... 24 8.3 The phonoreceptor and neurons of hearing & the vestibular system EMPTY ...... 25 8.4 The chemoreceptor neurons of taste and smell...... 25 8.4.1 Brief summary of the literature, glossary ...... 28 8.4.1.1 Recent chemoreception literature...... 28 8.4.1.2 The problem of defining terms ...... 32 8.4.1.2.1 Is the bitter tasting CaCl2 a salt?...... 33 8.4.1.3 Brief Glossary ...... 33 8.4.1.4 Overview of chemoreception problem...... 37 8.4.1.5 Attempts to establish a structure-odor or structure-response relationships...... 38 8.4.2 The stimuli processed by the chemical sensors...... 39 8.4.2.1 The sensitivity of the gustatory receptor neurons...... 40 8.4.2.1.1 Synopsis of the Chemical Theory of the Neuron ...... 41 8.4.2.1.2 Electrolytic Theory of the Neuron & chemoreception . . . 43 8.4.3 Architecture of the external chemoreceptor modalities ...... 45 8.4.3.1 Top level architecture of the olfactory modality ...... 46 8.4.3.2 Expanded architecture of the olfactory modality...... 48 8.4.3.2.1 The vomeronasal organ and auxiliary olfactory bulb . . . 52 8.4.3.2.2 Afferent neural paths to the main olfactory bulb ...... 52 8.4.3.2.3 Schematic of the human main olfactory bulb ...... 53 8.4.3.3 Top level architecture of the gustatory modality...... 55 8.4.3.4 The path of stimulants to the external chemoreceptors...... 57 8.4.3.5 Common elements of the chemoreception modalities...... 58 8.4.3.5.1 Binding sites on chemoreceptor membranes...... 59 8.4.3.5.2 Transduction response of chemoreception...... 60 8.4.4 Global aspects of potential stimulant sensing chemistry ...... 60 8.4.4.1 Proteins as potential SSC’s in olfaction ...... 62 8.4.4.1.1 The role of histidine-containing dipeptides ...... 63 8.4.4.2 Lipids as potential SSC’s in olfaction...... 63 8.4.4.2.1 Dipole potential and moment of phospholipids...... 67 8.4.4.3 Coordination chemistry fundamentals ...... 67 8.4.4.3.1 Coordinate chemistry of the transition elements ...... 70 Signal Generation & Processing 8- 205 8.4.4.3.2 Chelation and the formation of metal complexes ...... 70 8.4.4.3.3 Organometallic chemistry fundamentals ...... 70 8.4.5 Extending the lock & key concept of stereochemistry...... 71 8.4.7 A structural shorthand to describe odorophores and gustaphores...... 71 8.4.7.1 Initial definition of one-dimensional taste and smell spaces .....73 8.4.7.2 Complex structural groups within chemical stimulants ...... 73 8.5 The gustatory modality...... 73 8.5.1.1 Chemical families pertinent to gustatory receptor identification ...... 83 8.5.1 Overview of the gustatory modality...... 85 8.5.1.1 Extending the chemoreception concepts of Shallenberger, Kier and Beets ...... 94 8.5.1.2 The anatomy of the peripheral portion of the gustatory modality ...... 95 8.5.1.3 The morphology of the taste bud and sensory neuron...... 96 8.5.1.3.1 The structure of the “sweet” lemma of the microvilli . . 101 8.5.1.3.2 The electrophysiology of the “sweet” gustatory sensory neuron ...... 104 8.5.1 ??? Renewal of the gustatory sensory neurons FIX COUNTER . . . 106 8.5.1.1xxx How are taste sensations defined? ...... 106 8.5.2.2 What are the structural constraints on tasting sweet? . . 107 8.5.2.3 The gustatory response versus molarity of stimuli .....108 8.5.2 Analysis of perceived gustatory sensations–Multidimensional scaling applied to gustation ...... 108 8.5.2.1 Dendrographic representation...... 108 8.5.2.2 ROC analysis...... 109 8.5.2.2 Two dimensional representation applied to gustation . . . 111 8.5.2.3 Multidimensional scaling applied to gustation...... 111 8.5.2.4 Changes in multidimensional presentation ...... 114 8.5.2.5 Conclusions from analysis of the data...... 116 8.5.2.6 The basis functions of gustation ...... 120 8.5.2.7 The proposed human sensory space of gustation...... 123 8.5.2.8 The family of Neural Response Functions ...... 124 8.5.3 Hypothesis of gustatory transduction ...... 127 8.5.3.1 Previous theories of gustatory transduction...... 130 8.5.3.2 The AH,B & AH,B,X coordination chemistry of the gustatory channel...... 130 8.5.3.3 Proposed transduction mechanism for “desireable/sweet” ...... 135 8.4.4.6xxx Sugars as potential SSC’s in gustation ...... 140 8.5.4 Operation of the gustatory sensory receptors ...... 141 8.5.4.1 Operation of the “sweet” gustatory sensory neuron ...... 143 8.5.4.2 Operation of the “super sweet” sensory neuron & AH,B,X...... 144 8.5.4.3 Operation of the “acidic” gustatory sensory neuron ...... 146 8.5.4.4 Operation of the “alkaline” gustatory sensory neuron ...... 149 8.5.4.4.1 The perception of sodium as sweet at low concentrations ...... 153 8.5.4.5 Operation of the “bitter” gustatory sensory neuron ...... 154 8.5.4.6 CaCl2 & MgCl2 as gustaphores...... 162 8.5.4.7 The “water” gustatory sensory response ...... 164 8.5.4.8 The “browned flavors” sensory response ...... 164 8.5.4.9 The role of amines & amino acids in the taste sensation TIE 8.6.2.5 ...... 164 8.5.4.10 The putative “umami” sensory response ...... 165 8.5.4.11 Transduction of the hydrophilic sugars ?NEW HOME ...... 167 8.5.5 Defining the gustatory sensory neurons and receptors...... 168 8.5.5.1 Artificial and Anti-sweeteners ...... 171 8.5.5.1 The nominal gustatory receptor EDIT...... 174 8.5.5.2 Blocking agents...... 182 8.5.5.3 Neuro responses to gustatory stimulants and blocking agents . . 182 8.5.5 The electrophysiology of the gustatory sensory neuron ...... 184 8.5.5.1 Circuit description of the gustatory sensory neuron EDIT...... 184 206 Neurons & the Nervous System

8.5.5.2 Circuit parameters of the olfactory sensory neuron EDIT ...... 189 8.5.5.3 Analog waveforms generated by xxx’s ...... 192 8.5.6 Potential stage 2 & 3 neural projection in gustation...... 192 8.5.6.1 Potential neural code for gustation ...... 195 8.5.6.2 Variation in stage 3 response with concentration MOVE...... 197 8.5.7 Potential stage 4 signal processing (information extraction) EMPTY.....198 8.5.6 Perceptual performance in gustation ...... 198 8.5.4xxx Interpreting the rectilinear & 3-D taste spaces ...... 199 ...... 199 8.5.6 A dichotomy: the labeled-line and across-neuron-pattern theories ...... 199 8.5.7 The labeled-line and across-neuron-pattern theories resolved ....199 8.5.8 Correlating genes to gustatory receptors...... 203 8.4.4.9 Rationalization of the lipid versus protein versus sugar debate in gustation EMPTY...... 203 8.5.9 Man-made taste sensors ...... 203 Signal Generation & Processing 8- 207

Chapter 8 List of Figures 12/25/11 Figure 8.1.1-1 A scheme depicitng the cluster of olfactory receptor (OR) genes...... 9 Figure 8.1.1-2 ADD Transcription and translation in genetics...... 10 Figure 8.1.1-3 Alternate splicing ADD ...... 10 Figure 8.1.1-4 Chemical signals within the body as described by one author...... 14 Figure 8.1.1-5 The generalization of the “second messenger” concept...... 16 Figure 8.1.1-6 An initial comparison of the Electrolytic and chemical theories of the neuron . . . 17 Figure 8.1.1-7 Summary of transduction mechanism in sensory modalities ...... 22 Figure 8.2.1-1 Three terminal active biological device, the Activa ...... 24 Figure 8.2.1-2 The three-terminal active biological device, the Activa, with an open terminal...... 24 Figure 8.3.1-1 Pneumatic/mechanical representation of the transmission of vibrations...... 25 Figure 8.4.1-1 ADD The histology of the olfactory sensory neuron of the moth...... 30 Figure 8.4.2-1 Threshold and recognition values versus pH of ionic materials ...... 40 Figure 8.4.2-2 Receptor potentials of a single rat taste cell to salts ...... 41 Figure 8.4.2-3 The morphology of the olfactory chemoreceptor and proposed neural circuitry . . 44 Figure 8.4.3-1 Top level architecture of mammalian olfactory modality ADD...... 47 Figure 8.4.3-2 Schematic of the mammalian olfactory modality...... 49 Figure 8.4.3-3 Gross neural circuitry of the olfactory and gustatory systems REVIEW ...... 50 Figure 8.4.3-4 Neuron density in olfactory epithelia of selected species...... 51 Figure 8.4.3-5 A Purkinje cell of the cerebellum...... 52 Figure 8.4.3-6 Neuronal schematic of the main olfactory bulb ...... 54 Figure 8.4.3-7 Top level architecture of the gustatory modality ...... 56 Figure 8.4.3-8 ADD The taste (gustatory) pathway...... 57 Figure 8.4.3-9 Artificial saliva used by Hellekant ...... 58 Figure 8.4.3-10 A proposed dendrogram of the chemoreceptors from the receptor perspective . . 59 Figure 8.4.3-11 The observed transduction characteristic of the chemo-sensory neurons...... 60 Figure 8.4.4-1 Basic structural features of the phospholipids...... 64 Figure 8.4.4-2 Drawing showing the complexity of a real lemma ...... 66 Figure 8.4.4-3 Two forms of coordination number 4 chemistry...... 69 Figure 8.5.1-1 The E/D waveforms of gustation in the bullfrog ADD ...... 75 Figure 8.5.1-2 Proposed schematic of the gustatory system ADD...... 78 Figure 8.5.1-3 Schematic of mammalian neural paths in gustation...... 79 Figure 8.5.1-4 Provisional block diagram of the gustatory modality ADD...... 81 Figure 8.5.1-5 A normalized etiology of chemicals relating to proposed gustatory sensory receptors ...... 84 Figure 8.5.1-6 Summary: sensory receptors of gustatory modality...... 89 Figure 8.5.1-7 Summary: performance of the gustatory modality...... 91 Figure 8.5.1-8 The first-order gustaphores of taste ...... 92 Figure 8.5.1-9 Proposed taste sensation space for a mammalian species...... 93 Figure 8.5.1-10 The transition from behavioral to fundamental perspectives in gustation...... 94 Figure 8.5.1-11 Semi-schematic representation of the tongue...... 95 Figure 8.5.1-12 Morphological features of the mammalian taste bud...... 97 Figure 8.5.1-13 Tracing of a gustatory sensory neuron showing internal lemma ...... 98 Figure 8.5.1-14 Two views of the colax and microvilli of a gustatory sensory neuron ...... 100 Figure 8.5.1-15 The cross section of “sweet” microvilli EDIT lemma ...... 101 Figure 8.5.1-16 The electrophysiology of the gustatory “sweet” microvilli MOD ...... 103 Figure 8.5.1-17 Candidate cytology & electrophysiology schematics of the gustatory sensory neuron DUMMY ADD...... 105 Figure 8.5.2-1 Cluster dendrogram of 31 hamster PbN neurons...... 109 Figure 8.5.2-2 Two-dimensional histogram of hamster taste preferences...... 111 Figure 8.5.2-3 Multidimensional scaling of gustatory stimulants in monkey ADD...... 113 Figure 8.5.2-4 Changes in three-dimensional taste spaces of one rat caused by amiloride.....116 Figure 8.5.2-5 The foundation for the chromaticity diagram of tetrachromatic vision ...... 118 Figure 8.5.2-6 A proposed taste sensation space based on an incomplete experimental database ...... 119 Figure 8.5.2-7 A two-dimensional taste space with alternate axes applied ...... 121 Figure 8.5.2-8 Proposed taste sensation space for a mammalian species...... 124 Figure 8.5.2-9 Proposed 2-D neural response function of X, NRFX ...... 125 208 Neurons & the Nervous System

Figure 8.5.3-1 XXX modified multidimensional analysis based on the polar head of lipids EMPTY ...... 129 Figure 8.5.3-2 Proposed coordination chemistry of the S-best sensory neurons...... 130 Figure 8.5.3-3 Comparison of AH,B,X geometries ...... 132 Figure 8.5.3-4 Sweet tasting non-sugars and the their AH,B relationships...... 133 Figure 8.5.3-5 Proposed sensitivity of the desirable/”sweet” sensory channel ADD...... 135 Figure 8.5.3-6 The numbering system of the simple sugars...... 136 Figure 8.5.3-7 Two gangliosides associated with the neural system...... 138 Figure 8.5.4-1 The “super sweet” tripartite glucophores of two sugars...... 145 Figure 8.5.4-2 Galactose in the plane of O-3 & O-4 ...... 146 Figure 8.5.4-3 Potential sensing of the hydrogen ion in the H-best sensory receptor ADD.....147 Figure 8.5.4-4 Phosphatidylserine shown in polar form ADD...... 147 Figure 8.5.4-5 Serine as the potential sensory receptor ligand...... 148 Figure 8.5.4-6 A carboxylic acid dimer ...... 148 Figure 8.5.4-7 The sodium ion at hydration levels of 2 and 6 ...... 150 Figure 8.5.4-8 A potential hydrated sodium “dimer ADD ...... 151 Figure 8.5.4-9 Myo-inositol, a sugar alcohol, and a fully hydrated sodium...... 151 Figure 8.5.4-10 Two perspective views of inositol from the software package, Molecules 3D Pro ...... 153 Figure 8.5.4-11 The natrophore of hydrated sodium and inositol in a coordinate bond...... 153 Figure 8.5.4-12 The diversity of bitter compounds...... 154 Figure 8.5.4-13 Denatonium benzoate, the most bitter compound ...... 156 Figure 8.5.4-14 Quinine, the preferred bitter taste in the laboratory...... 157 Figure 8.5.4-15 Corilagin, a tannic acid & a complex sugar ester...... 157 Figure 8.5.4-16 Caffeine, 1,3,7-trimethylxanthine and its parent...... 158 Figure 8.5.4-17 Two structures believed to cause the bitter taste sensation ADD...... 158 Figure 8.5.4-18 Candidate sensory receptor performance for the “bitter” channel ...... 160 Figure 8.5.4-19 Candidate picric sensory receptor and quinine coordinate bonding RESCALE ...... 161 Figure 8.5.4-20 Calcium cation fully coordinated with water ...... 162 Figure 8.5.4-21 The potential gustaphores of CaCl2 and MgCl2 ADD...... 163 Figure 8.5.4-22 A selected group of stimulants represented as “umami” type...... 167 Figure 8.5.5-1 The serine ligand coordinate bonding with organic acids and amino acids .....169 Figure 8.5.5-2 EDIT A dimer situation with galactose [spell?] as both sensory receptor and stimulant ...... 170 Figure 8.5.5-3 ED A dimer situation with galactose as both sensory receptor and stimulant . . . 172 Figure 8.5.5-4 Concept: A stacked situation with the sensory receptor galactose interfacing with the glucophore of an undefined stimulant...... 173 Figure 8.5.5-5 The galactose-based receptor in context with the sensory microvilli ...... 174 Figure 8.5.5-6 The proposed molecular operation of the microtubules in ORN’s...... 177 Figure 8.5.5-7 Aspartame showing the axis of its AH,B,X “supersweet” glucophore...... 179 Figure 8.5.5-8 Aspartame bonding to the picric sensory receptor ADD ...... 180 Figure 8.5.5-9 The structure of saccharin...... 181 Figure 8.5.5-10 Dendrogram of 44 parabrachial neurons ADD...... 184 Figure 8.5.5-11 Candidate circuitry of the olfactory sensory neurons...... 185 Figure 8.5.5-12 Proposed cytological and electrolytic description of the olfactory sensory neuron ADD & MODIFY ...... 188 Figure 8.5.5-13 Odorant induced currents at various holding potentials from a newt, Cynops pyrrhogaster ADD ...... 191 Figure 8.5.5-14 “Parametric” In-vitro patch clamp recordings from embryonic rat...... 194 Figure 8.5.5-15 Integrated chorda tympani responses to 0.5 M chlorides ...... 197 Figure 8.5.5-16 Dependence of subjective intensity of taste sensation with concentration ADD ...... 197 Figure 8.5.5-17 Records of total chorda tympani responses to water and taste stimuli...... 201 Signal Generation & Processing 8- 209 AUTHOR INDEX Davson...... 36 Donnan ...... 2, 12, 39, 58, 59 Goldman ...... 2, 12, 21, 22 Hodgkin...... 3, 27, 28, 34, 47, 50, 73 Huxley...... 3, 27, 28, 34, 47, 49, 50, 73 Katz...... 27, 28, 47, 51 Matthews...... 13, 20, 21, 26 Nernst ...... 2, 12, 34, 60, 69, 70 Ranvier ...... 6, 9, 46, 47, 63, 71 Tien ...... 15, 24 210 Neurons & the Nervous System

(Active) SUBJECT INDEX (using advanced indexing option) 9(2) + 2...... 43, 44, 98 95% ...... 12 across-neuron pattern...... 196 action potential...... 41, 75, 109, 190, 192, 193, 195-198 Activa . . . 4, 6, 24, 25, 67, 86, 101-103, 123, 132, 139, 142, 143, 149, 166, 176-178, 184, 185, 187, 189, 190, 195 active diode ...... 185 adaptation...... 3, 30, 41, 86, 110, 124, 127, 143, 164, 197 ageusic...... 31 AH,B . . . 71, 77, 87, 89, 91, 92, 94, 101, 107, 130-133, 137-139, 143-151, 155, 157-161, 164-166, 168, 170, 171, 175, 178-180, 199 AH,B,X ...... 71, 87, 91, 92, 130, 132, 144-146, 161, 168, 179, 180, 199 allosteric...... 42 AME...... 42 amiloride ...... 76, 114-116, 182, 196, 197 amplification ...... 3, 25, 27, 37, 45 amygdala ...... 48, 52, 55, 77, 78, 80, 81 anatomical computation...... 2 anomeric carbon...... 35, 168 AOB...... 52 attention...... 16, 73, 149 axon segment ...... 76 axonemal ...... 43 axoplasm ...... 19, 21, 43, 189, 190, 192, 194, 195 band gap...... 23 Bayesian...... 16, 39, 76, 183, 203 bilayer . . . 20, 21, 63, 65, 67, 83, 87, 93, 101, 103, 128, 136, 139, 140, 142, 143, 147, 149, 160, 174-176, 178, 187 bilayer membrane...... 20, 21, 160, 174, 178 Brodmann...... 48 C/D...... 20, 23, 76, 103 cAMP...... 18, 46, 76, 192 carnosine ...... 62, 63, 70 cerebellum ...... 52 cladogram ...... 199 class A ...... 195 computation ...... 2, 186 computational...... 48, 69, 110, 180 conduction velocity ...... 55 continuum ...... 112 coordinate bond ...... 71, 73, 87, 90, 94, 139, 148, 149, 153, 157, 160, 169, 170, 173, 174, 178, 179, 199 coordinate chemistry ...... 28, 61, 63, 67, 68, 70, 71, 85, 89, 128, 150, 197 cribriform plate...... 189 Cu(I)...... 168 Cu(II) ...... 60, 168 dendrolemma ...... 76, 140, 142, 176, 177 determinants...... 23, 39, 45, 46 diode...... 21, 101, 103, 139, 177, 185, 190, 192 dipole moment...... 67, 83, 93, 123, 142, 143, 146, 171, 175, 177, 178, 185-187 dipole potential...... 67, 71, 83, 86, 93, 103, 130, 132, 134, 145, 146, 149, 174-176, 178, 179 DNA...... 8, 9, 13, 15, 37, 62 dynamic range ...... 178 E/D...... 5, 20, 74, 75, 116, 143, 196-198 electrometer ...... 137, 176 electrostenolytic process ...... 17, 18, 21, 103, 176, 187 enantiomers ...... 168 EPC’s...... 23 epitopes ...... 39 Signal Generation & Processing 8- 211 ethmoid bone ...... 46 fascicle...... 53 Fehling’s reagent ...... 167 fMRI...... 198 GABA ...... 15, 18, 131 ganglion neuron...... 77, 79, 194, 196, 197 ganglioside...... 136-143 genetics...... 7-11, 13, 15, 155, 158 genome...... 11, 12 globoside ...... 101, 103, 127, 130, 140, 143, 177 glomeruli ...... 51-54, 59, 79, 189, 192 glucophore ...... 87, 91-93, 138, 166, 169, 173, 179 glutamate ...... 15, 29, 37, 86, 93, 94, 106, 107, 115, 124, 128, 165-167 glycophore ...... 92, 107, 132, 142 glycosylation ...... 63 GPCR...... 8, 11, 21, 45, 132 gustophore ...... 59, 72 heterodimer ...... 8, 11 Hodgkin condition ...... 74 hole...... 13, 154 homogeneous...... 160 hormone...... 59 hydronium ...... 103, 142, 176 hydronium liquid crystal ...... 176 hypothalamus ...... 48, 55, 77, 78, 80, 81 h-neurons...... 112 inositol...... 18, 35, 46, 77, 90, 128, 141, 151, 153, 167 Jacobson’s organ ...... 37, 52 knockout ...... 8 labeled-line...... 7, 17, 196, 199, 200 Langmuir ...... 67 liquid-crystal ...... 5, 6 lock and key...... 71 London dispersion ...... 107, 131 LOT ...... 142 major nerves...... 73 masking ...... 41, 182 microtubule ...... 44, 177 microvilli .... 2, 3, 32, 78, 86, 87, 98, 100-103, 123, 127, 128, 139, 140, 142, 143, 149, 158, 160, 166, 168, 169, 171, 174, 182 MOB ...... 52 modulation ...... 42, 77, 143 multi-dimensional ...... 31, 73, 90, 111, 116, 117, 119, 122, 144, 165, 199 myelin...... 85, 137 myelinated ...... 76, 77, 79 Myelination ...... 79, 96, 97 N1...... 116 narrow band...... 123 neural coding ...... 32, 55, 107, 108, 114, 121, 183, 190, 193, 196 neural response function ...... 124, 125, 127 neurite...... 36, 50 neurites...... 36, 40, 194 neuromodulator ...... 15 neuropil ...... 35 neurotransmitter ...... 4, 6, 14, 15, 18, 24, 63, 104, 127 neurotrophins ...... 194 neuro-affector...... 21 neuro-facilitator ...... 166 nitric oxide...... 13, 14 Node of Ranvier...... 76, 77, 79, 97 noise...... 19 n-neurons...... 112, 119 212 Neurons & the Nervous System n-type...... 176, 177 olfactophore ...... 46 operculum...... 77, 80 Orangutan ...... 31 orbitofrontal cortex...... 55, 78 P/D equation...... 23 parametric...... 36, 60, 168, 189, 194, 195 parietal lobe ...... 57, 77, 80, 114, 198 patch-clamp ...... 36, 192 percept ...... 122 perceptual space...... 114 perigeniculate...... 196 phonoexcitation/de-excitation ...... 20 phonoreceptors ...... 20, 23 piezoelectric...... 2, 4-6, 16, 23, 76 piriform cortex...... 55 pnp...... 176, 189 podites ...... 21 poditic ...... 52, 53, 190, 195 pons ...... 47, 78 pseudogenes ...... 8, 11 p-type...... 176, 177 quantum-mechanical ...... 3-5, 20, 22, 25, 44, 55, 60, 74, 76, 127, 128, 177, 178, 184, 185, 197 rafts ...... 98, 101 residue ...... 15, 33, 140 resonance...... 149, 168 saliency map...... 3, 29, 35, 36, 48, 80, 82, 114 SAR ...... 7, 38, 39, 71, 143 second messenger ...... 13, 15, 16, 18, 21, 35, 42, 43 stage 0 ...... 32 stage 1 ...... 2, 19, 32, 55, 75, 185, 196, 197 stage 2 ...... 2, 3, 5, 7, 46-48, 51, 52, 55, 58, 59, 80, 82, 86, 114, 192, 196, 198 stage 3 ...... 2, 7, 51, 54, 55, 75, 76, 79, 96, 97, 137, 185, 192, 194, 196-198 stage 4 ...... 2, 7, 31, 32, 36, 46-48, 52, 55, 59, 77, 80, 82, 110, 192, 197, 198 stage 5 ...... 32, 36, 55, 56, 80, 82, 106 stage 6 ...... 106 stage 7 ...... 19 synapse...... 4, 6, 17, 36, 76, 185 s-neurons ...... 112 temporal lobe...... 37, 47, 77, 80, 82, 198 tetrahedron...... 69, 119, 122, 123, 131 thalamic reticular nucleus...... 47, 55 thalamus...... 31, 37, 47, 48, 55, 57, 77, 80, 198 topography ...... 55, 80 topology ...... 142 transduction . . . 2, 4, 5, 7, 9, 10, 13, 15, 16, 18-23, 26, 28, 29, 31, 32, 37, 38, 40, 42-46, 55, 59-63, 67, 74-76, 81, 83, 85, 86, 91, 94, 96, 101, 102, 104, 107, 117, 123, 127-130, 135, 139-142, 146-149, 154, 155, 159, 167, 176-179, 183, 184, 187, 189 transistor action ...... 24, 25 transition dipole moment ...... 185-187 transition group ...... 62 translation...... 10, 165 tufted cell...... 185 type 1...... 21, 55, 103, 175 type 2...... 21, 101-103, 139, 143 type 3...... 21 type 4...... 21, 101-103, 123, 127, 128, 139, 141, 158, 169, 176, 178, 182 type 5...... 21 umami...... 29, 31, 81, 85, 86, 93, 94, 106, 107, 109, 120, 124, 165-167, 182 vagus nerve ...... 56 Van der Waals ...... 131 Signal Generation & Processing 8- 213 verification...... 178 vomeronasal organ...... 27, 29, 48, 52 white matter...... 85, 140 xxx . . 1, 3-6, 9, 18, 19, 24, 25, 27, 29, 31, 34, 39-42, 44, 46, 52, 55, 59, 60, 62, 72, 74, 75, 78, 81, 82, 85, 90, 92, 101, 107, 114, 115, 123, 129, 134-137, 139-141, 158, 160, 163, 169, 175, 176, 178, 182, 189, 190, 197, 203 [xxx . 11, 13, 21, 24, 25, 40, 41, 43, 45, 48, 50, 62, 71, 73, 80, 81, 86, 94, 103, 111, 119, 120, 126, 127, 133, 136, 140, 141, 144, 145, 147, 151, 153, 158, 160, 163, 165, 168, 169, 182, 185, 189, 190, 192-194, 199