Choline acetyltransferase is expressed by non-starburst amacrine cells in the ground squirrel retina

Brain Research 964 (2003) 21–30 www.elsevier.com / locate / brainres

Research report

Choline acetyltransferase is expressed by non-starburst amacrine cells in the ground squirrel retina ´ Cuenca a , *, Ping Deng b , Kenneth A. Linberg c , Steven K. Fisher c , Helga Kolb b Nicolas a

´ , Facultad de Ciencias, Universidad de Alicante, 03080 Alicante, Spain Departamento de Biotecnologıa Department of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, UT 84132, USA c Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106, USA b

Accepted 13 November 2002

Abstract We have used immunostaining techniques to reveal a new type of amacrine cell that is immunoreactive for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, in the Ground Squirrel (Spermophilus beecheyi) retina. Cryostat sections and double immunostained wholemount preparations were examined by confocal microscopy. This new ChAT type III cell is distinct in morphology and neurotransmitter content from the well know ‘starburst’ amacrine cells (types I and II) that are so well represented in the ground squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. The type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or exhibit calcium-binding proteins like the well-known starburst type. As well, type III cells do not occur as a mirror-symmetric pair with normally placed and displaced varieties. The type III cell is probably a small field amacrine type branching broadly in upper sublamina b of the inner plexiform layer, and is most likely A6 of the Ground Squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. Type III cells are ideally placed in the architecture of the Ground Squirrel retina to influence ON directionally selective ganglion cell types. Published by Elsevier Science B.V. Theme: Sensory systems Topic: Retina and photoreceptors Keywords: Retina; Amacrine cell; Choline acetyltransferase; Immunostaining

1. Introduction In the vertebrate retina, antibodies to choline acetyltransferase (ChAT) have been used to identify a cholinergic amacrine cell type that is known as the ‘starburst cell’ [4–6,9,16,19,21,23,28–30,32,34,35,37]. These amacrines are densely overlapping in distribution and characterized by very distinctive, radially symmetric dendritic trees. They are organized as mirror symmetric pairs on either side of the inner plexiform layer (IPL). One of the mirror pair has its cell body located in the amacrine cell layer with dendrites in sublamina a (OFF sublamina of the IPL). The other cell of the pair has its cell body displaced to the ganglion cell layer and its dendrites stratify in sublamina b *Corresponding author. Fax: 134-965-909-352. E-mail address: [email protected] (N. Cuenca). 0006-8993 / 02 / $ – see front matter PII: S0006-8993( 02 )04049-0

Published by Elsevier Science B.V.

(ON sublamina of the IPL). These amacrine cells are known to be involved in driving directionally selective ganglion cells in rabbit retina [2,13,18,19,33,38]. Recently the starburst amacrine cells have been implicated in optokinetic eye movements as well [38]. Perhaps just as important as their role in directional selectivity is their role in laying down the architecture of the IPL during development [11,26,31,39,40]. A second type of cholinergic amacrine cell has been reported to exist in some non-mammalian species with cone dominated retinas [7,12,22], although it is questionable in the tree shrew retina [28]. We ask whether the ground squirrel retina, a mammalian cone dominated retina where cones outnumber rods by 7:1 [15], could also bear a population of this second type of cholinergic amacrine cells. We here address this question by performing a confocal microscope study on ground squirrel sectioned or

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wholemounted retinas using immunostaining procedures to reveal ChAT immunoreactive cell types. The results of this paper have been published previously in abstract form [7].

2. Materials and methods

2.1. Immunostaining of wholemount retina Ground squirrel eyes (Spermophilus beecheyi) were used for this study. Animal handling and tissue acquisition protocols followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Nine retinas were used for this study; five were processed for vertical sections and four for wholemount double labeling. The retinas were dissected from ground squirrel eyecups and were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for 2 h and then washed in 0.1 M PB before being cryoprotected in 15% sucrose for 0.5 h, 20% sucrose for 1 h and 30% sucrose overnight at 4 8C. The next day wholemount retinas were put through a freeze–thaw procedure. For wholemount double labeling, the retinas were incubated in 10% normal donkey serum in 0.1 M PB 0.5% Triton X-100 for 1 h at 4 8C. Without washing, the retinas were then incubated in a mixture of two primary antibodies: goat anti-ChAT (Chemicon), rat anti-glycine (David Pow, University of Brisbane, Australia) or rabbit anti-parvalbumin (SWant) each at a concentration of 1:500 in 0.1 M PB 0.5% Triton X-100 for 4 days at 4 8C under shaking. After further washes in PB, secondary incubation took place in a cocktail of donkey anti-rat IgG coupled to fluorescein isothiocyanate (FITC), or donkey anti-rabbit IgG coupled to FITC and donkey anti-goat IgG coupled to tetramethyl rhodamine (TRITC) (Jackson Immuno) for 1 h at a 1:100 dilution, with shaking at room temperature. For single labeling, we used goat anti-ChAT (Chemicon) and donkey anti-goat IgG coupled to FITC as the secondary antibody. The retinas were washed, mounted, and coverslipped for viewing by confocal microscopy.

2.2. Cryostat sections and immunofluorescence techniques Fixed retinas were cryoprotected in sucrose overnight. Next day they were embedded in OCT and cut into 12-mm thick radial sections on a cryostat, and mounted on glass slides. Cryostat sections were double or single immunostained for combinations of different antibodies. Sections were incubated overnight, under agitation, in a mixture of two primary antibodies with goat anti-ChAT (Chemicon) as the one. Thus we used mouse anti-calbindin (Sigma), rabbit anti-calretinin (SWant), rabbit anti-parvalbumin (SWant) and rabbit anti-GABA and rat anti-glycine (gifts or David Pow, Brisbane, Australia) with the anti-ChAT.

They were then washed in 0.1 M PB and transferred to a cocktail of the secondary antibodies donkey anti-rat IgG coupled to FITC, and donkey anti-mouse IgG coupled to TRITC, donkey anti-rabbit IgG coupled to FITC, donkey anti-goat IgG coupled to TRITC. All secondary antibodies were used at a 1:100 dilution in 0.1 M PB 0.5% Triton X-100 for 1 h. Finally the sections were washed in 0.1 M PB, mounted in watermount (Vector Labs) and coverslipped for viewing under confocal microscopy (Zeiss LSM 510). TIFF images were enhanced using Adobe Photoshop software and printed on an Epson Photoquality printer. Omitting the primary antibody in the immunostaining procedures was done as a control. No labeling was ever found.

3. Results

3.1. ChAT immunoreactivity In cryostat vertical sections ChAT-IR was seen in three types of amacrine cells differing from each other in cell body position, dendritic stratification and overall distribution in the IPL (Fig. 1A). Type I cells had bodies in the amacrine cell layer, on the border between the inner nuclear layer (INL) and the IPL. Their cell body diameters were 9.5360.21 mm (mean and standard deviation), their primary dendritic processes were very fine (Fig. 1A, arrows) and the overlapping of their dendrites formed a prominent band in the outer portion of the IPL in sublamina a (Fig. 1A). Type II displaced cells had slightly larger cell bodies, with diameters of 10.4960.81 mm, and were found in the ganglion cell layer (GCL). Their dendritic branching made another thick plexus in the inner part of the IPL in sublamina b (Fig. 1A). Fig. 1B shows the displaced ChATIR amacrine cells in wholemount view. Arrows indicate the fine primary dendrites emerging from the cell body (Fig. 1B arrows). Both normally placed and displaced type I and II cells correspond to ‘starburst’ amacrine cells and will be called starburst cells from here on in this paper. Type III cells had their cell bodies in the middle of INL, above the type I starburst cell bodies (Fig. 1A). Their bodies were smaller (8.8460.12 mm diameter) and their dendrites stratified in the middle of the IPL. It was difficult to follow discrete dendrites from type III cell bodies into the IPL, but the small beads in the middle of the IPL are presumed to belong to them. Because there were no dendrites arising from these cell bodies going to the outer plexiform layer and because of their large cell body size as compared with bipolar cells, we definitely consider them amacrine cells. In wholemount views of ChAT-IR labeling, these three cell types and their plexi are easy to identify. The

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Fig. 1. (A) Vertical cryostat section of ground squirrel retina immunostained with antibodies to ChAT. Three types of ChAT-IR cells can be seen. Type I normally placed ‘starburst’ cells lie in the amacrine cell layer and have fine dendrites leaving the cell body (arrows) to join the thick plexus of processes on the S1 / 2 border of the IPL. Type II displaced ‘starburst’ cells are in the ganglion cell layer. Type III cells lie in the INL and contribute fine dendrites (arrowheads) to the middle broad area of beaded processes in the S3–S4 area of the IPL. (B) Wholemount view of ChAT-IR displaced ‘starburst’ cells. They have dendrites leaving the cell body (arrows). Scale bar 20 mm.

QuickTime movie (in the online version of this paper)1 and Fig. 2 show ChAT-IR cells in through focus at different levels of the same retinal area. Thus two populations of ChAT-IR amacrine cells can be identified in the INL. Fig. 2A shows the starburst cells in the amacrine cell layer, with their overlapping, beaded processes forming a dense plexus in sublamina a of the IPL (Fig. 2B) leaving holes to allow other processes to pass through. Focusing in the middle of the INL (Fig. 2C), type III ChAT-IR cells can be distinguished. Type III cell bodies are larger than the surrounding cells (mostly bipolar cells). Their terminals can be seen as a broad band in the middle of the IPL (Fig. 2D). In the GCL, displaced starburst cells are visible (Fig. 2E). Their dense and thick plexus (Fig. 2F) formed of their overlapping beaded processes, also has large voids in it, like the normally placed starburst cells’ plexus. Three ChAT-IR bands were clearly seen in the IPL in Fig. 1A. Fig. 3 shows a plot of the intensity of the ChAT immunoreactivity in the IPL, as seen by the plot profile utility (NIH Image program). At the most distal level of the IPL, a prominent band extends from 10 to 30% of the total 1

Legend for the quicktime movie: The QuickTime movie shows ChAT-IR cells in through focus at different levels of the same wholemount retinal area starting at the GCL and ending in the INL. Displaced starburst (type II cells) cell bodies come into view in the GCL and have a dense and thick plexus formed of their overlapping beaded processes in lower IPL. The plexus has large voids in it. Focusing further in the middle of the IPL, type III ChAT-IR cell dendrites can be distinguished as a broad band of beads. The starburst cells (type I cells) have overlapping, beaded processes forming another dense plexus, leaving holes in it to allow other processes to pass through, in sublamina a of the IPL. Type I starburst cell bodies appear in the lower INL and then the new type III cells become visible. The small inset shows the approximate plane of focus in a vertical section as the through focus movie proceeds.

thickness of the IPL (defining 0% as the border of the INL / IPL and 100% as the border of the IPL / GCL). This plexus corresponds to the dendritic branching of the normally placed starburst amacrine cells on the strata S1 / 2 border. The second prominent band extends from 70 to 90% (S4 / S5 border) corresponding to the dendritic overlapping of displaced starburst amacrine cells. At the 45– 65% level, a diffuse band of immunoreactive spots is seen (Fig. 3). Occasionally some processes arising from the cell body of type III amacrine cells can be seen to cross levels 10–40 to make this punctate plexus at the 45–65% level (Fig. 3).

3.2. Density counts The topography of ChAT-IR amacrine cell distribution was evaluated in retinal wholemounts. The density of normally placed starburst amacrine cells peaks at 4100 cells / mm 2 in temporal retina, beneath the optic nerve head, and decreases towards the periphery, with minimum densities of 800 cells / mm 2 in superior periphery and 1100 cells / mm 2 in inferior peripheral retina (Fig. 4A). The topography and distribution of the displaced starburst cells is almost identical, with peaks in the temporal retina (4700 / mm 2 ) and minima in superior peripheral retina of 800 / mm 2 (Fig. 4B). The density plots for type III cells show a very similar distribution. Again, the peak density is in the temporal central streak area of the retina, corresponding to the density peak of cones at 2 mm from optic nerve head described by Kryger et al. [15]. Like starburst cells, the fall off in density occurs more rapidly in the superior retina than in the inferior retina (Fig. 4C).

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Fig. 2. Wholemount views of two focal levels of ChAT-IR cells. (A, B) Normally placed starburst cell bodies in the INL and plexus of dendrites on the S1 / 2 border of the IPL. (C, D) Type III cells above the starburst cells in the INL with their diffuse dendritic branches in S3–S4 of the IPL. (E, F) Displaced starburst cells in the ganglion cells layer and their dendritic plexus on the S4 / 5 border of the IPL. Scale bar: 20 mm.

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Fig. 3. Fluorescence intensity profile of the dendritic plexi of the ChAT-IR cell types as seen in vertical view of the IPL. Normally placed and displaced starburst cells have intense plexi in S1 / 2 and S4 / 5 of the IPL, respectively. The less fluorescent diffuse beaded arrangement of the type III cell dendrites span the S3 to S4 levels of the IPL.

3.3. Colocalization Fig. 5 shows colocalization of ChAT-IR cells with the

calcium-binding proteins calbindin (CB), calretinin (CR), parvalbumin (PV) and the inhibitory neurotransmitters GABA and glycine in cryostat vertical sections.

Fig. 4. Density maps of the ChAT-IR cell bodies counted throughout a wholemount retina. (A) Densities of normally placed ChAT starburst amacrine cells. (B) Densities of displaced ChAT starburst amacrine cells. (C) Densities of type III ChAT amacrine cells. The peak density for all three cell types is at the temporal central area of the visual streak. The line above is the elongated optic nerve head in all cases.

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CB-IR was seen in horizontal cells, some bipolar cells and at least two types of amacrine cells in the INL. In the GCL, some cells were immunostained with CB. Double labeling with anti-ChAT shows colocalization in one type of the weaker CB-IR amacrine cells in the INL, corresponding to normally placed starburst amacrine cells (Fig. 5A, arrows, yellow). Because all the CB-IR cells in the GCL colocalize with ChAT, we identified this population as displaced starburst amacrine cells and not ganglion cells (Fig. 5A, arrowheads, yellow). Both bands of ChAT-IR dendrites in the IPL were double labeled, confirming the double label of ChAT cells with CB. ChAT-IR type III cell bodies and their dendrites in the middle of the IPL do not colocalize CB (Fig. 5A, curved arrows, red). Double labeling with antibodies against ChAT and PV indicates that starburst amacrine cells and their corresponding plexi are stained with both antibodies (Fig. 5B, yellow). However, type III ChAT cells only stain for ChAT (Fig. 5B, red cells). Fig. 6C also shows a wholemount view of normally placed starburst ChAT cells colocalizing PV. Immunostaining vertical sections for ChAT and CR shows CR label in horizontal cells, bipolar and many subtypes of amacrine cells, but none of them colocalize ChAT. The ChAT-IR starburst cells and both of their plexi exhibit a small to minimal amount of CR-IR (Fig. 5C and D). The type III cells exhibit no CR-IR at all (Fig. 5C and D). In double labeling experiments for GABA and ChAT, we found double label in both normally placed and displaced starburst amacrine cells, and in their corresponding bands in the IPL (Fig. 5E, yellow). ChAT-IR amacrine cells in the middle of the INL, i.e. type III cells do not colocalize GABA (Fig. 5E, red). Vertical sections processed for glycine and ChAT show starburst amacrine cells and both of their plexi faintly colocalizing glycine (Fig. 5F, orange). On the other hand, ChAT-IR type III cells and processes in the middle of the INL are strongly co-labeled with both antibodies (Fig. 5F, yellow, arrows). Wholemount views show the glycine / ChAT colabeling even more clearly. Focusing on the amacrine cell layer in the INL, we found several subtypes of glycinergic amacrine cells. One population expresses strong immunoreactivity for glycine (Fig. 6A, intense green) and another shows much weaker immunoreactivity (Fig. 6A, mottled green). Glycine-IR does indeed colocalize with ChAT in the the starburst type I amacrine cells

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(Fig. 6A). However, in the middle of the INL several cells types express strong glycine and colocalize ChAT (Fig. 6C). They are the population of ChAT-IR type III cells (Fig. 6C). Other small glycine labeled cell bodies could be bipolar cells (Fig. 6C). In the GCL, all the cells containing weak glycine were also identified as the displaced starburst type II amacrine cells. (Fig. 6B).

4. Discussion In this paper we have been able to demonstrate that a new type of amacrine cell appears to be immunoreactive for choline acetyltransferase, the synthesizing enzyme for acetylcholine. This new amacrine, here called the ChAT type III cell, is distinct in morphology and neurotransmitter content as compared with the heretofore well know ‘starburst’ amacrine cells (types I and II) that are so well represented in the ground squirrel retina [16]. The new type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or immunostain for any of the calcium-binding proteins as do the well-known starburst types. Type III cells do not come as a mirrorsymmetric pair with normally placed and displaced varieties. The type III cell is probably a small-field amacrine cell branching broadly in upper sublamina b of the IPL.

4.1. GABA and glycine in cholinergic amacrine cells In most retinas studied to date there is good evidence that starburst cholinergic cells synthesize, store and release the inhibitory neurotransmitter GABA as well as the excitatory transmitter acetylcholine [3,8,14,20,24,25,36]. A prominent difference between the new type III cell and the starburst cell is that the former contains no GABA but instead has a high affinity for glycine. The coexistence of two classical transmitters, one excitatory and the other inhibitory, in any retinal neural type was quite novel in the 1980s but is now considered almost the general rule. It has become apparent that different transmitters may be packaged differently in the various neurons and have separable actions upon postsynaptic dendrites by virtue of different postsynaptic receptor sites. It has been proposed that GABA released by the starburst amacrine cells influences the release of acetylcholine from these cells themselves through GABA B autoreceptors [41,42]. In terms of the action of the new glycinergic / cholinergic type III cells,

Fig. 5. (A) Double immunostaining for ChAT (red, rhodamine) and calbindin (CB) (green, fluorescein). CB locates in horizontal cells, bipolar cells and many amacrine cells. Double labelling occurs only in starburst amacrine cells and their plexi. (B) Double immunostaining for ChAT (red) and parvalbumin (PV) (green). Only the starburst cells and their plexi are double labeled. Red ChAT type III cell bodies remain only ChAT labeled. (C, D) Double label for ChAT (red) and calretinin (green). Horizontal, bipolar, amacrine and ganglion cells label for CR. Starburst amacrine cells contain weak CR-IR, and type III cells do not contain CR at all. (E) Double labeling for ChAT and GABA. The starburst cells contain both labels (yellow cells and plexi), whereas the type III cells do not contain GABA at all (red cells and middle diffuse red dendrites in the IPL). (F) Double labeling for ChAT and glycine. Note that the starburst cells label faintly with glycine (orange cells and plexi), but the type III cell bodies label intensely for glycine (yellow cell bodies in the INL). Scale bar: 20 mm.

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Fig. 6. Wholemount views of double labeling of the retina with ChAT (red, rhodamine) and glycine (green, fluorescein). (A) Weak double labeling occurs in the starburst cells at the level of the amacrine / IPL border of the INL. (B) Weak double labeling occurs in displaced starburst cells in the ganglion cell layer. (C) At a level in the middle of the INL some cells double label for ChAT and glycine. They are type III ChAT cells. Many small bipolar cells are glycine-IR. (D) Wholemount view of double labeling with ChAT (red) and parvalbumin (green) at the amacrine cell layer level. Normally placed starburst amacrine cells contain both labels (yellow). Scale bar: 25 mm.

these could be well situated so as to influence ACh release from both varieties of starburst cells in a manner similar to the proposed role for the glycinergic DAPI-3 cells of the rabbit retina [42]. Here we have also been able to see faint glycine and strong GABA in the common starburst amacrines of the ground squirrel. This finding complicates even further the theory of GABA and ACh interactions for the final release of ACh on the recipient ON–OFF directionally selective

ganglion cell. Both inhibitory transmitters are also seen in the mirror symmetric cholinergic amacrine cells in the salamander retina [8]. Interestingly, in the tree shrew, which bears a cone dominant retina like the ground squirrel, Sandman et al. [28] could not demonstrate GABA in the starburst cells at all. We suggest that these and other species need be reinvestigated for colocalization of all these transmitter candidates in order to sort out these disparate findings.

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4.2. Type III cells occur in diurnal species with a visual streak organization of the retina Type III cholinergic cells have now been described in birds, reptiles, and the cone dominant retina of the ground squirrel [7,12,22]. All these species are highly diurnal. The normal mirror symmetric starburst amacrine cells are particularly well developed in these species. Although the rabbit, in which these starburst cells were first described [9], is also a diurnal species, its retina has a greater rod population and rod pathway development than does the retina of a bird, reptile or ground squirrel. So the correlation of cholinergic cells with diurnality is not the whole story, particularly when taking into account diurnal primates. Monkey and human retinas contain cholinergic starburst cells, but they are not of as high a density or comparable dendritic complexity as those of rabbit or ground squirrel retinas [27]. However, diurnal primates have a foveate retinal organization, and it appears that starburst cell populations are a speciality of diurnal species, mammalian or non-mammalian, that have a visual streak based retinal topography. So the commitment of a large cholinergic amacrine population correlates better with visual streak based retinas than with diurnality. Along with the visual streak, of course, goes the development of directionally selective ganglion cell physiology. It may be no accident that these retinas also have need for further cholinergic amacrine populations above and beyond the starburst type, and hence have developed the type III population. We think that it is highly likely that the rabbit retina also has a type III population that hitherto has remained undetected.

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amacrine cells can be further segregated based on their expression of somatostatin, which identifies some of the type III cholinergic amacrines [10]. In the squirrel retina, somatostatin-IR amacrine cells are evenly distributed throughout the retina, have their cell bodies in the innermost portion of the INL, and form three bands of dendrites of similar width in strata S1, S3 and S5 [17]. In contrast, we have seen that type III ChAT cells have a peak density in the temporal central streak area of the retina, their cell bodies are in the middle of the INL, and their dendrites branch in strata S3 and S4. Therefore, squirrel type III cholinergic amacrine cells are almost certainly a different cell type from the somatostatin-IR amacrine cells. Type III amacrine cells branch in the center of the IPL, in a position to have synaptic input to ganglion cells G1, G2, G8 and G9 of the ground squirrel retina [16]. None of these ganglion cells correspond to the ON–OFF DS ganglion cell of ground squirrel, which by homology to the rabbit ON–OFF DS cell [1] is probably G11. G11 has a bistratified dendritic tree matching the levels of the two starburst cells dendritic plexi. However, the new type III cholinergic amacrine cell is in an ideal position to synapse upon the ON DS ganglion cell type which, in rabbit at least, stratifies precisely in S3 of the IPL [1]. The ground squirrel morphological equivalent of the ON–DS ganglion cell of rabbit is probably G8 [16]. So in conclusion, although we have no information on the light responses and correlative morphological labeling of any ground squirrel ganglion or amacrine cells, it appears that once again a putative cholinergic amacrine cell type is precisely balanced in the architecture of the IPL to influence another directionally selective ganglion cell, in this case the simple ON–DS ganglion cell.

4.3. Role of the cholinergic type III amacrine cell Present knowledge on the function of these type III ChAT amacrine cells is scarce, given that their existence in the retina of different species is beginning to be uncovered. Yet we can begin to speculate how they might interact in the neuropil of the IPL because of their dendritic branching pattern. Type III cells are broadly stratified with dendrites primarily in sublamina b from S3 through S4 (Fig. 3). This means that in all likelihood type III cells are ON type amacrines in physiological response to light. Although they would be in a branching position to receive some synaptic input from the deepest branching OFF bipolar cells they are more likely to receive their major bipolar input in strata 3 and 4 from ON center bipolar cells. In morphology, the type III cholinergic / glycinergic amacrine cell is most similar to the A6 cells described by Linberg et al. [16]. A6 cells have a dendritic tree span slightly smaller than starburst cells, called A5a and b, in ground squirrel [16]. This fits well with their population density, which we discovered here, to be about the same as one or the other of the ‘starburst cell’ populations (Fig. 4). In the chick retina, it has been shown that non-starburst

Acknowledgements Supported by Research to Prevent Blindness to HK and DGESIC PB98-0972 to NC.

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