Distribution of Retinogeniculate Cellsin the Tammar Wallaby in Relation to

Decussation at the Optic Chiasm

B.M. WIMBORNE, R.F. MARK,* AND M.R. IBBOTSON

Developmental Neurobiology Group, Research School of Biological Sciences,Australian National University, Canberra, ACT 2601, Australia

ABSTRACTPartial decussation of the optic nerve in mammals is related to the laterofrontal

placement of the eyes. To investigate this relationship in the wallaby (Macropus eugenii),injections of wheat germ agglutinin–conjugated to horseradish peroxidase were made into onedorsal lateral geniculate nucleus to label retinal ganglion cell bodies in both retinas.Contralaterally, labelled ganglion cells were present across the nasotemporal axis, except forthe far temporal retina where they were absent or very sparsely scattered compared with thedensity of labelled cells at similar nasal eccentricities in the same retinas. Ipsilaterally,labelling was confined to the temporal retina. Cell counts confirmed a visual streak and anarea centralis in the contralateral projection. Diameters of labelled cells ranged from 9 µm to30 µm with a hint of three categories of cells based on size. Only the large a-type cells wereeasily separated. Measurement of the acceptance angles of the eye in the anaesthetisedanimal showed about 15% of the horizontal visual field of each eye projects into a region ofbinocular overlap giving a binocular field of 50°. The uniocular visual field extends from 225°(nasally) to 1162° (temporally) in azimuth, giving the wallaby a monocular visual field widthof 187° and a total visual field width of 324°. In elevation, field ranges from 70° inferiorto 1120° superior, encompassing 190° in the vertical plane. The wallaby shows partialdecussation of optic nerve fibres projecting to the lateral geniculate nucleus that could allowstereopsis, plus an extensive panoramic field. J. Comp. Neurol. 405:128–140, 1999.r 1999 Wiley-Liss, Inc.

Indexing terms: retina; LGNd; ganglion cells; visual field; marsupial

In the vast majority of mammalian species, the opticnerves do not decussate completely at the chiasm, allowinga portion of their axons to turn back and innervate theipsilateral side of the brain, thus taking to each hemi-sphere signals from the same side of the visual field. Thismorphology forms the basis for binocular vision, present inall mammals with the possible exception of whales (Petti-grew, 1986).

Partial decussation varies between mammalian speciesand ranges from a few percent of optic axons in rodentsand lagomorphs to one third or more in primates andcarnivores (Polyak, 1957). After the original suggestion byCajal (1911), partial decussation has often been related tothe evolution of frontally placed eyes and binocular vision(Vidyasagar et al., 1992), on the assumption that binocularvision, through the provision of stereopsis, is a trade-off forthe loss of an extensive visual field enjoyed by animalswith lateral eye placement. Nevertheless, the facts are notalways in accord. For instance, the rat possesses lateraleyes and has a panoramic field of 320°. This range exceeds

that of the cat, which has frontal eyes by 135°, yet the rat’sbinocular field of 80° is close to the 99° of the cat (Hughes,1977a).

Although little is known about why decussation patternsdevelop, Cajal (1911) proposed that total decussationpresent in inframammalian vertebrates represents a phy-logenetic phase preceding partial decussation and is re-lated to the centralisation of the nervous system whichleads to the creation of the brain (Polyak, 1957). Lund(1978) points to a number of factors that influence decussa-tion. It is known, for instance, that decussation patternspredate visual experience, although to what extent timing

Grant sponsor: Strategic Initiative Fund; Grant sponsor: AustralianNational University.

*Correspondence to: Prof. R.F. Mark, Developmental Neurobiology Group,Research School of Biological Sciences, Australian National University,GPO Box 475, Canberra ACT 2601, Australia.

Received 15 April 1998; Revised 24 September 1998; Accepted 28September 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 405:128–140 (1999)

r 1999 WILEY-LISS, INC.

of axonal growth is relevant has yet to be determined.However, the pattern of decussation is not irrevocablyfixed and can be altered through lesions of the visualpathway. Decussation patterns can also be affected bygenetic factors, exemplified by the excessive decussation inalbino mammals (Guillery et al., 1995). Finally, the posi-tion of a cell in the retina is relevant to whether its axonwill cross at the chiasm. Also important is the cell type andwhere it projects in the brain (Stone and Dreher, 1973).

The tammar wallaby (Macropus eugenii) is a relativelysmall (4–7 kg) kangaroo belonging to an advanced order ofdiprotodonts that includes other wallabies, kangaroos,wombats, opossums, and the koala. Eye placement in thetammar is neither frontal nor lateral, lying between thetwo extremes. Wye-Dvorak (1984) showed with [3H]prolinelabelling of the eye followed by autoradiography of centraltargets of the optic nerve that the monocular segment ofthe dorsal lateral geniculate nucleus (LGNd) was foundrostrally and that the binocular segment was caudal.Further investigation of the topography of the tammar’sretinal projections to the LGNd and superior colliculuswas made by Flett et al. (1988) who used small laserlesions of the retina followed by intravitreal injections ofhorseradish peroxidase. This method produces a fillingdeficit sharply cut out from the otherwise densely labelledterminals of retinal ganglion cells in the LGNd correspond-ing in position to the projection of the ganglion cell bodiesin the retina damaged by the laser lesion. They concludedthat the dorsal retina projects ventrally in the LGNd andthat the ventral retina projects dorsally, whereas the nasalretina projects rostrally and the temporal retina projectscaudally. The binocular region of the nucleus is caudal andreceives only from the temporal retina of both eyes.

Physiologic retinotopic organisation in the tammar wasinvestigated by Wye-Dvorak et al. (1987) who mapped thelateral geniculate by using unit recording. They reportedthat the lateral geniculate has a monocular visual fieldranging from 230° nasal to 1179° temporal and 73°superior to 249° inferior in elevation. According to theirfindings, ganglion cells with receptive field positions be-tween 29° and 179° projected to the contralateral LGNd,whereas those with visual fields ranging from 0° to 30°projected to the ipsilateral LGNd. There has been littleanatomical research, however, into the organisation of thetammar’s retinogeniculate projections, particularly withrespect to decussation at the chiasm.

In both marsupial and placental mammals, the ganglioncell layer of the retina is composed of two types of neuron,ganglion cells, the axon of which ends in the brain, anddisplaced amacrine cells, which do not have long axons andhave synaptic targets in the retina (Rodieck, 1973; Hughes,1985; Wong et al., 1986; Dunlop et al., 1988). Cells in thetammar’s ganglion cell layer were investigated by Wong etal. (1986) who mapped their distribution and classifiedthem morphologically. They concluded, as expected, thatthe layer was composed of two main neuronal cell popula-tions, true ganglion cells and displaced amacrine cells.Glial cells and all the displaced amacrine cells, they claim,can be recognised by anatomical criteria by which theymay be classified as glia, bar cells, coronate cells, and othermicroneurons. By using such discriminators to restricttheir analysis to ganglion cells, they confirmed the exis-tence of both a visual streak and an area centralis of highganglion cell density as had been reported previously byTancred (1981). They also attempted, with partial success,

to classify ganglion cells by size in various regions of theretina.

The present study uses back-filling of retinal ganglioncells by injecting horseradish peroxidase into the lateralgeniculate nucleus, to extend these findings. We examinethe distribution, size, and density of the tammar’s retinalganglion cells that specifically innervate the LGNd andcompare the proportion of cells with crossed axons with theextent of binocular overlap present in the map of the visualfield. We also consider whether the size and morphology ofthe tammar’s retinal ganglion cells might provide a basisfor establishing separate classes.

MATERIALS AND METHODS

Animals

Tammar wallabies were purpose-bred for these experi-ments. Six mature adults of either sex, each weighingbetween 4 and 7 kg were used. Procedures were authorized bytheAnimal Experimental Ethics Committee of theAustralianNational University, Protocol Number R.DN.40.95.

Surgery

At the beginning of the experiments, each animal wasanaesthetised with an intramuscular injection of ket-amine hydrochloride (Parnell Laboratories [Aust.] Pty.Ltd., Silverwater, NSW, Australia; 20 mg/kg) and 0.3 ml ofthe muscle relaxant xylazine (Rompun, Bayer AustraliaLtd., Botany, NSW, Australia). Continuing anaesthesiawas maintained with periodic intravenous injections of 5%thiopentone sodium (Pentothal, Boeringer Ingelheim Pty.Ltd., Artarmon, NSW, Australia; 0.5–1.0 ml) as needed.

The animal was placed in a stereotaxic head holder andlocal anaesthetic ointment, lignocaine (Xylocaine 2% jellyAstra Pharmaceuticals, N. Ryde, NSW, Australia) wassqueezed into each external auditory meatus before insert-ing ear-bars that were shaped and tapered to lodge in thecanal without reaching the tympanum. The height of amouth bar was adjusted so that the line joining the innerand outer canthi of each eye was horizontal. A smallcraniotomy was made 10 mm posterior of Bregma and 10mm lateral of the midline to expose the cortex.

The electrocardiogram rate was monitored continuously,and normal body temperature maintained by using athermistor-controlled heating blanket. End-tidal CO2 wasmonitored with a Datex Normocap medical gas analyser(Datex Instrumentarium OY, Helsinki, Finland).

As soon as the tracer injections were done, the woundwas sutured, dusted with Tricin antibiotic powder (Pitman-Moore Australia Ltd., N. Ryde, NSW, Australia), and thewallaby given a 300-mg intramuscular injection of theantibiotic Depocillin (procaine penicillin Intervet (Austra-lia) Pty. Ltd., Lane Cove, NSW, Australia). The uncon-scious animal was placed in a hessian bag and allowed torecover in a darkened room before returning it to a pen.

Injection of tracer

Location of the LGNd was determined on the basis ofcoordinates set-out in Wye-Dvorak et al. (1987) and Henryand Mark (1992) and by recording from LGNd cells byusing tungsten-in-glass microelectrodes inserted at rightangles to the intercanthal line. A hand held light sourcedisplayed on a tangent screen was used to stimulate cellsas the microelectrode slowly penetrated the LGNd. For the

DISTRIBUTION OF WALLABY RETINOGENICULATE CELLS 129

purpose of the experiment only binocular cells were ofinterest. When an appropriate cell was located its stereo-taxic co-ordinates were recorded and the position of itsreceptive field plotted. The recording electrode was thenreplaced with a 1-µl Hamilton syringe filled with wheat-germ agglutinin conjugated with horseradish peroxidase(WGA-HRP) (2–4% solution in normal saline). By usingthe stereotaxic coordinates of the recording electrode aninjection was made of 0.2–0.5 µl of tracer. To ensureadequate uptake of tracer a series of injections of the aboveamount was made within the binocular visual region of theLGNd. The location of the injections for each experimentalanimal is indicated in Figure 1.

Processing for WGA-HRP reaction

After a survival period of between 48 and 72 hours theanimals were initially anaesthetised with an intramuscu-lar injection of ketamine hydrochloride together withxylazine in the doses cited above, followed by deep anaes-thesia with a slow intravenous injection of enough thiopen-tone sodium to arrest breathing. The animal was thenperfused transcardially with solutions of 0.5% saline (for 1minute), 1% paraformaldehyde/1.25% glutaraldehyde in0.1 M phosphate buffer (pH 7.4) (for 20 minutes) and 30%sucrose buffer (for 20 minutes).

During the fixation stage of perfusion 0.5 ml of fixativewas injected into each eye. Eyes were then enucleated,hemisected at the ora terminalis, and placed in fixative.After 30 minutes, the vitreous was removed, the eyecupseverted, and the retinae dissected from the sclera in 0.1 Mphosphate buffer (pH 7.4). The retinae were then reactedfor WGA-HRP visualisation with the tetramethylbenzi-dine method (Mesulam, 1982). Subsequently, each retinawas floated onto a 5% gelatinised slide and radial cutswere made so that the tissue would lie flat. The whole-mounts were covered with filter paper (one piece of What-mans No. 50 and two pieces of No. 2 wetted with phosphatebuffer) and a second glass slide. Elastic bands were used tohold each pair of slides tightly together. The flat-mountedretinae were then stained with cresyl violet, dehydratedthrough 50%, 70%, and 100% isopropanol, and cleared inxylene before being cover-slipped with mounting solution(DePeX, BDH Laboratory Supplies, Poole, England).

The brain was removed from the skull and stored for24–48 hours in 30% sucrose in 0.1 M phosphate buffer. Itwas then blocked and sectioned coronally at 60 µm on asledge freezing microtome. Selected sections were reactedwith tetramethylbenzidine for WGA-HRP visualisation,mounted on gelatinised slides, counterstained with thi-onin, cover-slipped, and the LGNd inspected for evidenceof injection sites.

Mapping of isodensity contours

To assess the relative density of neurons projecting tothe LGNd, counts of cells labelled with WGA-HRP weremade at a sample of locations across the retinal flat-mounts. A 1 mm grid was placed over each retina and asample counted at every intersection of the grid lines. Cellswere counted within a 100 3 100 µm sampling frame withcells intersecting the top and right margins of the frameedges being included. Isodensity contours were then con-structed by using a thin plate spline technique to interpo-late across the data points (see Methods section in Mark etal., 1993).

Measurement of soma sizes and numbers

In one animal, retinal cells were sufficiently filled withtracer to permit measurement of cell bodies. A 1 mm gridwas placed over each retina and at the intersection of thegrid lines a sampling frame measuring 100 3 100 µmvisible through a drawing tube attached to the microscope,was used to sample labelled cells. Cells which extendedacross the top and right margins of the sampling framewere included in the count. Cells were viewed through anoil immersion objective and drawings made at each samplelocation. Total magnification of the microscope was 31,250.Cell sizes were assessed by estimating the diameter of acircle with the same area as the soma. In our one sample,cell diameters were measured at 83 locations in thecontralateral retina and at 46 locations in the ipsilateralretina. The number of cells sampled in the two retinaetotalled 818 and 236, respectively.

An estimate of the number of neurones of each type inthe retina was made by summing the numbers in all thesamples from different random locations across the retina.

Visualisation of cells in theganglion cell layer

One mounted retina was used in this experiment. Afterfixation and a brief wash in phosphate buffer, the retinawas counterstained with 0.1% toluidine blue in phosphatebuffer, pH 5, for 1 hour, before being dehydrated through50%, 70%, and 100% isopropanol, cleared in xylene, andcover-slipped. Counterstaining facilitated visualisation ofcells in the ganglion cell layer. Criteria for distinguishingganglion cells from displaced amacrine cells (bar cells,coronate cells, other microneurons) were those used byWong et al. (1986). According to these, ganglion cells thathave relatively large amounts of cytoplasm and containsubstantial amounts of Nissl substance are distinguishedfrom displaced amacrine cells, which have little cytoplasmcontaining Nissl substance.

To count the cells, a 1 mm grid was placed over the retinaand cells counted within a 100 3 100 µm sampling framelocated at every intersection of the grid lines. Cells weresampled at 60 locations.

Measurement of the field of view

Measurement of the wallaby field of view was made onone anaesthetised animal. Having placed the wallaby in astereotaxic device, the position of its head was tilted sothat the palpebral fissure was horizontal. An ‘‘Aimark’’projection perimeter was then centred on the midpointbetween the eyes, and a back projecting ophthalmoscopewas used to make a series of angular measurements at themost peripheral locations in the visual field where lightcould enter the eye through the centre of the pupil. Thelight beam was back projected, and each angle was re-corded on the projection perimeter. The points were thenjoined to form a boundary map of the field of view. Thelocation of the optic disc was plotted at intervals throughthe procedure to check that the eyes had not moved.

RESULTS

Extent and density of retinal label

Ganglion cells labelled with WGA-HRP were present inboth retinae. The heaviest concentration occurred in theretina contralateral to the injected LGNd and, although

130 B.M. WIMBORNE ET AL.

Fig. 1. Schematic diagrams showing the location of wheat germagglutinin-horseradish peroxidase injections (filled circles) that wereplaced in the right dorsal lateral geniculate nucleus (LGNd) of sixexperimental animals. In each case, a number of separate injectionswere placed at various depths and at different anterior-posterior andmedial-lateral locations to maximise uptake of the tracer. Lateral-

Medial, distance from the midline; Anterior-Posterior, distance fromBregma; Ventro-Dorsal, distance from cortical surface. All distances inmillimeters; dashed lines, isoelevation; stippling, approximate binocu-lar region. A small rim of monocular representation found in therostral LGNd is not shown.

labelling was apparent across much of the tissue, celldensity was low in the most temporal region. By contrast,much of the ipsilateral retina did not contain any labelling.Labelled cells were present only in a relatively smallregion of the temporal or temporal-ventral retina. Draw-ings of a typical pair of retinae that illustrate thesefindings are presented in Figure 2.

Labelled ganglion cells were counted systematically inretinal whole-mounts of three experimental animals.About90% of labelled cells occurred in the contralateral retinaand 10% in the ipsilateral retina, indicating that thecontralateral retina accounts for approximately 90% ofcells projecting to the LGNd (crossed fibres), whilst theipsilateral retina accounts for about 10% (uncrossed fi-bres). In the extensively labelled contralateral retina, acell count was also made on the nasal and temporal sides ofa vertical line bisecting the area centralis. About 20% ofcells were located on the temporal side of the line and 80%on the nasal side.

The extent of ventrodorsal labelling in the retina de-pended on the depth of injections in the LGNd. Relativelysuperficial injections resulted in labelling that was con-fined to the ventral half of the retina; with deeper injec-tions, labelled cells spread into the dorsal half. However, inno experiment was the dorsal half completely labelled,which suggests that projections from the dorsal retinaconverge on a very small region of the LGNd. To illustratethese findings, the drawings in Figure 3 compare thedepths and spread of injections in the LGNd of threeanimals with the resulting distribution of labelled cells inthe retinae.

Based on samples of cells counted, isodensity contourmaps were prepared to provide a picture of the relativedensities of retinal ganglion cells projecting to the LGNd.Three typical examples of isodensity contour maps of theretina are presented in Figure 4 where contour linesrepresent steps of 10 cells/0.04 mm2.

It can be seen in the contralateral retina that thegeneral pattern of the isodensity maps is one of approxi-mately concentric contours emanating from a region ofhigh cell density, the area centralis. This region is locatedadjacent to the optic disk head on the temporal side.Taking the total area of high density it covers an area ofapproximately 2 mm2 and has an uncorrected density ofabout 2,500 ganglion cells/mm2.

In addition, in the contralateral retina only, the contoursform an elongated horizontal band that extends from thearea centralis toward the nasal edge of the retina. Thisnarrow strip of relatively high cell density constitutes thevisual streak. On the basis of our sample of cell counts, weestimate that cells of the visual streak, including the areacentralis, make up approximately a third of the populationof retinal ganglion cells.

In the ipsilateral retina, the area of labelled cells islocated only in the far temporal region. The ipsilateralprojection conforms roughly to the area of the extremetemporal region of the contralateral retina that is almostwithout labelled cells.

Morphology of labelled retinal cells

The morphology of one group of cells projecting to theLGNd was clearly evident by the presence of conspicuous

Fig. 2. Camera lucida drawing of ipsilateral and contralateralretinas from the same animal showing the disposition of labelledretinal ganglion cells (black dots) that resulted from an injection of

wheat germ agglutinin-horseradish peroxidase into the contralaterallateral geniculate nucleus. The dashed line indicates the heavilypigmented ventral half of the retina. Scale bar 5 5 mm.

132 B.M. WIMBORNE ET AL.

Fig. 3. Drawings of the lateral geniculate nucleus (LGNds) andretinas of three experimental animals showing the depth of theinjection (filled circles), its spread within the LGNd (black area), andthe resulting distribution of retinal labelling. The results demonstrate

that the extent of ventrodorsal labelling in the retinas is dependent onthe depth of the tracer injection in the LGNd. Scale bar 5 5 mm(applies only to the retinas).

DISTRIBUTION OF WALLABY RETINOGENICULATE CELLS 133

Fig. 4. Camera lucida drawings of three representative pairs ofretinas to which isodensity contours have been fitted to samples oflabelled retinal ganglion cells that project to the lateral geniculatenucleus. The contour lines, which were constructed by using a thin

plate spline technique to interpolate across the data points, representsteps of 10 cells/0.04 mm2. The region of highest cell concentrationcovers about 4 mm2 in the contralateral retina and has a peak densityof about 2,500 cells/mm2. Scale bar 5 5 mm.

amounts of WGA-HRP in their cytoplasm and processes.These cells, which were characterised by large somas andextensive dendritic branching, were found in relativelysmall numbers. The photomicrograph in Figure 5 showsseveral of these neurons in association with smaller cells.

The somas of the large cells were frequently bulbous inshape, often with several dendritic branches extendingfrom the cell body. In some cases, it was possible to followthe course of a dendrite for over 300 µm. Varicosities,which may be associated with synaptic contacts, werevisible along the dendrites.

The remainder of the labelled cells comprised a popula-tion of smaller neurons of varying diameters. Apart fromthe general spherical shape of these cells, no distinctmorphologic features could be detected. In some instances,dendritic branching was apparent, but it was rarely pos-sible to trace the course of a dendrite for more than a fewmicrons.

Cell diameters

In Figure 6, samples of cell diameters from the contralat-eral and ipsilateral retinae have been interpolated byusing a cubic spline algorithm. The figure also contains aninterpolation of cell diameters sampled within the visualstreak. Soma diameters ranged in size from 10 µm to 30µm. There is evidence for the presence of three peaks,especially in the contralateral retina from which thelargest sample was drawn. In the contralateral retina andwithin the visual streak, peaks relate to soma diameters ofapproximately 15–16 µm, 19–20 µm, and possibly 24–25µm. In the ipsilateral retina, two less-prominent peaks arevisible at diameters of 16–17µm and 19–20µm.

It is possible that the concentration of cells at thesepeaks is indicative of the modal values of three classes of

cell. An estimate of the proportion of cells in each class iscontained in Table 1.

Total number of neuronsin the ganglion layer

On the basis of our count of one retinal whole-mountstained with toluidine blue, we estimate that the ganglioncell layer of the whole retina contains about 566,110 cells.

Fig. 5. High-magnification photomicrograph of labelled retinal ganglion cells that resulted frominjections of WGA-HRP into the LGNd. Labelled cells of differing sizes are present. Dendritic processeswith granular label come mainly from the largest cells. Scale bar 5 10 µm.

Fig. 6. Graphical representation of three samples of labelledretinal ganglion cell diameters taken from the contralateral (Contra.)and ipsilateral (Ipsi.) retinas and interpolated by using a cubic spinealgorithm. Evidence for the presence of three peaks, which mayrepresent three classes of retinal ganglion cells, exists in the contralat-eral retina. In the ipsilateral retina, the peaks are not as distinct, butthe sample size is smaller.

DISTRIBUTION OF WALLABY RETINOGENICULATE CELLS 135

This count includes ganglion and displaced amacrine cells.Approximately 30%, or 169,830 cells, could be classified asdisplaced amacrine cells, and the remaining 396,280 neu-rons were categorised as ganglion cells of which 6% hadcell bodies exceeding 20 µm in diameter.

Measurement of monocularand binocular visual fields

Because the perimetric method used to assess thewallaby uniocular field takes into account the influence ofthe nose and optic adnexa, our result is a measurement ofthe relative or obscured field of view. Assuming an absenceof ocular divergence, the wallaby monocular visual fieldranges from 225° nasal to 1162° temporal in azimuth, and270° inferior to 1120° superior in elevation. This rangegives the animal a uniocular field of view covering 187° inthe horizontal plane and 190° in the vertical plane. Thetammar’s complete field of view encompasses about 324° inazimuth. These findings are illustrated in the perimeterchart at Figure 7.

Only the uniocular field of view needs to be looked at inassessing the extent of binocular overlap. According to ourmeasurements, assuming there is no ocular convergenceor divergence, the uniocular visual field extending from 0°(the midline) to 225° nasal accounts for about 15% of theanimal’s field of view. On this basis, we estimate thatbinocular overlap covers about 50° in azimuth. In eleva-tion, however, the region of binocular overlap is consider-ably larger. It extends over 110°, ranging from 240° inferiorto 170° superior (see Fig. 7).

DISCUSSION

In this paper, we have outlined the organisation ofretinal cells projecting to the LGNd, examined their distri-bution densities across the retina, and measured theirsizes. In addition, we have differentiated displaced ama-crine cells from ganglion cells in the ganglion layer andmapped the animal’s field of view. The organisation ofganglion cells projecting from the ipsilateral (uncrossedprojections) and contralateral (crossed projections) retinaeto the LGNd is consistent with the tammar’s frontolateraleye placement. A high concentration of cells in a smalldiscrete region of temporal retina confirms the presence ofan area centralis, and a long horizontal stretch of retinaextending into the nasal half where cell density remainshigh represents the visual streak. Although only one celltype could be clearly distinguished on the basis of morpho-logic variation, the distribution of soma sizes is consistentwith the separation of neurons into three classes, whichmay reflect functional differences. Our results also showthat approximately a third of the cells in the ganglion celllayer are displaced amacrines.

Retinal projections to the LGNd

The organisation of ipsilateral and contralateral projec-tions in the tammar is similar to that seen in the visualpathway of many mammals in which uncrossed (ipsilat-eral) projections arise from ganglion cells in the temporalretina, whereas crossed projections, on the other hand,arise primarily, although not exclusively, from the nasalretina. This pattern is comparable to that found in thequokka, although with the quokka, a few uncrossed projec-tions are located in the nasal retina close to the nasotempo-ral division (Harman and Jeffery, 1992). By contrast, smallnumbers of ipsilateral projections from the nasal retinahave been reported in rodents (Drager and Olsen, 1980;Jeffery, 1984; Dreher et al., 1985) and ferrets (Morgan etal., 1987). These were not evident in the tammar.

Our finding that approximately 10% of the wallaby’sganglion cell population projects ipsilaterally is similar tothe proportion in both the quokka (Dunlop and Beazley,1985) and the ferret (Morgan et al., 1987). These cells,which lie in the temporal division of the ipsilateral retina,subserve binocular vision, and their proportion of the totalganglion cell population should be reflected in the degreeof binocular overlap present in the visual field. Ourestimate that the proportion of ganglion cells in thetemporal retina that project contralaterally is approxi-mately 20% compares with virtually none in the monkey(Stone et al., 1973; Bunt et al., 1977), less than 10% in theferret (Morgan et al., 1987), 25% in the cat (Stone, 1966),30% in the quokka (Harman and Jeffery, 1992), and up to85% in rodents (Drager and Olsen, 1980; Jeffery et al.,1981).

Isodensity contour maps of retinal ganglion cells havebeen prepared for a number of marsupials and the pres-ence of a distinct visual streak and an area centralis in thetammar wallaby is consistent with a pattern found inother Australian marsupials such as the pademelon, Tas-manian devil, brush-tailed possum (Freeman and Tancred,1978; Tancred, 1981), and quokka (Beazley and Dunlop,1983). However, not all marsupials conform to this configu-ration. For example, a visual streak without any areacentralis occurs in the hairy-nosed wombat (Tancred,1981) and an area centralis without a visual streak isfound in North and South American opossums (Hokok andOswaldo-Cruz, 1979; Rapaport et al., 1981a). At a behav-ioural level, in the case of the tammar, the existence of avisual streak would aid in the detection of movement thatbreaks the continuity of the horizon (Wong et al., 1986).

Studies of variations in the distribution of ganglion cellsin the retinae of several Australian marsupial speciesparticularly kangaroos and possums led to considerabledebate over the evolution of the visual streak (Hughes,1977a; Freeman and Tancred, 1978). According to Wong etal. (1986), differences in the steepness of the cell densitycontours between the two species, rather than the pres-ence of a visual streak, may be highly significant. Forinstance, ganglion cell contours in the possum visualstreak have a predominantly circular configuration whilsta steeply contoured streak characterises the retina of therabbit (Wieniawa-Narkiewicz, 1983) and plains kangaroo(Hughes, 1975). In their study of the tammar’s retinal celllayer, Wong et al. (1986) concluded that the tammar’svisual streak is closer to that of the plains kangaroo thanthat of the brush-tailed possum. Our finding that thetammar’s retina incorporates a visual streak with rela-

TABLE 1. Cell Classification by Size1

Cell Size (µm)

10–17 18–23 24–30

Contralateral retina (%) 51 41 8Visual streak (%) 47 45 8Ipsilateral retina (%) 56 38 6

1The temporal and nasal divisions of the retina were defined as lying on either side of animaginary vertical line passing through the area centralis. The mean sizes of cells ineach division were assessed at 17.54 µm and 17.90 µm, respectively. These sizes werenot significantly different from the mean diameter of 17.43 µm for cells in the ipsilateralretina.

136 B.M. WIMBORNE ET AL.

tively steep contours and an area centralis projecting tothe LGNd is consistent with their conclusion.

Morphology and size of retinal ganglion cells

In the cat, Rowe and Stone (1976) classified retinalganglion cells into three classes on the basis of soma size —small were less than 14 µm, medium were 14–20 µm, andlarge were over 20 µm — which were identified with the W,X, and Y classification of Stone and Fukuda (1974) andthus with g, b, and a cells. The three classes have distinctanatomical and physiologic characteristics, including dif-fering morphologies, axon conduction velocities, and cen-

tral projections (Fukuda and Stone, 1974; Boycott andWassle, 1974; Kelly and Gilbert, 1975; Rowe and Stone,1976; Wassle and Illing, 1980). In many other mammalianspecies ganglion cells may be divided into groups based oncell size, for example the rat (Dreher et al., 1985), rabbit(Provis, 1979; Oyster et al., 1981), tree shrew (Debruyn etal., 1984), monkey (Perry et al., 1984), and man (Stone andJohnston, 1981).

There is some evidence that retinal ganglion cells ofmarsupials may also be divided into three classes. In thecase of the North American opossum, Rapaport et al.(1981a,b) used soma diameter and axon conduction veloci-

Fig. 7. A perimeter chart for an adult tammar wallaby indicatingthe animal’s field of view and degree of binocular overlap. Themonocular field extends from 225° nasal to 1162° temporal in azimuth,

and 270° inferior to 1120° superior in elevation. Binocular overlapcovers 50° in azimuth and 110° in elevation. The location of the opticdisk heads are represented by the two large black dots.

DISTRIBUTION OF WALLABY RETINOGENICULATE CELLS 137

ties to distinguish three classes of cells, whilst Freemanand Watson (1978) used conduction velocity to categorisethree cell types in the brush-tailed possum. In their studyof the quokka, Beazley and Dunlop (1983) concluded on thebasis of soma diameters that the animal may possess threebroad classes of retinal ganglion cells.

The range of diameters of retinal ganglion cells variesconsiderably between classes of marsupial. For instance,those of the South American opossum range from 5 µm to20 µm (Hokok and Oswaldo-Cruz, 1979); the wombat, 11µm to 33 µm (Tancred, 1981); and the quokka, 7 to 24 µm(Beazley and Dunlop, 1983). We found soma diameters inthe tammar ranging from 9 to 30 µm, whereas Wong et al.(1986) reported a similar range from 10 µm to 28 µm.

Anatomical diversity in retinal ganglion cells of thetammar wallaby by Wong et al. (1986) was not as clear-cutas in the cat. Nevertheless, on the basis of cell diameters,they reported the existence of three distinct populations ofganglion cells, after excluding glial cells and displacedamacrine cells, which maintains the possibility that thewallaby retina, like that of the cat, contains three similarfunctional classes.

In our experiments, the morphology of the largest reti-nal ganglion cells suggests that they constitute a uniqueclass. We consider them to be a-type cells. Smaller cells,however, although comprising the majority, were not sepa-rable into different classes on the basis of their morphol-ogy.

We found evidence for the existence of three classes ofretinal ganglion cells on the basis of cell size. In the cubicspline interpolation of cell size data for the contralateralretina, peaks occur at modal values of 15–16 µm, 19–20µm, and 24–25 µm. Cells within these three size categoriesconstitute 25%, 22%, and 4%, respectively, of the totalpopulation. Over 50% of cells fall within the range 10–17µm and about 40% within the range 18–23 µm. Cells at theupper end of the range, 24–30 µm, make up 6–9% of thetotal. These size ranges are larger than those of thequokka, for which Beazley and Dunlop (1983) suggestedthree classes of 7–12 µm, 12–18 µm, and 18–24 µm. Thepercentages of cells in each class in the tammar retina,however, are not dissimilar to the proportions of catganglion cells that have been classified by Wong andHughes (1987) into small (49%), medium (47%), and large(4%).

Our data indicate with some certainty that up to 9% ofretinal ganglion cells projecting to the LGNd are composedof a-type cells, and the remaining neurons probably repre-sent two populations that account for about 40% (medium)and 51% (small) of the total population. These cells may becomparable to b and g categories as found in cat retina.The finding that there were no significant differences incell sizes between ipsilateral and contralateral projectingcells contrasts to Harman and Jeffrey’s (1992) finding forthe quokka in which ipsilateral projecting cells weresignificantly larger.

Field of view and binocular overlap

According to Hughes (1977a), the optic field (‘‘the solidangle subtended at its anterior nodal point by that regionof visual space from which light may be refracted throughthe lens’’) is of relatively constant size in most species,whilst the retinal field (‘‘that portion of its optical fieldwhich is encompassed by the external projections of theretinal margins’’) displays considerable species variation.

In considering an animal’s behavioural range of vision, theretinal field is of primary importance.

Our finding that the perimetric determination of theophthalmoscopically observed limits of the wallaby retinaluniocular field is about 190° may be compared withanimals with frontally placed and laterally placed eyes.For instance, the monocular retinal fields of cat and man(animals with frontal eyes) are 143° and 179°, respectively(Hughes, 1977a), whilst those of the rabbit and rat (ani-mals with lateral eyes) are 192° (Pisa, 1939; Hughes, 1971,1972) and 205°, respectively (Lashley, 1932; Hughes,1977b).

The considerable difference between the visual fields ofanimals with frontal and lateral eyes is even betterillustrated in variations in their cyclopean retinal field,which is defined by Hughes (1977a) as ‘‘that portion of itscyclopean optical field which is encompassed by the exter-nal projection of the retinal margins of either eye.’’ Thecyclopian retinal field extends for 360° in azimuth in therabbit (Pisa, 1939; Hughes, 1971), 320° in both rat (Hughes,1977b) and goat (Hughes and Whitteridge, 1973), and 300°in the grey squirrel (Hall et al., 1971; Lane et al., 1971). Asone might predict, however, the cyclopian retinal field ofanimals with frontally placed eyes is considerably reduced.For example, that of the cat is 186° (Hughes, 1976) andthat of man is 208° (Hartridge, 1919). In the tammar, thecyclopean retinal field encompasses 324°, giving it arelatively wide field of view.

Although both the rat and rabbit may be described ashaving lateral eyes, the extent of their binocular fieldsdiffer considerably. The rat’s binocular field extends over80° in azimuth (Hughes, 1977b), which is surprisinglyclose to that of the cat whose frontally placed eyes give it abinocular field of approximately 98° (Hughes, 1976). Onthe other hand, the rabbit’s binocular field is only 24°(Hughes, 1971), which suggests that in the rabbit a widepanoramic view is more essential than a large binocularfield. The tammar’s binocular field of 50° in azimuthaccounts for about 15% of its total field of view andprovides it with the substrate for good stereopsis.

The upper visual field in man, monkey, and cat isrelatively limited but is comparatively extensive in grounddwelling species such as rabbits and rats (Hughes, 1977a).In common with ground dwelling animals, the area of thetammar’s retina devoted to the upper visual field exceedsthat of the lower field. The elevated optics of grounddwellers are usually associated with aerial predators(Hughes, 1977a), and it seems feasible that the tammar’selevated optics may have evolved from the animal’s needfor protection from birds of prey such as eagles. Elevatedoptics would also be beneficial to the animal when its headis lowered for long periods during grazing.

In their study of the retinotopic organisation of theLGNd of the tammar, Wye-Dvorak et al. (1987) reportedthat the visual field of one eye ranges from 230° nasal to1179° temporal in azimuth and 173° superior to 249°inferior in elevation. Whilst the azimuth measurementsare not dissimilar to our own results, the elevation rangefalls well within the limits of our findings. It is believedthat this discrepancy in elevation is attributable to limita-tions in sampling within the LGNd, because Wye-Dvoraket al. point out that no recordings were made from the mostventral and caudal ends of the LGNd. In a retinotopic mapof the tammar’s striate cortex made from single unitrecordings, Vidyasagar et al. (1992) estimated the visual

138 B.M. WIMBORNE ET AL.

field elevation as 130° superior to 250° inferior. Againthese measurements fall within the limits of our findings,but Vidyasagar et al. reported considerable difficulty infinding units in the superior field above 130°.

Cells of the ganglion layer

Our estimate that the ganglion layer contains a total ofabout 566,000 cells is very similar to the 556,000 calcu-lated by Wong et al. (1986). We assessed one third of theganglion layer to be composed of displaced amacrine cells,which is close to the 35% estimated by Wong et al. (1986).Of the ganglion cell population, about 6% had somataexceeding 20 µm and are considered to be a-type cells. Intheir study of the tammar’s ganglion cell layer, Wong et al.(1986) concluded, on the basis of morphology and size, that4% of cells were ‘‘alphalike.’’ In Table 2, a comparison ismade of estimated retinal cell numbers in several species.

CONCLUSION

The tammar’s visual system appears to be well adaptedto the natural visual environment of grasslands and lowscrub in which it is normally found. The frontolateralplacement of the eyes give the tammar a comparativelywide view of 324°, whereas the existence of a horizontalstreak and an area centralis offer high acuity visionparticularly during panoramic scanning. In addition, itsbinocular field, although not extensive, provides the oppor-tunity for stereoscopic depth perception covering 50° inazimuth and 110° in elevation. The presence of an exten-sive upper visual field, which is common in ground-dwelling animals, may be especially useful during grazingand may be associated with defence against aerial preda-tors. In common with a number of mammals, visualinformation in the tammar seems to be conveyed to theLGNd by means of three classes of retinal ganglion cells,provisionally identified here by the size of the cell body.

ACKNOWLEDGEMENTS

The authors acknowledge the technical and histologicassistance provided by Margaret Porter and the invalu-able advice given by Dr. Lauren Marotte.

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