Localization of GABAA ReceptorSubunits �1, �3, �1, �2/3, �1, and �2 in the

Salamander Retina

JUN ZHANG,1 ANGEL L. DE BLAS,2 CELIA P. MIRALLES2AND CHEN-YU YANG1*

1Department of Neurobiology and Behavior, State University of New York atStony Brook, Stony Brook, New York

2Department of Physiology and Neurobiology, University of Connecticut,Storrs, Connecticut

ABSTRACTElectrophysiological studies have demonstrated that �-aminobutyric acid receptors type

A (GABAA) mediate important information processing in the retinas of salamander and othervertebrates. The pharmacology and physiology of GABAA receptors depend on their subunitcomposition. We studied the localization of GABAA receptor subunit isoforms �1, �3, �1, �2/3(antibody BD-17 and 62-3G1), �1, and �2 in salamander retina with immunocytochemicalmethods. All three �-subunit antibodies labeled similarly in the outer retina, especially theinner segments and synaptic terminals of rod photoreceptors (identified with protein kinaseC). Somatic labeling was observed in cell bodies of some horizontal cells, bipolar cells,amacrine cells, and cells in the ganglion cell layer (GCL). Puncta were present throughout theinner plexiform layer (IPL) for �1 and 62-3G1, but not for BD-17. �1-immunoreactivity (IR)stained a population of presumed OFF rod-dominated bipolar cells, including dendrites,soma, and axon terminals in the distal IPL. A subtype of GABAergic amacrine cell alsoexpressed �1-IR, with puncta sparsely distributed at the distal and proximal margins of theIPL. Both the OPL and IPL were labeled throughout for �3-IR, as opposed to the narrowdistribution of �1-IR in the IPL, suggesting that the two �-subunits are localized at differentsynaptic sites. Punctate �1-IR was observed in the OPL and IPL, whereas �2 was mostprominent in cone photoreceptors (identified with calbindin), including the terminal teloden-dria, in cell bodies of some horizontal cells, amacrine cells, cells in the GCL, and less intenselyin the IPL. In addition, several subunits were present in Muller cells. The differentiallabeling suggests the existence of GABAA receptor subtypes with different subunit composi-tions that mediate multiple GABAergic functions in salamander retina. J. Comp. Neurol. 459:440–453, 2003. © 2003 Wiley-Liss, Inc.

Indexing terms: GABAA receptor subunit; immunocytochemistry; salamander; retina

�-Aminobutyric acid (GABA) is the principal inhibitoryneurotransmitter in the vertebrate retina (reviewed byYazulla, 1986; Massey and Redburn, 1987; Wassle andBoycott, 1991; Freed, 1992; Wu and Maple, 1998). Immu-nocytochemical and autoradiographic studies localizedGABA to populations of amacrine cells, horizontal cells,some bipolar cells, as well as interplexiform cells (Lam etal., 1979; Yazulla, 1981; Yazulla and Brecha, 1981;Agardh et al., 1987; Yang and Yazulla, 1988; Pourcho andOwczarzak, 1989; Grunert and Wassle, 1990; Gabriel etal., 1992; Vardi and Auerbach, 1995).

The actions of GABA are mediated by three subtypes ofreceptor: GABAA, GABAB, and GABAC. These classes ofGABA receptors are differentiated on functional, molecu-

lar, and pharmacologic bases. Ionotropic GABAA andGABAC receptors are ligand-gated, chloride-ion channels;whereas metabotropic GABAB receptors modulate cal-

Grant sponsor: National Institute of Health; Grant number: R01 EY10322; Grant sponsor: National Institute of Neurological Disorders andStroke; Grant number: 39287.

*Correspondence to: Chen-yu Yang, Department of Neurobiology andBehavior, SUNY at Stony Brook, Stony Brook, NY 11794-5230.E-mail: chyang@notes.cc.sunysb.edu

Received 23 September 2002; Accepted 20 December 2002DOI 10.1002/cne.10626Published online the week of March 24, 2003 in Wiley InterScience

(www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 459:440–453 (2003)

© 2003 WILEY-LISS, INC.

cium and potassium channels by means of G-proteins andsecond-messenger pathways (reviewed by Slaughter andPan, 1992; MacDonald and Olsen, 1994; Bormann andFeigenspan, 1995; Sieghart, 1995; Lukasiewicz andShields, 1998a).

GABAA receptors are found on almost all types of neu-rons in the vertebrate retina (see Lukasiewicz andShields, 1998a, and Wassle et al., 1998, for reviews). Elec-trophysiological studies in amphibians have providedstrong evidence that GABAA receptors are of great impor-tance in the regulation of retinal information processing.In the outer retina, GABAA receptors are involved in neg-ative feedback from horizontal cells to cones (Wu, 1991)and may mediate feedforward inhibition to the dendritesof bipolar cells (Du and Yang, 2001). Both the feedbackand feedforward processes are thought to mediate thereceptive field surround response of bipolar cells (Dowlingand Werblin, 1969; Lasansky, 1973; Hare and Owen,1992; Wu, 1992). However, there is disagreement aboutthe GABAA mediated feedforward effect based on the ob-servation that there was little or no GABA sensitivity onbipolar cell dendrites (Lukasiewicz and Werblin, 1994; Wuand Maple, 1998), and GABA antagonists did not block thesurround response of bipolar cells (Hare and Owen, 1996).In the inner retina, GABAA receptors participate ininhibitory synaptic transmission from GABAergic ama-crine cells onto other amacrine cells, ganglion cells, and toa lesser extent, reciprocally onto bipolar cell axon termi-nals (Lukasiewicz et al., 1994; Zhang et al., 1997;Lukasiewicz and Shields, 1998a,b) and, thus, may modu-late the output signals of bipolar cell and shape ganglioncell responses (Lukasiewicz and Shields, 1998b; Shen andSlaughter, 2001), including the center-surround organiza-tion (Cook and McReynolds, 1998).

Molecular biology has demonstrated that GABAA recep-tors are derived from multiple subunits including �1–6,�1–3, �1–3, �, �, �, and � isoforms, which form subunitdependent heteropentameric receptors. In the central ner-vous system (CNS) as well as in the retina, the mostabundant pentamers are composed of ���-subunit vari-ants (Greferath et al., 1995; Hevers and Luddens, 1998)and the pharmacologic properties and locations of thereceptor depend on the composition of subunit variants(Greferath et al., 1995; Korpi et al., 2002). Immunocyto-chemical localization of GABAA receptor subunits hasbeen well described in the retinas of goldfish, chicken, rat,rabbit, cat, and human (Yazulla et al., 1989; Hughes et al.,1991; Vardi et al., 1992; Greferath et al., 1993,1994,1995;Vardi and Sterling, 1994; Koulen et al., 1996; Fletcher etal., 1998; for review, see Wassle et al., 1998). However,there is relatively little information about the localizationof GABAA receptor subunits in the retina of amphibiansincluding salamander. The only report was the localiza-tion of BD-17 (�2/�3-subunit) in the frog retina which wasexpressed predominantly in the synaptic sites in the innerplexiform layer (IPL) (Vitanova et al., 2001). Yang et al.(1992), by using in vitro autoradiography (ARG) insalamander, found [3H]muscimol binding in the outerplexiform layer (OPL) and IPL, but also observed thatGABAA receptors were coupled to benzodiazepines (BZs)in the IPL but not in the OPL, as there was no detectable[3H]flunitrazepam binding in the OPL. Recently, Wang etal. (2000) demonstrated the presence of GABAA receptorsin both plexiform layers with a fluorescent ligand bindingtechnique and concluded that there were multiple sub-

types. However, the subunit composition of these recep-tors has not been determined. The purpose of this studywas to identify the cellular and subcellular localization ofGABAA receptor subunits in the salamander retina. Aportion of these results has been presented in abstractform (Zhang et al., 2001).

MATERIALS AND METHODS

Animals

Larval tiger salamanders (Ambystoma tigrinum) 15–20cm in body length were maintained at 4°C on a 12-hourlight/dark cycle. Animals were dark adapted for 1–2hours. The animals were decapitated, and the eyes wereenucleated under dim red light. Care and handling ofanimals were approved by the Institutional Animal Careand Use Committee (IACUC) in accordance with the NIHguidelines.

Antibodies

Primary antibodies. Seven anti-GABAA receptorsubunit antibodies, including �1, �3, �1, �2/3 (antibodyBD-17, 62-3G1), �1, and �2, were used. BD-17 was a gift ofDr. Richards; �1 and �3 were purchased from AlomoneLaboratory (Jerusalem); all other antibodies were fromDr. De Blas’s lab.

Polyclonal anti-�1 was raised in rabbit against highlypurified peptide QPSQDELKDNTTVFTR(C), correspond-ing to residues 1–16 of mouse or rat GABAA receptor�1-subunit with additional C-terminal cysteine. Theanti-�3 was raised in rabbit against purified peptideQGESRRQEPGDFVKQ(C), corresponding to residues1–15 of human GABAA receptor �3-subunit with addi-tional C-terminal cysteine. Both antibodies were affinitypurified on the corresponding immobilized peptide, andtheir specificities have been described (Gao et al., 1993).The anti-�1 was raised in rabbit against a purified fusionprotein of the large intracellular loop of the rat �1, corre-sponding to amino acids 303–425 expressed in bacteria.The preparation and purification of the fusion protein andthe affinity purification of �1 were described previously(Moreno et al., 1994; Li and De Blas, 1997). This antibodyhas been used in studies in the CNS (Christie et al., 2002)as well as in the fish retinas (Yazulla, unpublished data).Monoclonal antibody BD-17, was raised in mouse againstthe purified GABAA receptor/benzodiazepine receptor/Cl-

channel complex. It was characterized (Richards et al.,1987) and determined to recognize �2/3 but not the �1-subunit (Ewert et al., 1992). This antibody has been usedextensively in the retina (Hughes et al., 1991; Vardi et al.,1992; Vardi and Sterling, 1994; Greferath et al., 1994,1995; Lin and Yazulla, 1994; Yazulla et al., 1997). Mono-clonal antibody 62-3G1, raised in mouse against the affin-ity purified GABAA receptor from bovine brain (Vitorica etal., 1988) recognizes the same epitope of the �2/�3-subunits as BD-17 (Ewert et al., 1992). It has been usedextensively for brain and retinal research (i.e., De Blas etal., 1988; Vitorica et al., 1988; Yazulla et al., 1989, 1997;Yazulla and Studholme, 1997; Miralles et al., 1999). Poly-clonal rabbit anti–�1- and �2-subunits were generated af-ter immunizing rabbits with the corresponding peptides(amino acid residues 359–370 of the rat �1 and N-terminalamino acids 1–15 of the rat �2-subunit of GABAA receptor)coupled to keyhole limpet hemocyanin through a cysteine

441GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA

that was added to the carboxy-end (Khan et al., 1993),they were affinity purified and their specificities werereported elsewhere (Khan et al., 1996; Miralles et al.,1999; Christie et al., 2002). For the double-labeling stud-ies, antibodies known to label distinct population of neu-rons were used. Polyclonal rabbit anti-calretinin, mono-clonal mouse anti-calbindin, polyclonal rabbit anti-PKCwere purchased from Chemicon. Polyclonal guinea piganti-GABA and monoclonal mouse anti-tyrosine hydroxy-lase (TOH) were from Eugene Tech (Eugene, OR).

Secondary antibodies. The secondary antibodieswere purchased from Jackson Immunoresearch Laborato-ries (West Grove, PA), including goat anti-rabbit immu-noglobulin G (IgG) conjugated to Cy3, goat anti-mouse IgGconjugated to Cy3, goat anti-rabbit IgG conjugated to flu-orescein isothiocyanate (FITC), goat anti-mouse IgG con-jugated to FITC, and goat anti-guinea pig IgG conjugatedto FITC.

Tissue preparation

Animals were decapitated and double pithed. Eight eyeswere hemisected, and eyecups or the posterior portion ofthe isolated retinas on Millipore filter paper were fixed.Depending on the sensitivity of the subunit antibodies tofixation, we used 4% paraformaldehyde in 0.1 M sodiumphosphate buffer (PB, pH 7.6) fixation for only 15–30minutes at 4°C for the highly sensitive anti 62-3G1 anti-body (Yang et al., 1992). Tissues for the other subunitantibodies were fixed in 4% paraformaldehyde for 1 hourat room temperature except the tissues for anti-�1 thatwas fixed in 4% paraformaldehyde plus 0.02% glutaralde-hyde for 80 minutes at room temperature. After severalwashes in 0.1 M phosphate buffer saline (PBS, pH 7.4),the eyecups or isolated retinas were cryoprotected withsucrose (15%, 30%) overnight at 4°C. The tissue was em-bedded in OCT compound (Tissue Tek, Mile, Inc.), verti-cally sectioned at 12 �m on a cryostat, and collected onchrome-gelatin–coated glass slides.

Immunocytochemistry

Single labeling. Slides were rinsed in PBS, blocked in5% normal donkey serum (NDS) or normal goat serum(NGS) in PBS for 1 hour, incubated overnight at 4°C inrabbit antisera �1 (1:300–600), �3 (1:300–600), �1 (1:8),�1 (1:8), �2 (1:10) in 2% PBS-NDS with 0.3% Triton X-100or mouse monoclonal BD-17 (1:5) and 62-3G1 (1:2–5) in2% PBS-NGS with 0.3% Triton X-100. After rinsing, sec-tions were incubated for 35 minutes at 37°C in Cy3-conjugated donkey anti-rabbit IgG (1:400) for �1, �3, �1,�1, �2, and Cy3-conjugated goat anti-mouse IgG (1:400) forBD-17 and 62-3G1. Slides were rinsed and cover-slippedwith Vectashield (Vector).

Double labeling. The sections were incubated over-night at 4°C in a mixture of two primaries: one from therabbit, the other from mouse. The dilution of the primarieswas the same as that for the single labeling. Sections wererinsed, followed by incubation for 35 minutes at 37°C witha mixture of two secondary antibodies, the Cy3-conjugateddonkey anti-rabbit IgG (1:400) and the FITC-conjugatedgoat anti-mouse IgG (1:100).

Control experiments were processed by omitting theprimary antibody for single labeling; no detectable stain-ing was observed. For double-labeling control, one pri-mary antibody and then the two appropriate secondary

antibodies were applied. Only the immunoreactivity spe-cific for the remaining primary antibody was detected.

Data analysis

Immunoreactivities were viewed with a Zeiss Axioskopepifluorescence microscope equipped with appropriate flu-orescence filters. An additional FITC narrow–band passfilter (D535, Chroma Technology Corp., Brattleboro, VT)was inserted when viewing FITC to further insure nocrossover from the Cy3. Sections, labeled for either FITCor Cy3 alone, showed no evidence of crossover fluorescencewhen viewed or photographed with the alternate filter set.Fluorescence photomicrographs were obtained withKodak T400 CN film (ASA 400) at exposures of 5 to 30seconds. For some double-labeling sections, paired colornegatives (Kodak Color Gold Max 400) were obtained fromthe same location, one with the Cy3 filter, the other withthe FITC filter. Selected areas of the double-labeled sec-tions were examined with the use of a confocal laser scan-ning microscope Bio-Rad Radiance 2000. Sections wereviewed through a Nikon 60 Planapochromatic oil objec-tive (n.a. 1.4), which had an optical x–y axis resolution of0.20 �m. Five to 17 optical sections were collected every 1�m along the z-axis with a resolution of 0.25 �m. Imageswere collected and merged by using the Bio-Rad LaserSharp 2000 software. For colocalization, the software wasBio-Rad Laser Pix. Figures were prepared by scanningphotographic prints on an HP 1200 flat bed scanner at 300dpi. The TIFF files were processed only for brightness andcontrast and composed with lettering using Adobe Photo-shop 6.0.

RESULTS

�-Subunit antibodies label rodphotoreceptors

Two monoclonal antibodies against the �2/3-subunits(62-3G1 and BD-17) produced similar labeling in the outerretina but not in the inner retina (Figs. 1, 2). Antibody62-3G1 was very fixation-sensitive. At 15 minutes fixation(Fig. 1), there was intense label over photoreceptor innersegments and the OPL. More scattered punctate label wasseen throughout the IPL and over cell bodies of horizontalcells, amacrine cells, and cells in the ganglion cell layer(GCL). Also, numerous cell bodies in the inner nuclearlayer (INL) appeared outlined by 62-3G1 immunoreactivity(IR). The presence of punctate label between ganglion cellsand at the inner limiting membrane suggests that much ofthe label in the INL was on Muller cell processes.

At 30 minutes of fixation (Fig. 2A), outer segments wereautofluorescent and there was an overall reduction inlabel in the inner retina. However, with the improvedhistology, it now appeared as if the label in the OPL wasdue to photoreceptor synaptic terminals; label in the innersegments remained prominent. BD-17 also labeled photo-receptors but more discretely than 62-3G1 (Fig. 2B) inthat the OPL was labeled less extensively with BD-17.Individual synaptic terminals could be discerned, com-plete with the axon projected to the outer nuclear layersimilar as that found in 62-3G1-IR with longer fixation(Fig. 2A). As with 62-3G1, the nucleus and outer segmentwere not labeled. Rather, the inner segment, located be-tween the outer segment and nucleus was labeled in-tensely. There was no evidence for BD-17 label in the IPL,

442 J. ZHANG ET AL.

and just a hint of vertical streaks in the IPL and GCL thatwere indicative of Muller cells.

The distal position of the nuclei of the labeled photore-ceptors suggested that they were rods (Mariani, 1986). Wefound that the rabbit polyclonal antiserum from Chemiconagainst the �-, �-, and �-subunits of PKC specifically la-bels all rods especially the terminals with telodendria inthe salamander retina (Zhang and Yang, 2001). Doublelabeling with PKC and BD-17 (Fig. 3, on color plate) showscomplete correspondence of these labels demonstratingthat rods label for BD-17 and, by extension, 62-3G1 as well.

Labeling for the �1-subunit had features common toboth 62-3G1 and BD-17 (Fig. 4). The inner segments andsynaptic terminals of rods appeared to be labeled, al-though more extensively than with BD-17. In addition, theIPL was labeled throughout, with hints of horizontal stri-ations and lamination, unlike 62-3G1 that had punctatelabeling in the IPL, but with no evidence of lamination.Granular, weak punctate label also was observed on cellbodies of some horizontal cells, bipolar cells, amacrinecells, and cells in the GCL.

�1-Subunit labels a rod OFF bipolar cell andGABAergic amacrine cell

A remarkable feature of the labeling with the �1-subunit was that a population of bipolar cells was labeled

Fig. 1. A–C: Immunoreactivity (IR) of 62-3G1 antibody. Tissueswere fixed for only 15 minutes. Punctate label was present in photo-receptor inner segment (arrowheads), outer plexiform layer (OPL),inner plexiform layer (IPL), cell bodies of horizontal cells (H in C),amacrine cells (A in A), and cells in the ganglion cell layer (asterisk inB). Scale bar 12 �m in A (applies to A–C).

Fig. 2. A comparison of 62-3G1 immunoreactivity (IR) with that ofthe BD-17-IR. A: 62-3G1-IR, at 30 minutes fixation, inner segments(arrowheads) and terminals (arrows) of rods were labeled, including adescending axon projecting to the outer plexiform layer (OPL). Rods(R) were recognized by the distal location of the soma in the innernuclear layer compared with cones (C). B: BD-17-IR, the labeling ofinner segments (arrowheads), descending axons and rod terminals(arrows) was very similar to that of the 62-3G1-IR. However, therewas no detectable label in the inner plexiform layer (IPL). Scale bar 10 �m in B (applies to A,B).

443GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA

in its entirety from the Landolt’s club to the synapticterminals in the distal sublamina a of the IPL (Fig. 5). Thedendrites in the OPL as well as the thin axon also werelabeled. In contrast to the uniform label of the bipolar cellbodies, there was a granular label that outlined scatteredlarge amacrine cells at the proximal margin of the INL

(Fig. 5B). Bipolar cells with axon terminals at the mostdistal margin of the IPL have been identified by Luciferyellow filling after electrophysiological analysis as roddominant OFF bipolar cells (Wu et al., 2000).

We investigated the possible relationship between thelabeled bipolar cell dendrites and the terminals of photo-receptors in two double-label experiments that selectivelylabeled cones or rods. Figure 6, left column, compares thedistributions of calbindin-IR, found in the somata andpedicles of cones, with the �1-subunit. The bottom panel ofthis column shows a composite of the two patterns and

Fig. 3. Double labeling of BD-17 immunoreactivity (IR) withPKC-IR as observed with confocal microscopy. All rods were labeledfor PKC-IR, including the inner segments, synaptic terminals, andtelodendria (arrows). A complete correspondence of double labeling ofrods for BD-17-IR and PKC-IR is shown in the merged image (bottompanel). Scale bar 10 �m.

Fig. 4. A: Immunoreactivity of �1-subunit was present in the innersegments and synaptic terminals of photoreceptors, similar to thatobserved with 62-3G1 and BD-17. B: At a higher magnification, sep-arated patches of label in the outer plexiform layer (OPL) resembledthe synaptic terminals of rods. Weak label was also present in cellbodies of horizontal cells (H), bipolar cells (B), amacrine cells (A), andcells in the ganglion cell layer (asterisk). In addition, the inner plexi-form layer (IPL) was labeled throughout, with hints of lamination.Scale bars 10 �m in A,B.

444 J. ZHANG ET AL.

illustrates that �1-labeled bipolar cell dendrites extendedto the distal region of the OPL, approached calbindin-IRcone pedicles, but did not overlap with them. Conversely,�1-labeled bipolar cell dendrites appeared to overlap thedistribution of rod synaptic terminals as labeled by BD-17(Fig. 6, right column), suggesting that �1-labeled bipolarcells were more likely to receive input from rods thancones. This finding, of course, would require ultrastruc-tural verification.

There is mounting evidence that populations of bipolarcells in salamander retina are GABAergic (Yang and Ya-zulla, 1988, 1994; Yang, 1997, 1998; Yang and Wang,1999), raising the question as to whether GABAergic bi-polar cells also express the �1-subunit of GABAA recep-tors. Double labeling showed that �1-labeled bipolar cellswere not GABA-IR (Fig. 7, left column). Calretinin-IRlabels the vast majority of bipolar cells in salamanderretina, including most of the GABA-IR bipolar cells (Yangand Zhang, 2000; Deng et al., 2001). Surprisingly, how-ever, �1-labeled bipolar cells were not calretinin-IR either(Fig. 7, right column). This finding suggests that the �1-IRbipolar cells were a distinct subtype that differ neuro-chemically from GABA-IR, calretinin-IR, and GABA-IR/calretinin-IR bipolar cells.

On occasion, we observed amacrine cells with large so-mas in the proximal of the INL that were �1-IR. Theseamacrine cells also were GABA-IR (Fig. 7, left columnarrowheads) and could have been the large dopaminergicamacrine cells (Watt et al., 1988). However, double label-ing for �1-subunit and TOH-IR showed that they weremutually exclusive populations (Fig. 8, arrowheads). Thesomas clearly were not double-labeled. Also, their pro-cesses, although predominantly in distal sublamina a ofthe IPL were interlaced rather than overlapping. TOH-IRamacrine cells are mainly stratified in the most distal IPL,with some processes extending to the proximal IPL (Fig.8), whereas the �1-IR amacrine cell processes appear justproximal to the TOH-IR processes. Based on these obser-

vations, �1-IR amacrine cells are GABAergic rather thandopaminergic.

There were no obvious �1-IR puncta detected as de-scending processes between the distal and proximal IPL,suggesting that the labeled processes in proximal IPLcame from somas located in the GCL. It is uncertainwhether they would be ganglion cells or displaced ama-crine cells.

�3-Subunit antibody labels the synapticlayers and Muller cells

The �3-IR was most prominent in the IPL with a bit lessintensity in the OPL (Fig. 9A). �3-IR also appeared invertically oriented streaks extending through the entireretina from the outer to inner limiting membranes, indic-ative of Muller cells. Label in the OPL was punctate andirregular with no shape that typified terminals of photo-receptors. The thickness of the labeled band in the OPLsuggests that the label was due to a combination of Mullerprocess, photoreceptor terminals, and processes of second-order neurons. �3-IR in the IPL appeared as numerouspuncta evenly distributed throughout the IPL, with nohints of lamination.

�1-Subunit

�1-IR was punctate and densest in the OPL (Fig. 9B) butcould not be assigned to any particular structure. Lessintense �1-IR was diffusely distributed throughout theIPL with no indication of lamination. Scattered punctawere present in the INL and GCL, but no somata could berecognized. It is possible that Muller cells contributed tothe overall labeling of �1-IR.

�2-Subunit labels cone photoreceptors

The �2-subunit labeled almost all cone photoreceptors(Fig. 10A). The labeled structures included the outer andinner segments, soma, pedicle, and telodendria. Threemorphologic types of cones were identified: large singlecones characterized by a short, conical outer segment anda long inner segment with an ellipsoid (Fig. 10A, arrow-heads), double cones with paired inner and outer seg-ments (Fig. 10A, arrows), and a small single cone with aslender inner and outer segments (data not shown). Dou-ble labeling for �2-IR and calbindin-IR showed that all�2-immunoreactive cones, including large single cones(Fig. 10B, arrowheads), double cones (Fig. 10B, arrows),and small single cones (data not shown) expressedcalbindin-IR. A curious observation was that, except forthe outer segments, cones that were extremely bright forcalbindin-IR were relatively weak for �2-IR. These ap-peared to be the accessory member of double cones (Fig.10A, arrows). The principal cone is identified by a small,less tapered outer segment and more distal placed ellip-soids (Fig. 10A,B, open arrows). The accessory cone (Fig.10, arrows) is identified by a longer outer segment and abroad inner segment in its inner half (Mariani, 1986;Sherry et al., 1998). Accessory cones appeared intenselystained by calbindin-IR (Fig. 10B, arrows) but less in-tensely with �2-IR, compared with principal cones (Fig.10A, open arrows).

Weak puncta of �2-IR were found throughout the IPL(Fig. 10A) and in cells in the GCL. They may be ganglioncells and/or displaced amacrine cells (Fig. 10A, asterisk).Very faint somatic labeling occasionally was detected in

Fig. 5. A,B: Immunoreactivity of the �1-subunit was observed in apopulation of presumed rod-dominant OFF bipolar cell (B in A,B)including Landolt’s club (LC in A,B), dendrite (arrowhead in B),descending axons that ramified at the most distal level of the innerplexiform layer (IPL; small arrows in A,B). A subgroup of largeamacrine cell (A in B) was also labeled. OPL, outer plexiform layer.Scale bar 10 �m in B (applies to A,B).

445GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA

horizontal cells (Fig. 10A, H). The results are summarizedin Table 1.

DISCUSSION

We have shown here the differential distribution of sev-eral isoforms of the most abundant subunits �, �, � of

GABAA receptors. All three �-subunit antibodies appearto label the inner segments and synaptic terminals of rodphotoreceptors. Cell bodies of some horizontal, bipolar,amacrine, and possibly ganglion cells were weaklystained. �1-IR stained a population of presumed OFF roddominated bipolar cell completely and a subtype ofGABAergic amacrine cell. �2-IR was found prominently in

Fig. 6. Double labeling of �1-immunoreactivity (IR) bipolar cellswith cone-specific marker calbindin-IR, and rod-specific marker BD-17-IR as observed with confocal microscopy. Left column: Upperpanel, an �1-IR labeled bipolar cell (B) with dendrite extended to thedistal region of the outer plexiform layer (arrow, arrowhead points toan �1-IR amacrine cell); middle panel, cones (C) were labeled forcalbindin-IR; lower panel, merged image shows that the �1-IR labeled

dendrite did not approach the calbindin-IR cone pedicle (arrow). Rightcolumn: Upper panel, arrows point to an �1-subunit labeled bipolardendrite (B); middle panel, BD-17-IR labeled rod descending axons(open arrows) and synaptic terminals (arrows); lower panel, themerged image shows the labeled dendrite appeared to approach andoverlap with BD-17-IR rod terminals (arrows). Scale bars 10 �m.

446 J. ZHANG ET AL.

cone photoreceptors, and in cell bodies of some horizontal,amacrine, and possibly ganglion cells. The distribution ofpuncta in the IPL were different for �1- and �3-IR, and for�1- and �2-IR, suggesting that the two �-subunits and thetwo �-subunits were not located at the same synapticsites.

�1-Subunit

In rod dominant retinas of mammals, rod bipolar cellsreceive input from rods and send messages directly to ONcone bipolar cells in sublamina b and indirectly to OFFcone bipolar cells in sublamina a through AII amacrinecells (Kolb and Famiglietti, 1974). The situation is quitedifferent in retinas of salamander, where bipolar cellsreceive input from both rods and cones (Lansansky, 1973,1978) and show different relative strengths of the input,rod-dominated, and cone-dominated (Hensley et al., 1993).The output of bipolar cells in the IPL follows the generalobservations of the segregation of ON and OFF pathwaysin sublaminas b and a, respectively (Hare et al., 1986). It

has been reported that the cone signals of bipolar cellswere predominantly sent to the central IPL and the rodsignals predominantly to the inner and outer margins ofthe IPL (Wu et al., 2000). The axon terminals of �1-IRbipolar cells described in this study ramified along thedistal margin of the IPL in a narrow stratification level,which matches well with the stratification level of type 1(or type 2) monostratified OFF bipolar cells that are mostrod-dominated as identified electrophysiologically by Wuet al. (2000). In addition, our double labeling results pro-vide support for the rod-dominant input to the �1-IR bi-polar cells. Ultrastructural analysis is necessary to verifythis issue.

The �1-IR seems to be distributed uniformly on thesoma, dendrites, the axon, and its terminals of the roddominant OFF bipolar cells. This finding is similar to thehippocampal pyramidal neurons, where the GABAA �1-subunit was equally distributed on soma, proximal anddistal dendrites, axon hillock, and the spines (Nusser et

Fig. 7. Double labeling of �1-immunoreactivity (IR) with�-aminobutyric acid (GABA)-IR and with calretinin-IR, as observedwith confocal microscopy. Left column: �1-IR labeled bipolar cells (B)were not double-labeled for GABA-IR. An �1-IR labeled amacrine cellwas double-labeled for GABA-IR (arrowheads). Right column: �1-IRlabeled bipolar cells were not double-labeled for calretinin-IR. Scalebars 10 �m.

Fig. 8. Double labeling of �1-immunoreactivity (IR) with anti-tyrosine hydroxylase (TOH)-IR as observed with confocal microscopy.Left column: An �1-subunit labeled amacrine cell (arrowhead) withprocesses predominantly in the most distal inner plexiform layer(arrows) was not double-labeled for TOH-IR. Right column: The �1-IRprocesses appeared to be interlaced with the TOH-IR processes,rather than overlapping. Scale bars 10 �m.

447GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA

al., 1996) but is different from the retinal dopaminergicneurons where the GABAA receptors containing �1-subunit were differentially located on large dendrites(Gustincich et al., 1999), suggesting a difference that isrelated to the neuron being an output neuron (pyramidalcell and bipolar cell) rather than a laterally projectinginterneuron (amacrine cell).

Yang and Zhang (2000) reported that approximately74% of the salamander bipolar cells were calbindin-IRalone, 4% were GABA-IR alone, and 22% were double-labeled. It is clear from this study that the �1-IR bipolarcells constitute a distinct bipolar cell population from thecalbindin and GABA-IR bipolar cells, and these appear tooccur also at a low frequency.

The source of GABAergic input to the dendrites of �1-IRbipolar cells could be the GABAergic horizontal cells(Yang and Yazulla, 1988). This possibility is consistentwith the ultrastructural observations that horizontal cellssynapse on bipolar cell dendrites in both mud puppy andsalamander (Dowling and Werblin, 1969; Lasansky,1973). The other possible source could be the GABA-IRinterplexiform cells (IPCs) described by Yang and Yazulla(1988) in this species. GABAergic IPCs making synapticcontact onto bipolar cell dendrites have been found inretinas of mammals (for example, Pourcho and Goebel,1983; Chun and Wassle, 1989; Greferath et al., 1994). Theexistence of �1 receptors on bipolar cell dendrites was inagreement with ultrastructural results in rabbit, rat, andmouse, demonstrating that �1-IR was found on the den-dritic tips of bipolar cells (Greferath et al., 1994, 1995;Havercamp and Wassle, 2000). However, it is contradic-tory to the observations in salamander (Lukasiewicz et al.,1994; Wu and Maple, 1998) in that bipolar dendriteslacked GABA sensitivity. Wu and Maple (1998) reportedthat the chloride current could only be elicited by focalapplication of GABA to the bipolar axon terminals but not

the dendrites, indicating that GABA receptors are not onthe bipolar cell dendrites but on axon terminals. Onepossible explanation for this discrepancy is that the fre-quency of �1-IR bipolar cells is low and, therefore, was notincluded in the study of these two groups. Du and Yang(2000) reported the presence of GABAA receptors on den-drites of bipolar cells in bullfrog. This finding may or maynot correspond to our �1-IR bipolar population; �1-IR com-prised only a small population of rod dominated bipolarcells, whereas the dendrites of all ON and OFF bipolarcells that they tested showed GABAA sensitivity.

The source of GABA input to the axon terminals of �1-IRbipolar cells likely comes from GABAergic amacrine cells.Several lines of electrophysiological evidence demon-strated that the axon terminals of bipolar cells insalamander receive GABAergic amacrine feedback inputthat is mediated predominately by GABAC receptors, butalso by GABAA receptors (Lukasiewicz et al., 1994;Lukasiewicz and Shields, 1998b). Here, we provide mor-phologic evidence for GABAA-mediated feedback based onthe observations that �1-IR was present in axon terminalsof a population of salamander bipolar cells. In our recentelectron microscopic (EM) work (Zhang and Yang, manu-script in preparation), conventional synapses were ob-served from GABAergic amacrine cells onto the bipolarprofiles in very distal sublamina a of the IPL, an areawhere �1-IR bipolar terminals are stratified. This findingis in agreement with the earlier report that amacrine cellsmade feedback synapses onto bipolar cell processes in thisspecies (Wong-Riley, 1974).

We suggest that the �1-IR bipolar cells possess highaffinity GABAA receptors to BZ, because the type IGABAA/BZ high affinity receptor contains an �1-subunit(Pritchett et al., 1989a,b). The time course of the GABAresponses of �1-IR bipolar cells may differ from thosebipolar cells containing other �-subunit variants, because

Fig. 9. Immunoreactivity (IR) of �3- and �1-subunits. A: Puncta of�3-IR were present in the outer plexiform layer (OPL), inner plexiformlayer (IPL) and in broad vertical streaks that extended through thefull thickness of the retina, this latter label due likely to Muller cells.B: �1-IR puncta were densest in the OPL with no particular structure

evident. Punctate label was less intense and more diffusely scatteredover the IPL, compared with �3-IR. No somatic labeling was detectedin the inner nuclear or ganglion cell layers for either antibody. Scalebar 10 �m in B (applies to A,B).

448 J. ZHANG ET AL.

it is reported that the GABAA receptor-gating kineticsdepend on the �-subunit isoform (Gingrich et al., 1995).

We found a population of GABAergic amacrine cell thatexpressed �1-IR. Despite the similarity with the dopami-nergic amacrine cell, the �1-IR and dopamine amacrinecell comprise separate types. Recently, Pang et al. (2002)correlated the distribution of axonal arbors of amacrinecells in the IPL with cellular physiology. The proximalposition of the soma and narrow stratification in the distal

IPL suggests that the �1-IR amacrine cell most closelyresembles the type 1 narrowly stratified OFF amacrinecell.

�3-Subunit

There was no obvious somatic labeling for �3-IR. There-fore, the types of retinal neuron expressing this subunitcan not be determined at this time. The vertical labeling,extending the full thickness of the retina, clearly indicates

Fig. 10. Double labeling of cone photoreceptors for �2-immunoreactivity (IR; A) and calbindin-IR (B). Both antibodies ap-peared to label all cone photoreceptors, including single cones (arrow-heads), principal member of double cones (open arrows), andaccessory member of double cones (arrows). Label was present in theentire photoreceptor, including outer and inner segment, soma, pedi-cle, and telodendria. Double cones, particularly the accessory mem-

ber, were strongly labeled for calbindin-IR (B, arrows). However, itappeared as if the accessory member was relatively weak for �2-IR (A,arrows), compared with the principal member (A,B, open arrows).Somatic �2-subunit labeling was occasionally found in horizontal cells(H) and cells in the ganglion cell layer (asterisk). Scale bar 10 �min B (applies to A,B).

449GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA

the presence of Muller cells. However, the uniformity anddensity of label in the OPL and IPL cannot be accountedfor solely by Muller cells processes. �3-IR was uniformlydistributed in a dense band across the whole depth of theIPL, whereas �1-IR was mainly stratified in distal sub-lamina a with a sparse band in proximal sublamina b.This finding suggests that the two subunits were not sub-stantially colocalized at synaptic sites and, thus, involvedifferent synapses. Similar observations were found in ratIPL in which the �1 and �3 were not extensively colocal-ized (Greferath et al., 1995). If the �� isoforms are thesame, different pharmacologic properties of receptors com-posed of �1�� or �3�� would be expected. It is known thatthe high affinity type I GABAA/BZ receptor contains an�1-subunit, whereas the low affinity type II GABAA/BZreceptor contains an �3-subunit (Pritchett et al., 1989a,b).Thus, in the salamander IPL, it is suggested that lowaffinity type II GABAA/BZ receptors outnumber the type Ireceptors.

�-Subunit

All three �-subunit antibodies labeled the rod photore-ceptors: the inner segments and terminals. By usingPKC-IR that labels salamander rods (Zhang and Yang,2001), the BD-17/PKC double-label showed complete cor-respondence of label in rods. Vitanova et al. (2001) re-ported weak BD-17 immunostaining in the OPL in frog,but they were uncertain as to the specificity of that label.By using reverse transcription-polymerase chain reactionmethods, single rods of rat retina displayed mRNA for �1-and �2-subunits (Grigorenko and Yeh, 1994), and in iso-lated rat photoreceptors, only �3 mRNA was amplified butnot the �1 and �2 (Vardi et al., 1998). Contradictory re-sults were found with the use of BD-17 antibody in rat(Greferath et al., 1993) in which the photoreceptor termi-nal membrane was not immunolabeled; the only label wasintracellular DAB reaction product, the same as reportedfor goldfish rods using monoclonal 62-3G1 (Yazulla et al.,1989). There is ultrastructural evidence demonstratingthat horizontal axon terminals synapse onto rod spherules

within the invagination (Linberg and Fisher, 1988); and arecent confocal microscopic double-label study alsoshowed that the vesicular GABA transporters were lo-cated at horizontal cell terminal tips entering rod spher-ules (Jellali et al., 2002). Normann and Pochobradsky(1976) recorded oscillations in rod and horizontal cellsthat they suggested was evidence for feedback to rods inthe toad retina. Although there has been no physiologicalevidence that GABAergic horizontal cells feedback to rodsin any vertebrate species. For the salamander, it is possi-ble that the chloride current of the GABAA receptor at therod spherule connecting to the very thin descending axonwould not be detected by whole cell recording at the rodsoma.

The two monoclonal antibodies BD-17 and 62-3G1 dif-ferentially labeled the salamander retina. Monoclonal an-tibody 62-3G1 stains clearly the OPL and IPL, especiallyon short-fixed sections, whereas BD-17-IR was detected inthe OPL but not in the IPL. In frog retina, the distributionof �2/�3-IR (BD-17) was dense throughout the IPL, butweak in the OPL (Vitanova et al., 2001), an apparentreverse of what we observed with BD-17 in thesalamander. Differential staining was also observed ingoldfish (Lin and Yazulla, 1994), in which 62-3G1 wasfound in both plexiform layers, whereas BD-17 was ex-pressed only in the IPL. The situation in mammalianretina is different still, with BD-17 rather than 62-3G1observed in the OPL (i.e., Hughes et al., 1991; Vardi et al.,1992; Vardi and Sterling, 1994; Greferath et al., 1994,1995; Yazulla et al., 1997). This differential labeling inretina remains a curiosity because both 62-3G1 and BD-17reportedly recognize the same epitope (Ewart et al., 1992)and show similar immunocytochemical sensitivity in thebrain (Richards et al., 1987; De Blas, 1988; Moreno et al.,1994). Among retinas, only in zebrafish do both antibodiesproduce the same labeling pattern (Yazulla and Stud-holme, 2001). It is possible that a portion of the �2 and/or�3 isoforms in the GABAA receptor were different in theOPL and IPL and that the BD-17 was more susceptible tofixation in the salamander IPL than in OPL.

TABLE 1. Summary of the Localization of GABAA Subunits in Salamander Retina1

�1 �3 �1 bd17 3G1 �1 �2

RodsIS �� �� ��SomaTerminals �� �� ���Telodendria

ConesIS ��Soma �Terminals ��Telodendria �

OPL �� �� �� 2 2 �� 2

INLHC soma � � �BC soma ��� �LC ���Dendrite ���Axon-stratification � indis sub aAC soma � � �� � � �

IPL � ��� ��� �� �� �Sub a �� top levelSub b �� most proxi

GCL � � � �Muller � � � � � �

1The number of ‘�’ in the table represents a qualitative estimation of relative labeling intensity among retinal structures in the same section for a particular subunit antibody.GABA, �-aminobutyric acid; IS, inner segment; OPL, outer plexiform layer; INL, inner nuclear layer; HC, horizontal cell; BC, bipolar cell; LC, Landolt’s club; AC, amacrine cell;IPL, inner plexiform layer; GCL, ganglion cell layer.2It seems that, in the OPL, the more discerned structures labeled were the synaptic terminals of photoreceptor.

450 J. ZHANG ET AL.

�-Subunits

Pharmacologic studies have shown that the �-subunit isessential for a GABAA receptor that is sensitive to BZmodulation (Pritchett et al., 1989a,b; Gunther et al.,1995). The �2-subunit is the most widely distributed�-subunit in the brain as well as in retina (Gerferath et al.,1995; Pirker et al., 2000). Replacement of �2 with �1 in a��� ternary receptor resulted in a reduced potentiation bycertain BZ agonists (Hevers and Luddens, 1998). In thisstudy, differences in labeling pattern of the two subunitantibodies were observed. The most prominent location of�2-IR was in cones, including the terminal telodendria,whereas the �1-IR was present in the OPL but could not beassigned to photoreceptor terminals. The �2-IR was weakand less dense in IPL, whereas for �1-IR, the puncta werepresent in the whole depth of the IPL. Thus, the �1- and�2-subunits could be located at different synaptic sites atleast in the IPL, although partial colocalization in the IPLcannot be excluded. Of note is the codistribution of the �3-and �2-subunits in both the OPL and IPL, indicative of BZreceptor action. GABAA receptors composed of �3��1-subunits would have different BZ pharmacology comparedwith that of GABAA receptors composed of �3��2-subunits.

The presence of �2-IR in cone photoreceptors is sup-ported by physiological evidence that GABAergic horizon-tal cells feedback to cones in this species (Wu, 1991). Inaddition, fluorescently labeled muscimol binding toGABAA receptors was found in salamander photoreceptorsoma and terminals (Wang et al., 2000). Although, there isno proper explanation for the presence of �2-IR in outersegments of cones, same observation has been reported inouter segments of cones in cat (Hughes et al., 1991). It hasbeen reported that photoreceptors were “sticky” to manyantibodies (Enz et al., 1996; Vitanova et al., 2001), but inthis study, �2-IR was only found in cones and not in themore numerous rods.

Salamander cones were classified according to the rela-tive spectral sensitivity to light. The red-sensitive coneswith pigment absorption peak at 610 nm occur most fre-quently, which include large single cone, double cone, andsmall single cone (Mariani, 1986; Perry and McNaughton,1991). Also, both principal and accessory members of thedouble cones were immunoreactive to the same L-coneopsin (Sherry et al., 1998). However, the accessory mem-ber of the double cones appeared to be weaker for �2-IR. Itis possible that the accessory cones are less sensitive to BZcompared with the principal members.

An earlier study, using in vitro autoradiography, re-ported that there was specific binding of [3H]flunitraz-epam in the salamander IPL but not in the OPL (Yang etal., 1992). However, here we reported that the �-subunitthat is necessary for BZ sensitivity was present also in theOPL. The reasons for this discrepancy are not clear. Onepossibility could be that, in the BZ binding study, tissueswere fixed overnight in 0.1–1% paraformaldehyde at 4°C,whereas in this study, fixation was limited to 1 hour.Thus, the combination of higher density of GABAA/BZreceptors in the IPL and a difference in the relative sen-sitivity of GABAA/BZ receptor to fixation could account forthe absence of BZ binding in the OPL. Because the mod-ulatory effect of BZ on GABAA receptors depends on the�-subunit variant and �-subunit variant, the difference

and complexity of GABAA/BZ receptors between OPL andIPL remains to be determined.

Axosomatic GABAA receptor

In this study, �1-, �2/3-, and �2-subunits were found incell bodies of some horizontal cells, bipolar cells, amacrinecells, and possibly ganglion cells. This finding is in agree-ment with physiological results that horizontal cells pos-sess GABAA receptors (Stockton and Slaughter, 1991; Ka-mermans and Werblin, 1992) and with morphologicobservations of axosomatic synaptic connections betweenamacrine cell process and cell bodies of bipolar, amacrine,and ganglion cells (Wong-Riley, 1974). The subunit label-ing was not always restricted to the synaptic sites; forexample, �1-IR was present in the cytoplasm of the rod-dominated OFF bipolar cells and the likely type I nar-rowly stratified OFF amacrine cells. Possibly, the recep-tors that were in transit to the synaptic sites or that werebeing degraded were labeled. Considering that the punc-tate label of �-, �-, �-IR in the frog IPL represents synapticsites (Vitanova et al., 2001), future EM work will resolvethe synaptic structure containing these subunits in rela-tion to specific cell types and, thus, will provide ultrastruc-tural evidence to support the physiological results regard-ing GABAA receptor function in the salamander retina.

CONCLUSIONS

We have shown in the salamander retina the differen-tial distribution of several isoforms of GABAA receptorsubunits �, �, and �, which are the most abundant subunitcomposition of GABAA receptors in retina and CNS. Thedistinct cellular and subcellular location of individual sub-units suggests a complex GABAergic pharmacology in thesalamander retina that includes photoreceptors and bipo-lar cells as well as the GABAergic interneurons: horizon-tal cells and amacrine cells.

ACKNOWLEDGMENTS

We thank Dr. Stephen Yazulla for critical reading of themanuscript and valuable suggestions. We also thankKeith Studholme and Yeva Fernandez for technical assis-tance.

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453GABAA RECEPTOR SUBUNITS IN SALAMANDER RETINA