European Journal of Neuroscience, Vol. 9, pp. 1536-1541, 1997 0 European Neuroscience Association

SHORT COMMUNICATION Evidence that Blue-on Cells are Part of the Third Geniculocortical Pathway in Primates

Paul R. Martin, Andrew J. R. White, Ann K. Goodchild, Heath D. Wilder and Ann E. Sefton Department of Physiology and Institute for Biomedical Research, University of Sydney, NSW 2006, Australia

Keywords: colour vision, Callithrix jacchus, magnocellular, parvocellular, koniocellular

Abstract Colour vision in primates is mediated by cone opponent ganglion cells in the , whose axons project to the dorsal lateral geniculate nucleus in the visual . It has long been assumed that cone opponent ganglion cells project to the parvocellular layers of the geniculate. Here, we examine the role of a third subdivision of the geniculocortical pathway: the interlaminar or koniocellular geniculate relay cells. We made extracellular recordings in the dorsal lateral geniculate nucleus of the common marmoset Callithrix jacchus, a New World monkey in which the interlaminar cells are well segregated from the parvocellular layers. We found that one group of colour opponent cells, the blue-on cells, was largely segregated to the interlaminar zone. This segregation was common to dichromatic (‘red-green colour-blind’) and trichromatic marmosets. The result calls into question the traditional notion that all colour information passes through the parvocellular division of the retino-geniculo-cortical pathway in primates.

Introduction The marmoset (CaEZithrix jacchus), in common with several other 1984; Ts’o and Gilbert, 1988; Hendry and Yoshioka, 1994). This species of New World monkeys (Jacobs, 1993), shows sex-linked connectivity has led to speculation about the role of the inter- polymorphism of colour vision. All marmosets express, and use laminar zone cells in transmitting cone opponent signals (Hubel and the signals from, short wavelength-sensitive (SWS) cones. Male Livingstone, 1990). Because the interlaminar zone is well segregated marmosets also express one of three alleles on the X chromosome from the main parvocellular layers in the marmoset, we have used coding for pigments in the medium-long (ML) wavelength-sensitive this species to test the hypothesis that cone opponent signals are range, and are dichromats. Females with a different allele on each X segregated to this subdivision of the retinogeniculate pathway. chromosome express a total of three different pigments, and show behavioural signs of trichromacy (Tovte et al., 1992). In other respects, the of this species bears strong resemblance Materials and methods to that of diurnal Old World monkeys and humans, with a well Adult marmosets (Callithrix jacchus) were obtained from the developed fovea (Wilder et al., 1996), midget and parasol ganglion Australian National Health and Medical Research Council (NHMRC) cell systems in the retina (Ghosh et al., 1996), and parvocellular and combined breeding facility, Adelaide. Animals were anaesthetized magnocellular subdivisions of the lateral geniculate nucleus (Kaas with 052% isofluorane in N20/02 and prepared for single-cell et al., 1978; Spatz, 1978). The functional properties of cells in the recording (for details see Yeh et al., 1995). All procedures conformed marmoset lateral geniculate nucleus (LGN) bear strong resemblance to the Australian NHMRC code of practice for the use and care of to their counterparts in the macaque LGN, with the important animals. Paralene-insulated tungsten or steel microelectrodes exception that red-green colour opponent cells are not present in (Frederick Haer, Brunswick ME) were used to isolate the activity of male animals or dichromatic females (Yeh et al., 1995). single cells in the LGN and peristimulus time histograms were In addition to the parvocellular and magnocellular layers, the constructed as described (Smith et al., 1992; Yeh et al., 1995). A interlaminarkoniocellular subdivision of the LGN (LeGros Clark, preliminary classification of cell type was made using hand-held 1941; Walls, 1953; Kaas et al., 1978; Jones and Hendry, 1989; stimuli, after which the temporal and spectral characteristics of the Casagrande, 1994; Hendry and Yoshioka, 1994) is also present in the cell were studied using a Maxwellian view visual stimulator system marmoset. These cells lie within and between the parvocellular and which was aligned on the axis of the cell’s receptive field (Smith magnocellular layers (LeGros Clark, 1941; Kaas et al., 1978) and et al., 1992; Yeh et al., 1995). The stimulus was a spatially uniform have been shown in the macaque to project directly to the colour- field 6.4 degrees in diameter, composed of the combined image of selective blobs in primary (Livingstone and Hubel, three light-emitting diodes (LEDs) which were intensity-modulated

Correspondence to: Dr Paul R. Martin, Department of Physiology F13, University of Sydney, NSW 2006, Australia Received 5 December 1996, revised I1 February 1997, accepted 21 February 1997 Blue-on cells and the third geniculocortical pathway 1537

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Interlaminar n [Ipml

AP 3.5 Maanocellular

Superficial AP 3.0 El

0 10 20 30 Cell diameter bml

FIG.1. Identification of interlaminar zone cells in mannoset LGN. (a) Coronal sections through the caudal aspect of the LGN of a trichromatic female marmoset, stained with cresyl violet to show the cell layers. Nomenclature follows that of Kaas et al. (1978). Ipm, interlaminar zone; ME, external magnocellular layer; MI, internal magnocellular layer; PE, external parvocellular layer; PI, internal parvocellular layer; S, superficial layer. (b) Soma diameter histograms measured from a swath along the axis shown in (a). Mean soma diameter indicated on each histogram: PE + PI, 12.2 pm; Ipm. 10.3 pm; ME + MI, 13.6 pm; S, 11.0 pm. The ME + MI measurements include cells from the interlaminar zone between the magnocellular layers. Vertical scale, 20 cells. (c) Section neighbouring (a), processed for calbindin-like immunoreactivity. Labelled cells are concentrated in the interlaminar zone and the superficial layers. (d) Representative sections at different rostrocaudal positions through the LGN, with the position of cells showing calbindin-like immunoreactivity. Numbers show the distance from the interaural axis in millimetres. 1538 Blue-on cells and the third geniculocortical pathway

Parvocellular

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lnterlaminar d

Magnocellular

0 1 2 3 4 1 2 3 4 Time [s] Time [s]

FIG.2. Response characteristics of cells in the marmoset LGN. (a, b) Each row of histograms shows the response of a single cell to two different stimuli, which are sketched in the top row. Laminar location of each cell is indicated. (a) Luminance modulation. The red and green LEDs were sine wave-modulated in-phase at 4 Hz, and contrast was varied with a sine envelope over a 4 s epoch. A minimum of 16 s of activity was recorded for each cell. All three cells responded to this stimulus. In (b), red and blue LEDs were modulated in counterphase to the green LED, with relative intensities calculated to provide differential modulation of SWS cones (Yeh et al., 1995). The cell located in the interlaminar zone is the only one which responds to this stimulus. (c) Amplitude and phase of the 4 Hz Fourier component for the interlaminar cell response to SWS stimulus. The response phase difference between the luminance and SWS conditions was close to 180 degrees. (d) Coronal section including an electrolytic lesion (arrow) at the recording site of the cell shown in (c). Scale bar: 1 mm.

at 4 Hz. The dominant wavelengths of the LEDs were 470 nm sectioned frozen at 30 pm and stained with Cresyl violet or processed ('blue'), 554 nm ('green') and 639 nm ('red'). The red and green for calbindin-like immunoreactivity (Jones and Hendry, 1989) using a LED luminance was matched by heterochromatic flicker photometry monoclonal antibody from Sigma, at 1:50 000 dilution. Cell soma (Smith et al., 1992; Yeh et al., 1995) and verified using a PR-650 size and calbindin-labelled cell distribution were measured using a spectrophotometer (Photo Research, Burbank, CA). Time-averaged computer-attached camera lucida system (Wilder et al., 1996). The retinal illuminance was close to 1000 human Td (CIE x,y = laminar depth and angle of each cell with respect to the hilus of the 0.618,0.361). The maximum luminance available from the blue diode LGN was mapped onto a drawing made from representative sections at was -7% that of red or green; for SWS modulation the red + the appropriate rostrocaudal level. Laminar assignment for nearly all green amplitude was reduced accordingly (Yeh et al., 1995). SWS cells was consistent with the -specific (ipsilateral or contralateral) modulation was between CIE x,y = 0.3,0.13 and xy = 0.46,0.521. excitatory retinal input. A few parvocellular cells were activated from Contrast was varied with a sine envelope over a 4 s epoch to obtain the 'wrong' eye; we interpret these cells as located in laminar leaflets a measure of cell responsivity (Smith et al., 1992; Yeh et al., 1995). (Kaas et al., 1978; Spatz, 1978) whose borders were not apparent in Depth values from the electrode micropositioner were recorded for our material. each cell and for small electrolytic lesions (20 FA X 20 s), which were made for at least two positions in each electrode track. Results At the end of a recording session, animals were overdosed with In Nissl-stained sections (Fig. la, b), regions containing relatively barbiturate and perfused with 4% paraformaldehyde. Brains were small somata were interposed between and below the main parvocellu- Blue-on cells and the third geniculocortical pathway 1539

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FIG. 3. Location of blue-on cells within the LGN. (ad) Profiles of the parvocellular (grey) and magnocellular (black) layers of the LGN at four rostrocaudal levels. Horizontal and vertical scale bars show distance from the midline, and depth below the cortical surface. Open circles shows the position of blue-on cells. For cell 1, an outer circle (radius 75 pm) shows the positional uncertainty of the histological reconstruction. For this track the position of other (non-blue-on) cells is also shown. The majority of blue-on cells is confined to the interlaminar zone between the parvocellular and magnocellular layers. (e) Receptive field centre positions of blue-on cells.

lar and magnocellular layers. Consistent with results obtained in the designed to modulate predominantly the SWS cones (Fig. 2b). Fourier macaque (Jones and Hendry, 1989; Hendry and Yoshioka, 1994). analysis of the blue-on cell’s response (Fig. 2c) showed a phase heavily labelled cells were seen in the interlaminar zone in sections difference close to 180 degrees for SWS-isolating compared with processed to reveal calbindin-like immunoreactivity (Fig. 1c). The luminance modulation, consistent with the identification of this cell cells were located predominantly in the caudal aspect of the LGN as blue-on, yellow-off. These characteristics strongly resemble those and in the superficial layer ventral to the magnocellular layers of blue-on cells in the macaque retina (Lee et al., 1990; Dacey and (Fig. Id). However, the anatomical segregation was not complete, Lee, 1994) and LGN (Wiesel and Hubel, 1966; Dreher et al., 1976; with occasional calbindin-labelled cells scattered throughout the Derrington et al., 1984). Figure 2d shows a section with an electrolytic parvocellular layers. These data demonstrate that the immunoreactivity lesion made at the site where a blue-on cell was recorded. The lesion and size distribution of cells in the interlaminar zones are homologous is immediately lateral to the internal magnocellular layer, in the with those described for other New World and Old World primates interlaminar zone. (Kaas et al., 1978; Casagrande, 1994). The position of each blue-on cell was measured and plotted onto The properties of cells within the LGN were studied in single-cell standard outlines (Fig. 3) drawn at the appropriate rostrocaudal level extracellular recordings in six female marmosets. A total of 84 cells through the LGN. Analysis was restricted to the caudal aspect of the was fully characterized and their position within the LGN recovered. LGN, where the fovea is represented and the lamination is most Of these, 48 were in the parvocellular layers, 13 were in magnocellular distinct (Kaas et al., 1978; Spatz, 1978). Two cells were located in layers, 22 were in the interlaminar zone and one was immediately the internal parvocellular layer. One cell was above the dorsal border dorsal to the main LGN layers. The vast majority of cells responded of the LGN. The remaining cells were either within the interlaminar to in-phase (yellow luminance) contrast modulation. Magnocellular zone (n = 6) or at the border with the ventral parvocellular layer cells showed the most vigorous responses, even at very low contrast (n = 4). We conclude that blue-on cells are largely confined to the levels (Fig. 2a). However, only the cells which were classified as interlaminar zones in the marmoset. blue-on using hand-held stimuli (n = 13) responded to a stimulus A minority of cells (17 from a total of 48) in the parvocellular 1540 Blue-on cells and the third geniculocortical pathway

a b (Rodieck, 1991), but such injections would normally include the 1 interlaminar zones between the parvocellular layers, thus labelling cells which project there. Retinal terminals within the interlaminar zone arise from mostly fine and medium-sized axons (Conley et al., 1987), and could thus include the medium-calibre axons of small bistratified cells (Rodieck, 1991; Dacey and Lee, 1994). Because the anatomical segregation of the interlaminar cells is less pronounced in the macaque than in the marmoset, any segregation of these cells 0 1 1 might have been obscured by the low sampling rate in single-unit C d studies to date (Schiller and Malpeli, 1978). The blue-on cells in the cat LGN are segregated in the ventral 1 x parvocellular C layers (Cleland et al., 1976; Wilson et al., 1976) and have been assigned to the ‘W’ stream of . Other 0Q properties of blue-on cells in primates are indeed like those of some CLar U 0.5 W-cells: they have bistratified morphology, relatively large receptive -._N E fields with coextensive On and Off regions, and poor sensitivity to b achromatic luminance modulation (De Monasterio and Gouras, 1975; z 0 Derrington et al., 1984; Lee et al., 1990; Dacey and Lee, 1994). The cortical terminals of intercalatedikoniocellular zone cells Time [s] Time [s] coincide with the position of cytochrome oxide-rich blobs in the FIG.4. Responses to red-green chromatic, luminance and SWS modulation. supragranular layers, where colour-opponent cells tend to be Graphs show normalized response amplitude of the 4 Hz Fourier component concentrated (Livingstone and Hubel, 1984; Ts’o and Gilbert, from histograms as in Figure 1. Maximum response amplitude for each cell 1988; Hendry and Yoshioka, 1994). Our results imply that blue-on (impulsesls) shown in upper right of each panel. Stimuli as in Figure 2, except cells in the geniculate project directly to the supragranular cortical ‘R-G’, where red and green LEDs were modulated 180 degrees out of phase. (a) Opponent cell, parvocellular layers. (b) Non-opponent cell, parvocellular layers, and give rise to the intriguing speculation that in the transition layers. (c) Blue-on cell, interlaminar zone. (d) Magnocellular cell. Throughout from dichromacy to trichromatic colour vision, the red-green opponent the 4 s stimulus epoch, responses to R-G modulation were apparent only in system has come to share (Livingstone and Hubel, 1984) or usurp cells in the parvocellular layers (a, b). Responses to SWS modulation were (Ts’o and Gilbert, 1988) the cortical pathways originally devoted restricted to blue-on cells (c). Residual responses to R-G or SWS modulation in some magnocellular and non-opponent parvocellular cells (b,d) could be to opponent processing in a primordial, blue-yellow system for accounted for by the difference between the human luminosity function and colour vision. the dominant pigment in the medium-long range for dichromat animals (TovBe et al., 1992; Yeh et al., 1995). Acknowledgements We thank R. Phillips for technical assistance, and B. Boycott, W. Burke, layers of some female marmosets showed responses indicative of B. Dreher and U. Griinert for helpful discussion and comments. Supported by Australian NHMRC and the Ramaciotti foundation. opponent interactions between two pigments in the medium-long wavelength-sensitive range (Fig. 4a). 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