THE JOURNAL OF COMPARATIVE NEUROLOGY 313:394-408 (1991)

GABA-Like Immunoreactive Cells Containing Nicotinic Acetylcholine Receptors in the Chick Retina D.E. HAMASSAKI-BRITTO, A. BRZOZOWSKA-PRECHTL, H.J. KARTEN, J.M. LINDSTROM, AND K.T. KEYSER Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0608, (D.E.H.-B., A.B-P., H.J.K., K.T.K.) and Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6142 (J.M.L.)

ABSTRACT The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 pm. Some double-labeled cells ranging from 7-21 pm were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells. Key words: ganglion cells, amacrine cells, neurotransmitters, receptor subunits

Gamma-aminobutyric acid (GABA) and acetylcholine (ACh) are both known to act as neurotransmitters in the vertebrate retina. The presence of GABA in different cell types of the inner nuclear layer (INL) and ganglion cell layer (GCL) of the vertebrate retina has been demonstrated by means of (1) autoradiographic studies employing (3H)GABA or (3H)-muscimoluptake, (2) immunohistochemical localization of the GABA synthesizing enzyme (L-glutamic acid decarboxylase, GAD) or of GABA itself (for a review see Yazulla, '86; see also Agardh et al., '86; Mariani and Caserta, '86; Mosinger et al., '86; Agardh et al., 87a,b; Ball, '87; Ryan and Hendrickson, '87; Glasener et al., '88; Yu et al., '88; Wassle and Chun, '88, '89; Caruso et al., '89, '90; Hurd and Eldred, '89; Pourcho and Owczarzak, '89; Shelton et al., 'go), and (3)in situ hybridization with a probe for GAD mRNA (Sarthy and Fu, '89a,b). The presumptive cholinergic neurons in the retina have also been extensively studied. Autoradiographic experiments with radioactive choline uptake (Baughman and Bader, '77; Masland and Mills, '79; Masland et al., '84), and immunohistochemical experiments with antisera against the rate-limiting enzyme in the synthesis of ACh, choline D

1991 WILEY-LISS, INC.

acetyltransferase, have consistently revealed labeled amacrine cells in the INL, and "displaced" amacrine cells in the GCL in the retina of several vertebrates (Eckenstein and Thoenen, '82; Eckenstein et al., '83; Ma and Grant, '84; Tumosa et al., '84; Millar et al., '85; Conley et al., '86; Voigt, '86; Spira et al., '87). Although these methods have made it possible to recognize the putative cholinergic neurons in the retina, the postsynaptic targets have been more difficult to characterize. Until recently, the autoradiographic localization of radioactive a-bungarotoxin was the primary method employed to detect nicotinic acetylcholine receptors (nAChRs). However, growing evidence has indicated that the a-bungarotoxin binding patterns do not represent an exact portrayal of the distribution of nAChRs in the nervous system (Whiting and Lindstrom, '86, '87). Thus the recent availability of antibodies directed against nAChRs (Whiting and Iindstrom, '86, '87; Lindstrom et al., '87; Whiting et al., '87, '911 has provided a considerable advance ~

Accepted August 20,1991. Address reprint requests to D.E. Hamassaki-Britto, Dept. of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0608.

NICOTINIC RECEPTORS IN GABA-IMMUNOREACTIVE CELLS in studying these receptors, as these antibodies do not bind bungarotoxin-binding proteins. Using antibodies against nAChRs, Keyser et al. ('88) described three distinct groups of cells in the chick retina exhibiting nAChR immunoreactivity: amacrine cells, ganglion cells, and displaced ganglion cells. Physiological studies have suggested the involvement of both GABA and ACh in the formation of complex receptivefield properties of ganglion cells (Masland and Ames, '76; Masland and Livingstone, '76; Caldwell et al., '78; Ariel and Daw, '82a,b; Daw et al., '89). Additional information has indicated that GABA has some effects upon the cholinergic system. For example, GABA agonists inhibit the lightevoked release of ACh from the retina (Massey and Neal, '79; Massey and Redburn, '82; Cunningham and Neal, '83; Massey and Redburn, '85, '87). Colocalization of GABA and ACh in amacrine and displaced amacrine cells has been recently described in mammals (Brecha et al., '88; Chun et al., '88; Kosaka et al., '88; Vaney and Young, '88). The equivalent studies have not yet been carried out in the avian retina. However, most of the morphological basis for the interaction between the cholinergic and GABAergic systems remains to be determined. Scant information is available concerning either the possible direct synaptic contacts of GABAergic cells onto cholinergic cells or the other presumptive pathways involved in the GABAergic modulation of the cholinergic system. Furthermore, it is not known whether the cholinergic system can reciprocally modulate the GABAergic system by means of direct or indirect connections of ACh-containing neurons with GABAergic neurons. The recent availability of antibodies directed against both nicotinic receptors and GABA has facilitated this type of investigation, and additional information about the retinal circuitry has been obtained employing this technique (Mariani et al., '87; Richards et al., '87; Keyser et al., '88; Hughes et al., '89). The present study was undertaken to investigate the possible presence of nAChRs in GABA-containing neurons of the chick retina. A monoclonal antibody against the a-3 ACh-binding subunit of the nAChR (mAb 315, Whiting et al., '91) was used to identify the presumptive cholinoceptive cells.

MATERIAL AND METHODS Retinae from seventeen 1-14-day-old white leghorn chicks (Gallus gallus) were used in this study. The animals were sacrificed with an overdose of ketamine (Parke-Davis) and xylazine (Haver), and their eyes rapidly removed. After cutting away the anterior pole and vitreous, the eyes were immersed in ice-cold 2% paraformaldehyde in 0.1 M sodium phosphate-buffered saline at pH 7.4 (PBS). Three hours later, the tissue was placed in a solution of 30% sucrose in PBS for at least 12 hours. The eyes were then frozen in embedding medium, cut perpendicularly to the vitreal surface (transverse sections) on a cryostat (10-ym sections), and collected on gelatin-coated glass slides. In some cases, the retinae were flattened, frozen, and cut parallel (20-ym sections) to the vitreal surface (horizontal sections) on a sliding microtome. The retinal sections were then processed for GABA or nAChR immunohistochemistry according to indirect fluorescence and avidin-biotin-peroxidasetechniques. The sections were washed in PBS for 30 minutes and then incubated with the primary antibodies. The primary antibodies

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consisted of either a rat monoclonal antibody against nAChR (mAb 315, Whiting et al., '91) diluted 1:lOO-1: 2,000, or a commercially available guinea pig antiserum against GABA (Eugenetech) diluted 1500-1:2,000. The latter antiserum was made against an hemocyanin conjugate and did not require glutaraldehyde as a fixative. The antibodies were diluted in PBS containing 0.3% Triton X-100, and sections were incubated for 12-24 hours at 4°C. After a 30-minute wash in PBS, the tissue was incubated with the secondary antibodies for 1hour at room temperature. The rabbit antirat IgG antisera (Jackson Labs) for nAChR, or goat antiguinea pig IgG antisera (Jackson Labs) for GABA were diluted 1:lOO-1:200 in PBS with Triton X-100. They were either biotinylated (avidin-biotin-peroxidase technique) or labeled with dichlorotriazinyl amino fluorescein (DTAF, fluorescence technique). For the fluorescence technique, the sections were subsequently washed in PBS for 30 minutes and coverslipped with a mixture of carbonate buffer and glycerin (9:1).For the avidin-biotinperoxidase technique, there was an additional step. After washing in PBS for 30 minutes, the sections were incubated in a mixture of biotin and avidin-peroxidase (ABC Elite Kit, Vector Labs) diluted 1 : l O O in PBS with Triton X-100 for 1 hour. Following a 30-minute wash in PBS, the tissue was incubated with 0.05% 3,3'-diaminobenzidine in PBS for 15 minutes. A 0.3%solution of hydrogen peroxide in distilled water was then added to make a final concentration of 0.01%. The reaction was allowed to proceed for 15 minutes. The tissue was then washed in 4 changes of PBS and coverslipped with a mixture of carbonate buffer and glycerin. A double-labeling fluorescence technique was also used that allowed the visualization of GABA-like and nAChRlike immunoreactivities (GABA-LI and nAChR-LI, respectively) in the same section through the use of two different fluorophores. The same immunofluorescence protocol as mentioned before was used for these double-labeling experiments, except that the tissue was incubated with a combination of GABA antiserum and the nAChR antibody in the same dilutions described above. A mixture of secondary antisera was then applied, containing (1)goat antiguinea pig IgG conjugated to DTAF, and (2) rabbit antirat IgG conjugated to tetramethyl rhodamine isothiocyanate (TRITC). In some cases, the fluorophores were reversed. Controls for the specificity of labeling included (1)omission of the primary antibodies, and (2) substitution of the primary antibodies with either rat (for nAChR) or guinea pig (for GABA) normal sera. Specific staining was abolished under each of these conditions. In addition to these controls, we also attempted to determine whether the GABA-LI neurons in the chick retina also contained GAD-LI. These double-labeling experiments were conducted with a sheep antiserum against GAD (kindly donated by Dr. W. Oertel; see Oertel et al., '81) and the procedures described above. The dilution of anti-GAD was always 1:1,000 and the dilution of the antisheep secondary (labeled with DTAF or TRITC) was 1 : l O O . Rhodamine-labeled microspheres (Luma Fluor Inc.; Katz et al., '84) were injected into the optic tectum of two chicks. The animals were anesthetized with ketamine (5 mg/100g of body weight, i.m.) and xylazine (1mg/100g, i.m.1, and a local anesthetic was injected under the scalp. The skull was opened and approximately 1 yl of rhodamine beads (1:5 in PBS) was injected through a Hamilton syringe into the dorsal optic tectum. Sterile bone wax was placed over the

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Fig. 1. Photomicrographs illustrating the distribution of GABA-LI cells in the chick retina (avidin-biotin-peroxidase technique). A. Transverse section of the central retina illustrating labeled somata in the outer third and inner third of the INL, and also in the GCL. B.

Horizontal section through the INL in the retina with the focus on a specific population of GABA-LI amacrine cells. C. Horizontal section of the retina through the GCL. Some cells in this layer can be observed giving rise to axon-like processes. Bars: A, 30 km; B, C, 15 pm

damaged area of the skull and the scalp was sutured. After 4 days, the chick was given an overdose of ketamine and xylazine, and the eye contralateral to the injection site was removed, fixed, and processed for GABA and nAChR immunofluorescent detection techniques, as described above. In these cases, secondary antisera conjugated to either DTAF acid (AMCA) were or 7-amino-4-methylcoumarin-3-acetic employed that permitted a triple-labeling visualization in the same section. Sections were examined with a microscope fitted for epifluorescence microscopy and differential interference microscopy. The density of the labeled cells in the INL and in the GCL was determined in photomicrographs taken from different regions of horizontally-sectioned retinae (each representing 100,000 p,m2).The percentage of stained cells was obtained by comparing the density of GABA-LI,

nAChR-LI, or double-labeled cells in relation to the density of cells in the same regions of a horizontally sectioned retina stained for Nissl. The counts were made mainly in retinae processed for indirect fluorescence technique to permit comparison to cell counts in the double-labeling experiments. However, counts for GAl3A-LI and nAChR-LI were also performed in a few retinae processed for avidinbiotin-peroxidase technique. Cell sizes were measured in photomicrographs taken from different regions of horizontal retinal sections and compared to calibration marks.

RESULTS The antibodies against GABA and nAChR employed in this study labeled specific groups of cells in the INL and GCL of the chick retina. GABA-LI cells in the outer third of

NICOTINIC RECEPTORS IN GABA-JMMUNOREACTIVE CELLS

Fig. 2. Two fluorescence photomicrographs of the same transverse section of the chick retina processed for GABA (A) and GAD (B) immunoreactivities. Examples of double-labeled cells (arrows) containing both GABA-LI (A) and GAD-LI (B) are observed in the INL and in

the INL, and their processes, which extended into the outer plexiform layer (OPL) , were consistently negative for nAChR-LI. In contrast, many cells located in the inner third of the INL and those in the GCL exhibited GABA-LI, nAChR-LI, or both. Considering the high variability in the intensity of staining, a conservative approach was adopted for the cell counts. Only the cells that stained well above background were included. Therefore, the cell counts below may represent an underestimation of the actual number of positive cells. Counts made in retinae processed with the avidin-biotin-peroxidase protocol showed higher numbers when compared to the counts made in material processed with the fluorescence technique. This is probably due to the fact that the latter technique is less sensitive. However, no qualitative differences were observed in the distribution of GABA-LI and nAChR-LI cells in retinae processed with either protocol. All the numbers considered in the present study were obtained in retinae processed with the fluorescent technique, since all the double-labeled cell counts had to be conducted in material processed with the fluorescence protocol.

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the GCL. Cells stained only for GABA-LI can also be observed. In this case, GABA immunoreactivity was visualized with DTAF and GAD immunoreactivity with TRITC. Bar: 30 pm.

Distribution of GABA-LI Several cell types in the chick retina exhibited GABA-LI. Labeled round cells were consistently observed in the outer third of the INL (Fig. la). Based on their location and the fact that their dendrites entered the OPL, they were tentatively identified as horizontal cells. These cells were not studied in detail. Round to oval-shape GABA-LI somata, presumably amacrine cells, were observed in all rows of the inner third of INL (Fig. la,b). Thus this distribution possibly also contributed to the underestimated counts of GABA-LI cell density, since these counts were made in horizontal sections of the INL. Approximately 3.5-5.0% of all cells in the inner third of the INL throughout the retina showed GABA-LI. No significant differences in cell densities (750-1,260 cells/mm2) were observed between the central and peripheral retina, although there was some bias toward lower densities in the extreme periphery. Soma sizes of the GABA-LI amacrine cells ranged from 5-12 km in the major axis, and they tended to be slightly larger in the periphery (mean -r- standard deviation, 7.7 5 1.4 km) than in the center of the retina (7.2 & 1.2 pm). In a few

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Fig. 3. Photomicrographs illustrating the distribution of nAChR-LI cells in the chick retina (avidin-biotin-peroxidase technique). A. Transverse section of the central retina showing labeled somata in the inner third of the INL and in the GCL. Processes from a ganglion cell can be followed arborizing in lamina 2 of the IPL. Three distinct labeled laminae are visible in this figure. B. Horizontal section through the INL

in the peripheral retina. Besides staining small amacrine cells, the nAChR antibody intensively labels large somata of presumptive displaced ganglion cells (arrow). c.Horizontal section through the GCL in the peripheral retina, where cell bodies of different sizes and proximal processes can be seen containing nAChR-LI. Bars: A, 70 pm; B, 40 pm; C, 30 pm.

instances, large cells (up to 15 pm) in the inner third of the INL were weakly labeled with GABA-LI. However, this staining was very inconsistent, and therefore these cells were not considered in this study. The high intensity of GABA-LI stainingin the inner plexiform layer (IPL) extending from the outermost to the innermost lamina precluded the identification of the dendritic arborization pattern of most individual amacrine cells. A layered organization was difficult to distinguish in the IPL, although in some cases a poorly defined lamination that included laminae 1 , 2 , 4 ,and 5 was detectable. A few cell bodies exhibiting GABA-LI were seen in the middle of the IPL. The GABA-LI cells in the GCL were present in lower numbers and exhibited less intensely stained somata than

those in the INL (Fig. la,c). About 0.8-1.9% of all cells in the GCL exhibited GABA-LI. Their soma sizes ranged from 7 to 21 pm. Larger cell bodies were observed in the periphery (12.6 t 2.0 pm, up to 21 pm) as compared to those in the center (11.1 & 1.8 Fm, up to 17 pm). These cells were distributed throughout the retina (63-225 cells/ mm2)and tended to occur in lower densities in the temporal region. In horizontal sections that included the GCL and the optic fiber layer, it was possible to identify axons that emerged from labeled somata (Fig. lc). Some of the GABA-LI cells in this layer were retrogradely labeled after the injection of rhodamine beads into the optic tectum. Although these data indicate that at least some of the GABA-positive cells in the GCL were ganglion cells, we

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Fig. 4. Two fluorescence photomicrographs of the same transverse section of the retina processed for GABA (A) and nAChR (B) immunoreactivities. These panels illustrate examples of doubly-labeled cells (arrows) containing both GABA-LI (A) and nAChR-LI (B) in the INL

and in the GCL. Cells stained exclusively for GABA-LI (A), and not for nAChR-LI (B), or vice versa are observed as well. In this case, GABA immunoreactivity was visualized with DTAF and nAChR immunoreactivity with TRITC. Bar: 35 pm.

cannot exclude the possibility that some of the stained perikarya in the GCL were displaced amacrine cells. Both the antisera against GABA and GAD stained groups of cells located in the INL and GCL of the chick retina. A higher number of cells exhibited GABA-LI as compared to those containing GAD-LI. Double-labeling experiments showed that most, if not all, GAD-LI cells also contained GABA-LI (Fig. 2). Although the opposite was not true (many GABA-LI cells did not exhibit GAD-LI),these results indicated that the GABA antiserum used in this study labeled basically the same classes of neurons that contained GAD-LI.

Distribution of nAChR-LI The pattern of labeling that resulted from staining with antibody mAb315, which was directed against nAChR a3 ACh-binding subunits, resembled the pattern seen after staining with either of two antibodies against structural subunits of nAChRs (mAb 210 and mAb270; Keyser et al., '88). Stained neurons were seen in both the INL and GCL (Fig. 3). Large, intensely stained neurons were observed in the innermost portion of the INL. They were not studied in detail, but Keyser et al. ('88) have shown that most of them are displaced ganglion cells (Fig. 3b). Another group of cells

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Fig. 5. Two fluorescence photomicrographs of the same tangential section through the IPL and INL in the peripheral retina. The GAl3A (A) and nAChR (B) irnrnunoreactivities were visualized with TRITC and DTAF, respectively. Examples of double labeled amacrine cells containing GAl3A-LI and nAChR-LI are shown (arrows). Bar:25 krn.

in the INL exhibited less intense nAChR-LI (Fig. 3a,b). These presumptive amacrine cells were distributed almost homogeneously throughout the retina (830-1,250 cells/ mm'), although a slight tendency to higher densities in the central region was observed. Their range of sizes appeared slightly smaller in the center (5-10 pm, 6.2 2 0.9 pm) than in the periphery (5-12 pm, 6.3 '-t 1.1 pm). In some instances, processes of single cells could be followed into laminae 1, 2, and 4 of the IPL. About 4.0-4.8% of the amacrine cells in the TNL were stained with the antibody against nAChR. Approximately 7.5-10.6% of all cells in the GCL contained nAChR-LI (Fig. 3a,c). These cells were found in all areas of the retina, but lower cell densities were observed in the temporal region (up to 788 cells/mm2) as compared to the rest of the retina (up to 1,325 cells/mm2).Although the

density of nAChR-LI cells in the GCL was low in the temporal retina, the percentage of labeled cells in that region was in the same range as in other peripheral retinal regions (around 10.5%). In contrast, there was a lower percentage of labeled cells in the central retina (about 7.5%). The nAChR-LI cells tended to be larger in the periphery (7-31 pm, 13.8 & 3.9 pm) than in the central retina (7-19 pm, 11.6 s 1.7 pm). Sometimes their dendrites could be followed to laminae 2 and 4 of the IPL (Fig. 3a). In horizontal sections, we could observe that some labeled cells in the GCL gave rise to labeled axons. Following rhodamine beads injection into the optic tectum, some nAChR-LI cells in the GCL were retrogradely labeled. This result indicated that at least part of the population of immunoreactive cells in the GCL were indeed ganglion cells.

NICOTINIC RECEPTORS IN GABA-IMMUNOREACTIVE CELLS

2 mm Fig. 6. Diagram illustrating the density of double-labeled cell bodies (cells/mm2)in the INL of the chick retina. Dorsal, ventral, temporal, and nasal portions of the retina correspond to the superior, inferior, left, and right regions in this drawing. Note a slightly higher density of double-labeled cells in the central area as compared to the periphery.

Double-labeling studies In the double-labeling experiments performed to demonstrate the presence of GABA-LI and nAChR-LI in the same cell body, different fluorophores (DTAJ? and TRITC) were employed. Regardless of which fluorophore was used to reveal either GABA-LI or nAChR-LI, the pattern of staining was the same, and double-labeled cells were observed in the INL and GCL. Small and medium cells in the INL were double labeled with antibodies against GABA and nAChR. The presumptive displaced ganglion cells that displayed intense nAChR-LI staining were occasionally observed to be very weakly stained for GABA-LI. Therefore, these cells were not considered to be double labeled in this study. In general, the GABA-LI amacrine cells were more intensely stained than the nAChR-LI amacrine cells. Yet cells in the INL clearly stained for GABA-LI and nAChR-LI were observed in both cross and horizontal sections of retina (Figs. 4, 5 ) . The percentage of double-labeled cells in the INL in relation to the total number of cells in this layer ranged from 1.3 to 1.7%. About 29.1-36.0% of the GABA-LI cells in the INL also contained nAChR-LI, and approximately 29.2-39.1% of the nAChR-LI cells in the same layer showed GABA-LI (Figs. 4,5). The double-labeled cells were distributed fairly uniformly throughout the retina, but their density (280390 cells/mm2)appeared slightly higher in the center (Fig. 6). Histograms illustrating the numbers and ranges of size of GABA-LI,nAChR-LI, and double-labeled cells in the INL are presented in Figure 7. Overall, GABA-LI cells tended to be slightly larger than the nAChR-LI cells in both the center (means: 7.2 pm and 6.2 km, respectively) and periphery (means: 7.7 Fm and 6.3 pm, respectively). The histograms in Figure 7 demonstrate that the soma sizes of double-labeled cells appear t o mirror the soma sizes of the

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nAChR-LI cells in the center (5-10 pm, 6.2 ? 1.2 pm), and the soma sizes of the GABA-LI cells in the periphery (5-12 Fm, 7.7 2 1.5 Fm). The nAChR-LI cells in the GCL were more intensely stained and were present in higher numbers than the GABA-LI cells in the same layer. A small percentage of the total number of cells in the GCL (0.1-0.7%) were stained both for GABA-LI and nAChR-LI (Figs. 4, 8). Higher percentages and densities of doubly labeled cells (72-77 cells/mm2)were observed in the nasal and inferior retina (Fig. 9). The temporal retina appeared to have a low density of double-labeled cells (6 cells/mm2) as compared to the center (45 cells/mm2)or to other peripheral regions. Approximately 19.4-37.0% of the GABA-LI cells in the GCL also contained nAChR-LI, and they were homogeneously distributed across the retina, except for the temporal retina. In this particular region, the percentage of GABA-LI cells containing nAChR-LI was lower (about 9.1%). The temporal region also contained lower numbers of nAChR-LI cells showing GABA-LI (0.7%). In other retinal regions, about 2.5-6.2% of the nAChR-LI cells also contained GABA-LI. The double-labeled cells in the periphery were slightly larger (7-21 pm, 12.5 2 2.1 pm) than those in the center (7-17 Fm, 11.6 2 1.6 bm, Fig. 10). In general, GABA-LI cells in the GCL seemed to be slightly smaller than the nAChR-LI in the retinal periphery (12.6 pm and 13.8 pm, respectively). The soma sizes of the double-labeled cells in the periphery resembled those of the GABA-LI cells. The results suggested that in this region mostly small and medium cells staining for nAChR-LI also contained GABALI . The pattern of arborization of the GABA-LI and nAChR-LI cells in the IPL appeared to be coincident in the layers 1, 2, and 4. However, we were never able to trace processes of double-labeled cells to their points of arborization in any of those layers in the IPL (Fig. 4). Following rhodamine beads injections into the optic tectum, a large number of cells in the GCL were retrogradely labeled. However, only a few of these presumptive ganglion cells were observed to contain both GABA-LI and nAChR-LI (Fig. 11).Because of the difficulty of injecting the entire optic tectum and therefore of retrogradely labeling most of the ganglion cells, we did not study the triple-labeled cells in detail. We cannot exclude the possibility that some of the GABA-LI/nAChR-LI cells in the GCL were displaced amacrine cells.

DISCUSSION Our data provide a description of a n anatomical substrate for cholinergic effects upon GABAergic cells in the chick retina. Substantial variability in the intensity of staining of the GABA-LI and nAChR-LI cells was observed throughout the retina. A conservative approach was then assumed for the cell counts, and only the unequivocally labeled cells were considered. Furthermore, all of the cell counts were conducted in tissue processed with the indirect fluorescence method, which is less sensitive than the avidin-biotinperoxidase or peroxidase-anti-peroxidasetechniques. Thus the numbers presented here are probably underestimates of the actual numbers of cells to be found in the retina.

Distribution of GABA-LI The pattern of distribution of GABA-LI described in this study is in agreement with previous immunohistochemical

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Fig. 7. Histograms illustrating the distribution of sizes of GABA-LI (top), nAChR-LI (middle), and double-labeled (GABA-LI/nAChR-LI, bottom) in the INL of the central versus the peripheral retina. The

distribution of sizes of double-labeledcells resembles the distribution of sizes of GABA-LI cells in the periphery, and the distribution of sizes of the nAChR-LI in the central retina.

studies of the chick retina, which used antisera against GABA or GAD (Mosinger et al., '86; Agardh et al., '87a; HokoG et al., '90). In general, the antisera against GABA and GAD appear to label the same type of cells in the retina, includingamacrine cells and cells in the GCL (Agardh et al., '87a). Cells in these layers have been recently shown to contain GAD mRNA in the cat and primate retinae (Sarthy and Fu, '89a,b). However, a discrepancy exists between the distribution of GABA and GAD in other retinal cell types (e.g., bipolar and horizontal cells), depending upon the

species in question (Agardh et al., '87a; Hurd and Eldred, '89; Sarthy and Fu, '89a,b). Furthermore, in many species there is a major difference between the number of GABAimmunoreactive cells compared to the number of GADimmunoreactive cells (Agardh et al., '87a). Several explanations have been proposed to clear up this point (see, e.g., Hurd and Eldred, '89). Our experiments revealed a similar distribution of the GAD and GABA immunoreactive cells in the chick retina, but a higher number of GABA-LI cells compared t o the GAD-LI cells, as described in previous

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2 mm Fig. 9. Diagram illustrating the density of double-labeled cell bodies (cellsimm') in the GCL of the chick retina. Dorsal, ventral, temporal, and nasal portions of the retina correspond to the superior, inferior, left, and right regions in this drawing. Note higher densities of double-labeled cells in the nasal and ventral retinae as compared to the temporal retina.

mammals investigated to date (Hendrickson et al., '85; Mosinger et al., '86; Pourcho and Owczarzak, '89; Wassle and Chun, '89; Grtinert and Wassle, '901, and in one non-mammalian species (tiger salamander; Yang and Yazulla, '88). The wide variability in the position of GABA-LI cells in the INL of the chick retina is consistent with the possible existence of additional GABAergic cell types in this species as well, such as bipolar and/or interplexiform cells. However, we were not able to observe processes emerging from possible bipolar and/or interplexiform cells that projected to the IPL or the OPL. Some somata in the IPL were seen exhibiting GABA-LI, analogous to those in the rat (Lin et al., '83; Mosinger et al., '86) and primates (Hendrickson et al., '85; Sarthy and Fu, '89b; Grunert and Wassle, '90). These cells may be equivalent to the "axon-bearing amaFig. 8. Two fluorescence photomicrographs of the same tangential crine cells" located in the middle of the IPL of the macaque section through the GCL and optic fiber layer in the peripheral retina processed for GABA (A) and nAChR (B) immunoreactivities. GABA-LI monkey retina (Dacey, '89). was visualized with DTAF and nAChR-LI with TRITC. An example of In the present study we detected some GABA-LI ganglion double-labeled cell containing GABA-LI (A) and nAChR-LI (B) can be cells after injection of a retrograde tracer into the optic observed in this figure (open arrows). A cell stained only for GABA-LI is pointed out in A (arrow). Many nAChR-LI cells can also be observed (B). tectum. In the past few years, the existence of ganglion cells containing GABA has been reported in several species (tiger Bar: 30 km. salamander: Yang and Yazulla, '88; turtle: Hurd and Eldred, '89; rat: Caruso et al., '89, '90; rabbit: Yu et al., '88; studies in chicks (Agardh et al., '87a; HokoG et al., '90). cat: Shelton et al., '90). Among them, Caruso et al. ('89) and However, as mentioned before, many GABA-LI cells in the Hurd and Eldred ('89) revealed GABAergic projections to the tectum in agreement with our present findings. The INL and the GCL were shown to also contain GAD-LI. In the present work, GABA immunoreactivity was ob- possibility remains, however, that some of the GABA-LI served in horizontal cells, amacrine cells, and some somata somata in the GCL were displaced amacrine cells, since not in the GCL. Several studies have indicated the presence of all of the cells in this layer were retrogradely labeled. GABA in bipolar cells of nonmammalian (frog: Mosinger et Approximately 30-35% of the cells in the GCL of the chick al., '86; Agardh et al., '87; mudpuppy: Mosinger et al., '86; retina appear actually to be displaced amacrine cells (Ehrtiger salamander: Yang and Yazulla, '88) and mammalian lich, '81). Also, many displaced amacrine cells in the GCL retinae (cat: Pourcho and Owczarzak, '89; Wassle and contain GABA in all species examined so far (Mosinger et Chun, '89; monkey: Griinert and Wassle, '90). Similarly, al., '86; Brecha et al., '88; Yu et al., '88; Caruso et al., '89; GABA-positive interplexiform cells have been found in all Grunert and Wassle, '90).

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Fig. 10. Histograms illustrating the distribution of sizes of GABA-LI (top), nAChR-LI (middle), and double-labeled cells (GABA-LIlnAChR-LI, low) throughout the GCL in the center and peripheral retina. A similar pattern can be observed for the cell sizes of GABA-LI and CLXBA-LIlnAChR-LI cells in the peripheral retina.

About 3.5-5.0% of the cells in the INL contained GABALI, and no marked differences were observed in the density or size of labeled somata in the central versus the peripheral retina. In the GCL, about 0.8-1.9% of the cells exhibited GABA-LI, with a higher density of stained cells in the central retina than in the periphery. The temporal region, in particular, contained low densities of stained

cells, and the stained cells were generally larger than elsewhere. In general, Nissl-stained chick retinae exhibited a lower density of cells in the same region, and the GABA staining seemed to follow this general distribution. According to other authors, about 30%of the cells in the INL of different species, including chicks, contain GABA (Mosinger et al., '86; Pourcho and Owczarzak, '89; Wassle and

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Chun, '89; Griinert and Wassle, '90). The number of GABA-containing cells in the GCL differs according to the species, but it can reach up to one-third of the cells in the cat, rabbit, rat, and chicken (Mosingeret al., '86). The lower percentage of GABA-LI cells in the INL and even in the GCL found in this study could be explained in part by the different immunohistochemical methods employed. In the present study, all measurements were made in the fluorescent-treated material. In contrast, other studies have used avidin-biotin-peroxidaseor peroxidase-anti-peroxidasetechniques, which could account for higher numbers of labeled cells reported. Another possible explanation of the limited numbers of GABA-LI cells could be the nature of the fixative used in our experiments. In order to obtain good nAChR labeling, we used 2% paraformaldehyde in PBS, which is not optimal for this GABA antiserum; the commercial antiserum against GABA we employed in these experiments had been previously used on tissue fixed in 4% paraformaldehyde. Although these procedures could account for the discrepanciesobserved, we cannot exclude the possibility that the different antisera used in these studies also contributed to the differences in the results.

Distribution of nAChR-LI

Fig. 11. Photomicrographs of a horizontal section of the chick retina 5 days after the injection of rhodamine beads into the contralateral dorsal optic tectum. This section was incubated with antibodies against GABA and nAChR, and the corresponding immunoreactivities were visualized with DTAF and M C A , respectively. Arrows indicate a ganglion cell retrogradely labeled with rhodamine beads (A), and stained for GABA-LI (B), and nAChR-LI (C) as well. There is some breakthrough of the rhodamine beads through the fluorescein (B) and AMCA (C) filters. Bar: 15 pm.

Our experiments employing a monoclonal antibody against the a3 ACh-binding subunit generated similar results to previous experiments using monoclonal antibodies against 62 structural subunits of the nAChR (Keyser et al., '88).Although the stainingwith both antibodies was not exactly identical, the similarity of labeling patterns with the monoclonal antibodies against a3 and p2, together with results showing a high level of expression of a3 mRNA in the retina (Whiting et al., '91), suggest that the acetylcholine receptors containing the a3 subunit represent the major nAChR subtype expressed in chicken retina. In our study, amacrine cells, ganglion cells, displaced ganglion cells, and possibly displaced amacrine cells exhibited nAChR-LI throughout their cytoplasm and processes. Immunoreactive dendrites of these cells were distributed in the laminae 1, 2, and 4 of the IPL. About 4.0-4.8% of the cells in the INL were nAChR-LI, whereas approximately 7.5-11.0% of the cells in the GCL exhibited nAChR-LI. No marked regional differences in sizes and densities were observed in the INL. In the GCL, however, lower densities were observed in the temporal retina, where the largest stained cells were also found. This is similar to the distribution observed for GABA-LI staining. Keyser et al. ('88), in contrast, reported that about 12-18% of the cells in the GCL exhibit nAChR immunoreactivity. This apparent discrepancy may be due to methodological differences. The earlier study employed an antibody against the p2 structural subunit of the nAChRs, and counts were made in avidin-biotin-peroxidaselabeled preparations. In contrast, we used an antibody against the a3 ACh-binding subunit of the nAChR, and the fluorescence technique. Alternatively, the difference in numbers may be due to the expression, by some cells, of other ACh binding subunits. For example, instead of 1x3, some cells may contain a2, a4, or other subunits in combination with p2. In the brain, p2 is known to be associated with an ACh receptor subtype that contains a4 subunits, and also with a subtype thought to contain a2 subunits (Schoepfer et al., '88). Furthermore, a previous study has shown that p2 subunits can form functional receptors in combinationwith a2,a3,or a4 subunits (Papke et al., '89). This could be responsible for the apparent

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greater number of cells that contain the p2 subunit compared to those that contain the a3 subunit. It is interesting to note that Keyser et al. ('88) were unable to find any cell in either the INL or GCL that exhibited both nAChR-LI and choline acetyltransferase-like immunoreactivity. This suggests that the GABAergic cells containing nAChR-LI described in the present study are not cholinergic. However, many GABAergic cells have also been shown to be cholinergic in other species (Brecha et al., '88; Chun et al., '88; Kosaka et al., '88; Vaney and Young, '88). Taken together, these reports and our findings suggest the existence of at least two subpopulations of GABA-LI cells in the chick retina: one that is cholinoceptive but not cholinergic, and another one that might be cholinergic. Double-labeling experiments are needed to verify the latter possibility.

Double-labelingstudies A small percentage of neurons in both the INL and the GCL were double labeled with antibodies against GABA and nAChR. The distribution of double-labeled cells in the INL followed the distribution of cells stained separately for each antibody. Except for a slight tendency toward higher densities of labeled cells in the central retina, these cell types were homogeneously distributed throughout the retina. Double-labeled cells had a similar size distribution when compared to the nAChR-LI cells in the central retina. In the peripheral retina, however, the soma sizes of the double-labeled cells mirrored those of the peripheral GABA-LI cells. Thus the results suggest that in the central retina mostly small and medium GABA-LI cells contained nAChR-LI, whereas in the periphery, small, medium, and large GABA-LJ cells contained nAChR-LI. In contrast, small, medium, and large nAChR-LI cells stained for GABA-LI in the central retina, whereas mostly medium and large nAChR-LJ cells stained for GABA-Ll in the peripheral retina. A small percentage of cells in the GCL were double labeled. Some of them were presumptive ganglion cells, as shown by injections of a retrograde tracer into the optic tectum. The temporal retina contained the lowest density of double-labeled cells, and the lowest percentages of GABA-LI cells containing nAChR-LI or of cholinoceptive cells containing GABA-LI. The soma sizes of the double-labeled cells were slightly smaller in the central retina. In the periphery, they seemed similar to the soma sizes of GABA-LI cells. This pattern was evident in the temporal retina, which contained the largest cholinoceptive cell bodies. These latter cells were never double labeled. Perhaps this fact contributed to the low percentages of GABA-LI cells containing nAChR-LI, and vice versa, observed in this region. The peculiarities of cellular distribution in the temporal region of the chick retina described in this study have not been previously reported. The significance of these findings is, at present, unknown.

Physiological correlates The existence of GABA-LI ganglion cells in the chick retina provides a morphological basis for a direct inhibitory input from the retina to the midbrain, as suggested by physiological studies of the pigeon tectum (Leresche et al., '86). The significance of the cholinergic inputs (mediated via nAChR) to these GABA-LI ganglion cells, however, needs to be investigated. As mentioned before, an interaction between GABAergic and cholinergic systems has been reported showing that

GABA agonists inhibited and GABA antagonists potentiated the light-evoked release of ACh from the retina (Massey and Neal, '79; Massey and Redburn, '82; Cunningham and Neal, '83; Massey and Redburn, '85, '87). This suggests that the cholinergic system receives an inhibitory input from GABA amacrine cells, direct or indirect, or even both (Massey and Redburn, '87). Recent studies demonstrated colocalization of ACh and GABA in amacrine cells (Brecha et al., '88; Chun et al., '88; Vaney and Young, '88) and showed that the release of ACh and GABA from the same amacrine cell are controlled by different mechanisms (O'Malley and Masland, '89). On the basis of these results and considering that GABA and ACh are both released upon stimulation, Brecha et al. ('88) proposed that ACh may exert an excitatory postsynaptic action, whereas GABA may exert an inhibitory presynaptic action. They did not exclude, however, postsynaptic actions of GABA. Taking into account the results presented in this work, the possibility exists that the cholinergic cells might also directly influence GABAergic cells postsynaptically. However, the main interest to date has been to investigate the GABAergic effects upon cholinergic cells, and no information concerning the possible cholinergic influences upon GABAergic cells is available. Thus no physiological data appears to be available to permit speculation on the possible functional significance of the nicotinic receptors present in GABA-Ll cells. In summary, our data showed different classes of presumptive GABA-LI cells in the chick retina that are also cholinoceptive. This demonstrates that a morphological basis for a possible interaction between cholinergic cells (through nicotinic receptors) and GABA-LI cells exists. Physiological experiments are needed to clarify which actions ACh exerts on retinal GABA-LI cells via nicotinic receptors.

ACKNOWLEDGMENTS The authors thank Ellie Watelet for excellent secretarial help, and Kevin Cox and Donna Harclerode for helpful technical assistance. We are also very grateful to Drs. Tom Shimizu, Thom Hughes, and Luiz Britto for critically reading this manuscript. This work was supported by NIH grants EY06890 (H.J.K.), NS11323 (J.M.L.), and EY07845 (K.T.K.). J.M.L. was also supported by ,the Council for Smokeless Tobacco Research and the Council for Tobacco Research. D.E.H.-B. was the recipient of a fellowship from FAPESP (Brazil).

LITERATURE CITED Agardh, E., B. Ehinger, and J.Y. Wu (1987b) GABA and GAD-like immunoreactivity in the primate retina. Histochemistry 86r485490. Agardh, E., A. Bruun, B. Ehinger, and J. Storm-Mathisen (1986) GABA immunoreactivity in the retina. Invest. Ophthalmol. Vis. Sci. 27:674678. Agardh, E., A. Bruun, B. Ehinger, P. Ekstrom, T. Van Veen, and J.Y. Wu (1987a) Gamma-aminobutyric acid- and glutamic acid decarboxylaseimmunoreactive neurons in the retina of different vertebrates. J Camp. Neural. 258:622-630. Ariel, M., and N.W. Daw (1982a) Effects of cholinergic drugs on receptive field properties of rabbit retinal ganglion cells. J. Physiol. 324:135-160. Ariel, M., and N.W Daw (1982b) Pharmacological analysis of directionally sensitive rabbit retinal ganglion cells. J. Pbysiol. 324:161-186. Ball, A.K. (1987) Immunocytochemical and autoradiographic localization of GABAergic neurons in the goldfish retina. J. Camp. Neural. 2.55317325.

NICOTINIC RECEPTORS IN GABA-IMMUNOREACTIVE CELLS Baughman, R.W., and C.R. Bader (1977) Biochemical characterization and cellular localization of the cholinergic system in the chicken retina. Brain Res. 138:469-485. Brecha, N., D. Johnson, L. Peichl, and H. Wassle (1988) Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase and y-aminobutyrate immunoreactivity. Proc. Natl. Acad. Sci. USA 85:61876191. Caldwell, J.H., N.W. Daw, and H.J. Wyatt (1978) Effects of picrotoxin and strychnine on rabbit retinal ganglion cells: Lateral interactions for cells with more complex receptive fields. J. Physiol. 276:277-298. Caruso, D.M., M.T. Owczarzak, and R.G. Pourcho (1990) Colocalization of substance P and GABA in retinal ganglion cells: A computer-assisted visualization. Vis. Neurosci. 5:389394. Caruso, D.M., M.T. Owczarzak, D.J. Goebel, J.C. Hazlett, and R.G. Pourcho (1989) GABA-immunoreactivity in ganglion cells of the rat retina. Brain Res. 476:129-134. Chun, M.H., H. Wassle, and N. Brecha (1988) Colocalization of (3H) muscimol uptake and choline acetyltransferase immunoreactivity in amacrine cells of the cat retina. Neurosci. Lett. 94:259-263. Conley, M., D. Fitzpatrick, and E.A. Lachica (1986) Laminar asymmetry in the distribution of choline acetyltransferase-immunoreactiveneurons in the retina of the tree shrew (Tupaia belungeri). Brain Res. 399:332-338. Cunningham, J.R., and M.J. Neal (1983) Effect of y-aminobutyric acid agonists, glycine, taurine and neuropeptides on acetylcholine release from the rabbit retina. J. Physiol. 336:563-577. Dacey, D.M. (1989) Axon-bearing amacrine cells of the macaque monkey retina. J. Comp. Neurol. 283:275-293. Daw, N.W., W.J. Brunken, and D. Parkinson (1989) The function of synaptic transmitters in the retina. Ann, Rev. Neurosci. 12205-225. Eckenstein, F., and H. Thoenen (1952) Production of specific antisera and monoclonal antibodies to choline acetyltransferase: Characterization and use for identification of cholinergic neurons. EMBO J. 1:363-368. Eckenstein, F., R.W. Baughman, M.V. Sofroniew, and J. Thibault (1983) A comparison of the distribution of choline acetyltransferase and tyrosine hydroxylase immunoreactivities in rat retina. SOC.Neurosci. Abs. 9:80. Ehrlich, D. (1981) Regional specialization of the chick retina as revealed by the size and density of neurons in the ganglion cell layer. J. Comp. Neurol. 195643-657. Glasener, G., W. Himstedt, R. Weiler, and C. Matute (1988) Putative neurotransmitters in the retinae of three urodele species (Triturus alpestris, Salamandra salamandra, Pleurodeles waltli). Cell Tissue Res. 2 5 2 3 17-328. Griinert, U., and H. Wassle (1990) GABA-like immunoreadivity in the macaque monkey retina: A light and electron microscopic study. J. Comp. Neurol. 297:509-524. Hendrickson, A, M. Ryan, B. Noble, and J.-Y. Wu (1985) Colocalization of [3H]muscimol and antisera to GABA and glutamic acid decarboxylase within the same neurons in monkey retina. Brain Res. 348:391-396. HokoG, J.N., A.L.M. Ventura, P.F. Gardino, and F.G. de Mello (1990) Developmental immunoreactivity for GABA and GAD in the avian retina: Possible alternative pathway for GABA synthesis. Brain Res. 532: 197-202. Hughes, T.E., R.G. Carey, J. Vitorica, A.L. de Blas, and H.J. Karten (1989) Immunohistochemical localization of GABA, receptors in the retina of the new world primate Saimiri sciureus. Vis. Neurosci. 2r565-581. Hurd, L.B., and W.D. Eldred (1989) Localization of GABA- and GAD-like immunoreactivity in the turtle retina. Vis. Neurosci. 3r9-20. Katz, L.C., A. Burkhalter, and W.J. Dreyer (1984) Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310:498-500. Keyser, K.T., T.E. Hughes, P.J. Whiting, J.M. Lindstrom, and H.J. Karten (1988) Cholinoceptive neurons in the retina of the chick An immunohistochemical study of the nicotinic acetylcholine receptors. Vis. Neurosci. 1.349-366. Kosaka, T.M. Tauchi, and J.L. Dahl (1988) Cholinergic neurons containing GABA-like and/or glutamic decarboxyase-like immunoreactivities in various brain regions of the rat. Exp. Brain Res. 7Or605-617. Leresche, N., 0 . Hardy, E. Audinat, and D. Jassik-Gerschenfeld (1986) Synaptic organization of inhibitory circuits in the pigeon’s optic tectum. Brain Res. 365383-387. Lin, C.-T., H.-2. Li, and J.-Y. Wu (1983) Immunocytochemical localization of L-glutamate decarboxylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decarboxylase, aspartate aminotransferase and somatostatin in rat retina. Brain Res. 27Ot273-283. Lindstrom, J.M., R. Schoepfer, and P. Whiting (1987) Molecular studies of

407

the neuronal nicotinic acetylcholine receptor family. Mol. Neurobiol. 1:281-337. Ma, P.M., and P. Grant (1984). Choline acetyltransferase and cholinesterases in the developing Xenopus retina. J. Neurochem. 42t1328-1337. Mariani, A.P., and M.T. Caserta (1986) Electron microscopy of glutamate decarboxylase (GAD) immunoreactivity in the inner plexiform layer of the rhesus monkey retina. J. Neurocytol. 15645-655. Mariani, A.P., D. Cosenza-Murphy, and J.L. Baker (1987) GABAergic synapses and benzodiazepine receptors are not identically distributed in the primate retina. Brain Res. 415r152-157. Masland, R.H., J.W. Mills, and S.A. Hayden (1984) The functions of acetylcholine in the rabbit retina. Proc. R. SOC.Lond. (Biol.) 223:121139. Masland, R.H., and J.W. Mills (1979) Autoradiographic identification of acetylcholine in the rabbit retina. J. Cell Biol. 81r159-178. Masland, R.H., and A. Ames (1976) Responses to acetylcholine of ganglion cells in an isolated mammalian retina. J. Neurophysiol. 39.1220-1235. Masland, R.H., and C.J. Livingstone (1976) Effect of stimulation with light on the synthesis and release of acetylcholine by an isolated mammalian retina. J. Neurophysiol. 39r1210-1219. Massey, S.C., andM.J. Neal (1979) The light evoked release ofacethylcholine from the rabbit retina in vivo and its inhibition by g-aminobutyric acid. J. Neurochem. 321327-1329. Massey, S.C., and D.A. Redburn (1982) A tonic y-aminobutyric acidmediated inhibition of cholinergic amacrine cells in rabbit retina. J. Neurosci. 2:1633-1643. Massey, S.C., and D.A. Redburn (1985) Light evoked release of acetylcholine in response to a single flash: Cholinergic amacrine cells receive ON and OFF input. Brain Res. 328:374-377. Massey, S.C., and D.A. Redburn (1987) Transmitter circuits in the vertebrate retina. Prog. Neurobiol. 28:55-96. Millar, T.J., I. Ishimoto, C.D. Johnson, M.L. Epstein, I.W. Chubb, and I.G. Morgan (1985) Cholinergic and acetylcholinesterase-containingneurons of the chicken retina. Neurosci. Lett. 74r281-285. Mosinger, J.L., S. Yazulla, and K.M. Studholme (1986) GABA-like immunoreactivity in the vertebrate retina: A species comparison. Exp. Eye Res. 42:631-644. Oertel, W.H., D.E. Schmechel, M.L. Tappaz, and I.J. Kopin (1981) Production of specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neurosci. 6:2689-2700. O’Malley, D.M., and R.H. Masland (1989) Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron. Proc. Natl. Acad. Sci. USA 86r3414-3418. Papke, R., J. Boulter, J. Patrick, and S. Heinemann (1989) Single channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neuron 3:589-596. Pourcho, R.G., and M.T. Owczarzak (1989) Distribution of GABA immunoreactivity in the cat retina: A light-microscopic and electron-microscopic study. Vis. Neurosci. 2425-435. Richards, J.G., P. Schoch, P. Haring, B. Takacs, and H. Mohler (1987) Resolving GABA,/Benzodiazepine receptors: Cellular and subcellular localization in the CNS with monoclonal antibodies. J. Neurosci. 7;18661886. Ryan, M.K., and A.E. Hendrickson (1987) Interplexiform cells in macaque monkey retina. Exp. Eye Res. 45:57-66. Sarthy, P.V., and M. Fu (1989a) Localization of L-glutamic acid decarboxylase mRNA in cat retinal horizontal cells by in situ hybridization. J. Comp. Neurol. 288:593-600. Sarthy, P.V., and M. Fu (1989b) Localization of L-glutamic acid decarboxylase mRNA in monkey and human retina by in situ hybridization. J. Comp. Neurol. 288:691-697. Schoepfer, R., P. Whiting, F. Esch, R. Blacher, S. Shimasaki, and J. Lindstrom (1988) cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor. Neuron 1,241-248. Shelton, L., S.B. Tieman, and K.R. Fry (1990) GABAergic ganglion cells in cat retina. SOC.Neurosci. Abs. I6:1217. Spira, A.W., T.J. Millar, I. Ishimoto, M.L. Epstein, C.D. Johnson, J.L. Dahl, and I.G. Morgan (1987) Localization of choline acetyltransferase-like immunoreactivity in the embryonic chick retina. J. Comp. Neurol. 26Or526-538. Tumosa, N., F. Eckenstein, and W.K. Stell (1984) Immunocytochemical localizations of putative cholinergic neurons in the goldfish retina. Neurosci. Lett. 48;255-259. Vaney, D.I., and H.M. Young (1988) GABA-like immunoreactivity in cholinergic amacrine cells of the rabbit retina. Brain Res. 438:369-373.

408 Voigt, T. (1986) Cholinergic amacrine cells in the rat retina. J. Comp. Neurol. 248:19-35. Wassle, H., and M.H. Chun (1988) Dopaminergic and indoleamineaccumulating amacrine cell express GABA-like immunoreactivity in the cat retina. J. Neurosci. 8:3383-3394. WBsle, H., and M.H. Chun (1989) GABA-like immunoreactivity in the cat retina: Light microscopy. J. Comp. Neurol. 279:43-54. Whiting, P., and J.M. Lindstrom (1986) Purification and characterization of a nicotinic acetylcholine receptor from chick brain. Biochem. 25:20822093. Whiting, P., and J.M. Lindstrom (1987) Purification and characterization of a nicotinic acetylcholine receptor from rat brain. Proc. Natl. Acad. Sci. USA 84:595-599. Whiting, P., R. Liu, B. Morley, and J.M. Lindstrom (1987) Structurally

D.E. HAMASSAKI-BRITTO ET AL. different neuronal nicotinic acetylcholine receptor subtypes purified and characterized using monoclonal antibodies. J. Neurosci. 7:40054016. Whiting, P., R. Schoepfer, W.G. Conroy, M.J. Gore, K.T. Keyser, S. Shimasaki, F. Esch, and J.M. Lindstrom (1991) Expression of nicotinic acetylcholine receptor subtypes in brain and retina. Mol. Brain Res. 10:61-70. Yang, C.Y., and S. Yazulla (1988) Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods. J. Comp. Neurol. 277:96108. Yazulla, S. (1986) GABAergic mechanisms in the retina. Prog. Ret. Res. 5:l-52. Yu, B.C., C.B. Watt, D.M. Lam, and K.R. Fry (1988) GABAergic ganglion cells in the rabbit retina. Brain Res. 439:376382.

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by me...
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