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Expression of Alpha 7 Nicotinic Acetylcholine Receptors by Bipolar, Amacrine, and Ganglion Cells of the Rabbit Retina

Nina A. Dmitrieva, Christianne E. Strang and Kent T. Keyser

Department of Vision Sciences and the Vision Science Research Center, University of Alabama at Birmingham, Birmingham, Alabama

Correspondence to: Kent T. Keyser, PhD, Vision Science Research Center, The University of Alabama at Birmingham, WORB, 626 Birmingham, AL 35294-4390. E-mail: ktkeyser{at}uab.edu

	Summary

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Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express ß2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of 7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express 7 nAChRs. At least three different populations of cone bipolar cells exhibited 7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of 7-positive amacrine cells. Some GABAergic amacrine cells also displayed 7 immunoreactivity; 7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of 7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on 7 nAChRs expressed by GCs themselves. (J Histochem Cytochem 55:461–476, 2007)

Key Words: cholinergic cells • immunohistochemistry • glycinergic cells • GABA • kinesin • synaptophysin • PSD-95 • -bungarotoxin

	Introduction

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THE VERTEBRATE RETINA utilizes two fast excitatory neurotransmitters: glutamate and acetylcholine (ACh). Glutamate is the primary neurotransmitter of the vertical pathways in the retina and carries signals from photoreceptors to bipolar cells, bipolar cells to ganglion cells (GCs), and then on to other parts of the brain. Amacrine cells contribute to the modulation of the vertical pathways by mediating lateral interactions between bipolar cell terminals and dendrites of ganglion cells (Sterling 1998; Kolb et al. 2001). Cholinergic amacrine cells in the mammalian retina comprise two mirror symmetrical populations of cells (Vaney 1984; Masland et al. 1984). One population, the OFF cells, is located in the inner nuclear layer (INL) with dendrites stratifying in a narrow band in the outer portion of the inner plexiform layer (IPL); the other population, the ON cells, is displaced to the ganglion cell layer (GCL) with dendrites arborizing in a narrow band in the inner portion of the IPL. ACh is released tonically (Masland and Livingstone 1976) from the dendrites of the ON cells at the onset of illumination and from the OFF cells at the cessation of light stimulation (Masland et al. 1984). However, the cellular mechanisms underlying the effects of ACh in the retina are only partly understood.

Functional studies in cat (Ikeda and Sheardown 1982; Schmidt et al. 1987) and rabbit retinas (Masland and Ames 1976; Ariel and Daw 1982a,b; Masland et al. 1984) revealed that application of ACh or acetylcholinesterase inhibitors influenced responses in many GC subsets, the visually driven responses of ON- and OFF-center GCs, and the responses of all GCs that are sensitive to motion. In vitro experiments with cat retina showed that intra-arterially applied ACh increased both the rod- and cone-driven b-wave amplitude of the electroretinogram, indicating increased depolarization of bipolar cells. ACh also induced a decrease in the optic nerve response, which reflects the summed activity of GCs (Jurklies et al. 1996). Electrophysiological studies revealed that the actions of ACh are mediated in part via nicotinic ACh receptors (nAChRs) (Masland and Ames 1976).

nAChRs are members of the family of ligand-gated ion channels. They are pentameric receptors comprised of subunits 2–6 and ß2–ß4 in /ß combinations or of subunits 7–9 in homomeric forms (McGehee 1999; Lindstrom 2000; Dani 2001). The subunit composition of nAChRs has been shown to determine the pharmacological and functional properties including agonist/antagonist affinity, open time, and desensitization rate. For instance, 7 nAChRs are characterized by their sensitivity to blockade by -bungarotoxin (Bgt, a protein from the venom of the banded krait), rapid desensitization, and relatively low sensitivity to activation by ACh. In contrast, heteromeric nAChR subtypes such as 3ß2 are Bgt-insensitive. Furthermore, 3ß2 nAChRs desensitize more slowly than the 7 nAChRs and are potently activated by ACh (Nelson and Lindstrom 1999). Optical recordings of intracellular free calcium changes in rabbit retinal GCs suggested that the actions of ACh on these cells are mediated solely by Bgt-insensitive nAChRs and are not attributable to indirect effects mediated by glutamatergic bipolar cells (Baldridge 1996). However, data from subsequent extracellular recordings, single-cell RT-PCR of rabbit retinal GCs (Reed et al. 2002; Strang et al. 2003,2005a), and the results of molecular and pharmacological studies of rat retina (Moretti et al. 2004) suggest that both Bgt-sensitive and -insensitive nAChR subtypes are involved in generating the light responses of some GCs. Both receptor subtypes are expressed directly by GCs, and nAChRs are expressed by upstream cells as well. Indeed, immunohistochemical (IHC) studies have demonstrated that in rabbit retina many GCs as well as several types of amacrine cells express Bgt-insensitive nAChRs containing ß2 subunits, some of which are in combination with 3 and possibly other subunits (Keyser et al. 2000; Dmitrieva et al. 2001,2003).

However, identification of cholinoceptive neurons expressing Bgt-sensitive nAChRs in mammalian retina has been more problematic. Pourcho (1979) described the distribution of Bgt-binding sites in mouse retina as shown by application of a conjugate of Bgt and horseradish peroxidase. In this study, labeled synaptic contacts were observed between amacrine cells and bipolar cells and at amacrine cell to amacrine cell synapses. No labeled contacts were described at amacrine to GC synapses, yet physiological data suggests that retinal GCs express either Bgt-sensitive or -insensitive nAChRs or both (Strang et al. 2005a). Thus, the identity of the retinal neurons that express solely Bgt-sensitive or both Bgt-sensitive and -insensitive nAChRs in mammalian retina and the subcellular localization of Bgt-sensitive nAChRs within the neurons are not clear. Furthermore, it remains to be determined whether these cholinoceptive neurons are the part of rod- and/or cone-driven retinal circuits. To address these questions, we examined the distribution of 7 nAChRs by means of single-, double-, and triple-label IHC using 7 subunit-specific antibodies together with antibodies against 3ß2 nAChRs and with specific markers for specific types of bipolar, amacrine cells and for synaptic sites in rabbit retina. As a test of the specificity of the 7 nAChR antibody used, we also performed studies using fluorescently labeled Bgt together with the 7 antibody. Our observations indicate that 7 nAChRs are implicated in information processing in multiple retinal circuits.

	Materials and Methods

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Tissue Preparation
Eyes were obtained from adult Dutch-belted rabbits. Animal treatment was in accordance with NIH guidelines and the policies of the Animal Resources Program of the University of Alabama at Birmingham. Rabbits were sacrificed with an overdose of sodium pentobarbital (120 mg/kg). Eyes were removed, the anterior pole of each eye was cut away, and the vitreous was removed completely. For IHC, the retinas from five individual animals were used. Eyes were hemisected, and the posterior eyecups were fixed by immersion in 1% paraformaldehyde with 0.34% L-lysine and 0.05% sodium-m-periodate (1% PLP) in 0.1 M phosphate buffer (PB), pH 7.2–7.4, for 2 hr at room temperature. Eyecups were then placed into 0.1 M PB with 30% sucrose for 24–48 hr, embedded in medium (OCT; Sakura Finetek, Torrance, CA), frozen, and sectioned (10–12 µm) on a cryostat. For Bgt-binding experiments, isolated retinas from four animals were incubated in fluorescently labeled Bgt, fixed in 1% PLP, and processed free floating for 7 IHC as described below.

For gel electrophoresis, retinas from four individual animals were removed from pigment epithelium. Protein from these retinas was analyzed by Western blots as described below.

Antibodies
Goat polyclonal antibody sc-1447 against the nAChR 7 subunit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody sc-1447 was raised in goat against a peptide with the sequence WKFAACVVDRLCLMAFSVFT, mapping at the cytoplasmic loop between the third transmembrane domain (TM) and TM4 and partially into TM4 of the human 7 subunit (NCBI GenBank accession number P36544). Specificity of this antibody in rabbit retina was tested in competition studies with blocking peptide (Santa Cruz Biotechnology) and in Western blot analysis.

To evaluate expression of 7 nAChRs by different types of neurons, we incubated rabbit retina with antibody sc-1447 in conjunction with other commercially available antibodies against different neurochemical markers. These antibodies have been previously used in rabbit retina to identify specific subpopulations of retinal neurons. Rabbit anti-NR1 subunit of NMDA receptor (Chemicon; Temecula, CA) and mouse anti PSD-95 (Affinity Bioreagents; Golden, CO) have been used to localize postsynaptic density protein PSD-95 and NMDA receptors in rat and monkey retinas. Results for each of the markers used in this study were consistent with those previously reported. The reference for each of the markers, types of retinal neurons they have been reported to label, and the working dilutions of antibodies are listed in Table 1 . All secondary antibodies were raised in donkey and specifically prepared for multiple-labeling studies (Jackson Immunoresearch Laboratories; West Grove, PA).

SDS-PAGE and Western Blotting
In four experiments, isolated retinas from two eyes were homogenized in five volumes of ice-cold lysis buffer (PBS), pH 7.4, containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (1:30; Sigma) and incubated at 4C for 30 min (Jones et al. 2001). Tissue homogenate was then centrifuged (15,000 x g for 20 min), the supernatant was retained and mixed with an equal amount of sample buffer and heated for 5 min at 95C. For gel electrophoresis, 15 µl of sample was loaded onto 10% SDS polyacrylamide gels and run at 100 V for 2 hr, after which the proteins were transferred to nitrocellulose membranes (Pierce Biotechnology). Nitrocellulose membranes were blocked with 7% non-fat milk (Bio-Rad) in PBS/0.1% Tween-20 (PBST) for 2 hr at room temperature, followed by incubation in goat antibody sc-1447 (1 µg/ml) in PBST /blocking solution overnight at 4C. After rinsing in PBS (3 x 10 min), the membrane was incubated in mouse anti-goat IgG (Jackson ImmunoResearch Laboratories) containing horseradish peroxidase diluted in PBST for 2 hr and washed three times. Immunoreactive bands were detected with a colorimetric detection Opti-4CN kit (Bio-Rad). Controls were performed by omitting the primary antibody from the procedure.

IHC
Indirect fluorescence was used to perform single, double, and triple labeling with different antibodies (see Table 1). Cryostat sections of rabbit retina were washed three times in PBS and then pretreated with 0.8% of 2-mercaptoethanol (Bio-Rad) in PBS/Triton solution for 1 hr at room temperature. We incorporated the mercaptoethanol treatment step to improve 7 recognition by sc-1447 antibody. 2-Mercaptoethanol reduces disulfide bonds in nAChRs (Watty et al. 1997), resulting in increasing the exposure of the antigenic epitope by either denaturation or dissociation of interacting proteins (Wilson and Bianchi 1999). It should be noted that mercaptoethanol retrieval of 7 antigenicity did not influence labeling patterns revealed by the other antibodies used in these studies. After the treatment, the tissue was rinsed at least four times in PBS, blocked in 10% donkey normal serum/PBS/Triton for 1 hr, and in avidin–biotin blocking solutions to eliminate nonspecific background binding. Sections were then incubated in primary antibody, sc-1447, diluted 1:50 in PBS/Triton for 24–48 hr at 4C. The tissue was next washed in three changes of PBS followed by incubation in secondary biotinylated donkey anti-goat IgG for 1 h. After incubation in the secondary antibodies, retinas were rinsed and incubated in streptavidin conjugated to Oregon Green 488 (green fluorescence; Molecular Probes, Eugene, OR) for 1 hr, rinsed, mounted in PermaFluor Aqueous mounting medium (Thermo Shandon; Pittsburgh, PA), and coverslipped.

In double- or triple-labeling IHC experiments, the tissue was exposed to a mixture of primary antibodies followed by a mixture of species-appropriate secondary antibodies made in donkey and tagged to Texas Red (red fluorescence) and to aminomethylcoumarin (AMCA, blue fluorescence). In double-labeling experiments with goat sc-1447 and rat MAb210, the sections initially were stained for MAb210 with Texas Red and then for sc-1447 with Oregon Green 488. For goat sc-1447 and mouse anti-kinesin, the tissue was labeled first for 7 by goat sc-1447 (Oregon Green 488) followed by labeling mouse antibody against kinesin with Texas Red. Control procedures involved systematically omitting each of the primary and secondary antibodies.

As an independent test of the specificity of the 7 nAChR antibody used, we used Alexa-Fluor 594-conjugated Bgt in conjunction with sc-1447. This allowed us to compare the pattern of -Bgt binding in the IPL with that of the anti-7 antibody. The Bgt-binding protocol was modified from that of Pourcho (1979). Live dark-adapted retinas were incubated in oxygenated Ames medium at 4C containing 6.7 µM Alexa-Fluor 594 conjugated to Bgt (Invitrogen/Life Technologies; Gaithersburg, MD) for 2 hr. To prevent competitive binding by ACh released by the population of OFF starburst amacrine cells in dark conditions, synaptic transmission was blocked by the addition of 2 mM cobalt chloride to the medium during incubations. After incubation, the retina was washed for hr in 4C oxygenated Ames medium followed by fixation in 1% PLP for 2 hr at room temperature. Following fixation, 200 µM vertical sections were cut and incubated in 0.8% 2-mercaptoethanol (Bio-Rad) in 0.1 M PBS/0.3% Triton for 4 hr at room temperature. Sections were blocked overnight at 4C with 10% donkey normal serum in 0.1 M PBS/0.3% Triton containing avidin blocking solution. Tissue was then incubated free floating with sc-1447 (1:200) in 0.1 M PBS/0.3% Triton containing 3% sodium azide and biotin blocking solution for 10 days at 4C. Incubation with the primary antibody was followed by four 30-min washes, incubation with biotinylated donkey anti-goat (1:200) for 2 days at 4C, four 30-min washes, and incubation with streptavidin-conjugated Alexa 488 (1:200) for 2 days at 4C. Some retinas were preincubated in 6.7 µM unlabeled Bgt in 4C oxygenated Ames medium for 20 min prior to incubation with the labeled Bgt, as described in the protocol above. This pretreatment abolished nearly all of the labeling, with only occasional flecks of fluorescence remaining (data not shown).

Sections were examined with a Leica TCS NT confocal laser scanning microscope (Leica Microsystems; Mannheim, Germany) using argon and krypton lasers. Each channel was scanned separately, and the images were saved as digital graphics files. Other sections were viewed with a Zeiss Axioplan 2 Imaging microscope (Göttingen, Germany) and images were collected with a Zeiss AxioCam MRm camera.

Cytofluorochrome masking as described in Strang et al. (2003) was used in some cases to provide an objective assessment of colocalization. Briefly, using the Leica software, a frequency distribution of the pixel colors and intensities, each 2-channels, was generated for optical sections. Pixels that represented simultaneous excitation of both channels (yellow) fell in the center of the frequency distribution. The simultaneous fluorescence of two fluorophores within one pixel was defined as indicative of colocalization and masked back onto the image as white pixels.

Brightness and contrast adjustments and figure preparation were completed with Adobe Photoshop (Adobe Systems; San Jose, CA).

	Results

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Specificity of the Antibody
Goat polyclonal antibody sc-1447 used in this study was raised against a 20-amino acid peptide from the extracellular carboxy terminal domain of human nAChR 7 subunit. This region is identical to the corresponding rat sequence. Anti-7 polyclonal antibody (sc-1447) has been used previously for immunostaining 7 nAChRs in transfected cells (line QT6) (Kassner et al. 1998), in rat hippocampal neurons (Kawai et al. 2002), and in chick spinal cord neurons (Roth and Berg 2003). In this latter study, specificity of sc-1447 was confirmed by colabeling with a previously characterized anti-7 antibody, MAb306 (Roth and Berg 2003). In Western blot analysis of proteins extracted from the hippocampal formation, MAb306 recognizes a band of 56 kDa (Fabian-Fine et al. 2001). A band of the same size was recognized by sc-1447 on Western blotting of rabbit retina, further demonstrating that sc-1447 and MAb306 bind to the same protein (Figure 1 ). The labeled band was eliminated when the primary antibody was omitted from the protocol. In our hands, IHC using the anti-7 antibody yielded a specific and repeatable pattern of labeling in the different layers of rabbit retina (Figure 2). Incubation of retinal tissue with sc-1447 preabsorbed with the corresponding 20-amino acid synthetic peptide used for immunization abolished staining in rabbit retinal neurons (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 1 Western blot analysis of rabbit retina probed with sc-1447 antisera. The sc-1447 antisera labeled a 56-kDa band corresponding to the 7 nicotinic acetylcholine receptor (nAChR) subunit.

View larger version (68K): [in this window] [in a new window]   Figure 2 Microscopic images of vertical sections of rabbit retina labeled with goat antibody against 7 nAChRs. Staining was observed in the somata of bipolar cells (b), amacrine cells (a), and ganglion (g) cells and in their dendrites in the OPL and in the IPL (A). The different patterns of arborizations of some labeled amacrine cells is shown in B–E. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bar = 4 µm.

Distribution of 7 nAChRs in Rabbit Retinal Neurons and Their Processes as Revealed by IHC and Bgt Binding

Somata of other labeled cells (32%), presumably amacrine cells, were found in the inner and in the middle or upper middle part of the INL. The presumptive amacrine cell dendrites could be traced into the IPL until they joined diffuse bands of labeled processes that ramified throughout the IPL (Figures 2B–2E). There appeared to be at least two different types of amacrine cells labeled by sc-1447: those that had a single primary dendrite that ramified in two or more levels of the IPL after branching (Figures 2B and 2C) and presumptive "flag cells" (MacNeil and Masland 1998) that sent a single oblique process with thin branching dendrites into the middle of the IPL (Figures 2D and 2E).

There were fewer labeled cells in the GCL. Immunoreactive GCs comprised 12.5% of the total number of immunoreactive cells. In some instances, dendrites of intensely labeled neurons in the GCL could be traced to the inner margin of the IPL (Figure 2A). However, because of diffuse labeling throughout the IPL, we were not able to systematically discern the arborization patterns of individual cells in the GCL. Occasionally, labeled cells in the GCL could be seen to give rise to labeled axons and were therefore identifiable as GCs.

It is worth noting that the intensity of 7 labeling was very low and almost undetectable in the retinal neurons in the area of optic nerve head. This pattern was consistent across retinas and reflects regional differences in the expression of 7 nAChRs in rabbit retina similar to the expression patterns of ß2-containing nAChRs (Keyser et al. 2000). Alternatively, the reduced labeling may be an artifact arising from poor penetration of the fixative or other reagents into this part of the retina. However, distribution of other cell-specific markers did not appear to be comparably affected.

As an independent test of the specificity of the sc-1447 labeling in the IPL, we carried out studies using Alexa-Fluor 594-tagged Bgt. This method yielded consistent punctate labeling on dendrites within the IPL (Figures 3A and 3B). Incubation of retinas with unlabeled Bgt prior to exposure to the Alexa-tagged Bgt abolished nearly all of the labeling, with only occasional flecks of fluorescence remaining (not shown). Double-labeling studies with the 7 antibody (Figures 3C and 3D) and the labeled Bgt revealed an excellent, but not 1:1, correspondence between puncta labeled by the two methods (Figures 3E–3G). There was consistently more antibody labeling than Bgt binding. The imperfect correspondence between the Bgt binding and sc-1447 labeling is addressed in Discussion.

View larger version (103K): [in this window] [in a new window]   Figure 3 Single optical confocal sections showing Alexa-Fluor 594-conjugated -bungarotoxin (Bgt) binding (A,B) and sc-1447 labeling (C,D) at the level of the IPL/GCL boundary at low and high magnifications, respectively. Overlay images (E,F) show correspondence between SC-1447 immunoreactivity and Bgt binding in processes within the IPL. White pixels in CF-masked images (G,H) highlight areas of colocalization. Bar = 10 µm.

Expression of 7 nAChRs by Different Subpopulations of Bipolar Cells and Amacrine Cells

View larger version (126K): [in this window] [in a new window]   Figure 4 Images of double- and triple-labeled vertical sections of rabbit retina using sc-1447 antiserum (green) and four different markers: anti-calbindin (red in A–C; blue in G), anti- PKC (red in D), anti-glycine (red in F,G), and anti-GABA (blue in K). All calbindin-immunorective cone bipolar cells were labeled with 7 antiserum (arrows in C). None of the rod bipolar cells exhibited 7 immunoreactivity (D). Some of the 7-positive bipolar cells were labeled with calbindin and glycine (arrows in E–H). Others were only immunoreactive for glycine antiserum (arrowheads in E–H), and the third population of 7-labeled bipolar cells was negative for both anti-glycine and anti-calbindin (asterisk in E,H). The majority of 7-bearing amacrine cells display glycine immunoreactivity (arrowheads, I, J, L), whereas a few cholinoceptive amacrine cells are positive for GABA (arrowheads in I,K,L). Bars: A–C = 7 µm; D = 15 µm; E–L = 7 µm.

Rod bipolar cells in rabbit retina are intensely immunoreactive for PKC (Young and Vaney 1991). Somata of rod bipolar cells are located in the outmost portion of the INL, give rise to several primary dendrites that branch in the OPL, and their axons descend unbranched through the whole depth of the IPL and arborize as multiple lobes in the innermost part of the IPL (Figure 4D). Double labeling of vertical sections with antibodies against PKC and 7 nAChRs showed no colocalization of the two markers, indicating that 7 nAChRs are not likely to be involved in the modulation of the rod pathway at the level of the first synapse. Thus, only cone but not rod bipolar cells were found to be cholinoceptive, and there were at least three different subsets of 7-expressing cone bipolar cells.

In mammalian retina including rabbit retina, 50% of cholinoceptive amacrine cells are glycinergic (Dmitrieva et al. 2003), whereas >35% contain GABA (Dmitrieva et al. 2001). To test whether or not 7-positive amacrine cells contain glycine or GABA, we performed triple-label IHC. Our results demonstrated that the majority of 7-expressing cholinoceptive amacrine cells displayed glycine immunoreactivity. Only a small population of amacrine cells were double labeled for 7 and GABA (Figures 4I–4L).

Among the 26 distinct morphological types of amacrine cells in rabbit retina, several types of glycine- and GABA-containing cells can be selectively labeled with specific antibodies (Table 1). Glycinergic AII amacrine cells that serve as a conduit for transferring the rod signal through the IPL are immunoreactive for parvalbumin, a member of a family of calcium-binding proteins (Casini et al. 1995; Bloomfield and Dacheux 2001). Dopaminergic amacrine (interplexiform) cells modulate AII–AII cell coupling under different light adaptation conditions (Bloomfield and Dacheux 2001) and can be labeled with antibodies against tyrosine hydroxylase (TH) (Brecha et al. 1984; Völgyi et al. 1997). GABA-containing cholinergic and nitrinergic amacrine cells and the glycinergic DAPI-3-positive amacrine cells in rabbit retina collect information from cone bipolar cells and other amacrine cells. These amacrine cells arborize with the dendrites of different types of amacrine and GCs (Brecha et al. 1988; Famiglietti 1991; Wright et al. 1997; Kim et al. 2000) and can also be selectively labeled. Cholinergic amacrine cells have been identified by immunostaining for choline acetyltransferase (ChAT) (Brandon 1987); nitrinergic cells have been consistently labeled with antisera against neuronal nitric oxide synthase (NOS) (Kim et al. 2000), and antibodies against the ß_2/3 subunit of GABA_A receptor prominently label DAPI-3 cells (Zucker and Ehinger 1998).

The 7 receptor labeling was never detected in somata and processes that exhibited parvalbumin, TH, or NOS immunoreactivity (Figures 5A –5D), further indicating that 7 nAChRs have the potential to modulate information transfer along cone, but not rod, pathways. Somata of ChAT-positive cells were also sc-1447 negative, but the processes of cholinergic cells in the IPL appeared to be labeled with anti-7 antibody (Figure 5E). Colocalization of ChAT and 7 in the processes could easily be seen in the IPL above optic nerve head where reduced 7 labeling of processes and somata was observed (Figures 5I–5K).

View larger version (109K): [in this window] [in a new window]   Figure 5 Double labeling of selective types of amacrine (red) and ganglion cells (GCs) with sc-1447 antibody (green) in rabbit retina. 7 Immunoreactivity was not detectable in the somata and dendrites of AII amacrine cells (A), dopaminergic amacrine cells (B), two types of nitrinergic amacrine cells (C,D), or in the somata of cholinergic amacrine cells (E). The distal dendrites of cholinergic cells appeared to show colocalization of two markers in the vertical section (arrowheads in F–H) and in the horizontal section (arrowheads in I–K). (L,M) Dendrites of some 7-expressing GCs (green) arborize in ON and OFF strata in the IPL of rabbit retina. (M,N) Dendrites of ON and OFF cholinergic amacrine cells labeled with anti-ChAT antisera are shown. (A–K) Single optical sections. (L,M) Stack of 3.5-µm sections. TH, tyrosine hydroxylase; NOS, nitric oxide synthase; ChAT, choline acetyltransferase. Bars: A = 12 µm; B = 15 µm; C,D = 12 µm; E–K = 10 µm; L–N = 20 µm.

Somata of glycinergic DAPI-3 cells in the INL, as well as many sites on their dendritic arborizations in the IPL, showed colocalization of GABA_A and 7 immunoreactivity (Figures 6A –6C).This might reflect the clustering of 7 nAChRs and GABA_A receptors on the same or adjacent membrane domains of DAPI-3 cells.

View larger version (123K): [in this window] [in a new window]   Figure 6 Confocal microscopic images of two vertical sections of rabbit retina. One section was exposed to both sc-1447 (green) and antisera against GABAA receptors (red). Somata of DAPI-3 amacrine cells (red) in the INL and some sites on their dendritic arborizations in the IPL were labeled with anti-7 antisera (arrows in A–C). Another section was exposed to a mixture of MAb210 (a marker for 3ß2 fnAChRs) and sc-1447 antisera (a marker for 7 nAChRs). Some of the somata of amacrine cells are double labeled, but only a few dendritic varicosities in the IPL demonstrated apparent colocalization (arrows in D–F). Bar = 10 µm.

Colocalization of 7 and 3ß2 nAChRs in Rabbit Retinal Neurons

Subcellular Distribution of 7 nAChRs
The functions of nAChRs rely on their channel properties and cellular and subcellular distribution (Sargent 1993; Le Novére et al. 2002; Lindstrom 2002). Previous studies have shown that in brain, 7-containing receptors can be found in diverse locations both at synaptic and non-synaptic sites (e.g., Berg and Conroy 2002). In rabbit retina, expression of 7 receptors clearly coincided with the axonal arborizations of calbindin-positive bipolar terminals at strata 4 (Figures 7A –7C). These terminals make ribbon synapses to dyads of postsynaptic dendrites that belong to either ganglion or amacrine cells (Strettoi et al. 1994). Thus, our data imply that 7 nAChRs are expressed by presynaptic elements at ribbon synapses of calbindin-positive bipolar cells.

View larger version (112K): [in this window] [in a new window]   Figure 7 Confocal images of three vertical sections of rabbit retina at the level of the IPL. Section shown in A–C was processed for 7 nAChRs (A) and calbindin (B). At stratum 4 of the IPL, the axonal arborizations of calbindin-positive cone bipolar cells were labeled with sc-1447 antisera (arrows in C). The section shown in D was processed for 7 nAChRs (green) and anti-kinesin (red), and the section shown in E was processed for sc-1447 antisera (green) and anti-synaptophysin (red). Yellow–orange patches in the IPL (D,E) might represent the colocalization or close apposition of 7 nAChRs with anti-kinesin and anti-synaptophysin (higher magnification areas, inserted panels). Bars: A–C = 2.5 µm; D = 7.5 µm (panel = 15 µm); E = 4 µm (panel = 8 µm).

Consistent with the localization of 7 nAChRs on calbindin-positive bipolar cells, many yellow–orange patches, indicating simultaneous fluorescence captured by the red and green channels, were visible in different strata of the IPL in vertical sections of rabbit retina processed either for 7 and kinesin (Figure 7D) or for 7 and synaptophysin (Figure 7E). Higher magnification areas (inset panels) revealed colocalization and/or close apposition of labeled sites. This pattern of association could mean that many 7 nAChRs are expressed at presynaptic elements of different bipolar cell (kinesin positive) synapses, as well as at presynaptic (synaptophysin positive) elements of amacrine cell synapses.

In rabbit retina, PSD-95 immunoreactive clusters coincided with punctate labeling of NMDR1 receptors and with all Glu2/3 receptor puncta in the IPL (Figures 8B –8D, 8E–8G). These results are identical to patterns of association shown to represent the postsynaptic aggregations of GluRs at glutamatergic synapses on the dendrites of amacrine and GCs in rat and monkey retina (Fletcher et al. 2000; Ghosh et al. 2001). Therefore, we considered the anti-PSD-95 stained clusters in the IPL as a reliable marker for postsynaptic densities in rabbit retina. In contrast to the results with markers for presynaptic elements, double-labeling studies with sc-1447 and PSD-95 detected little apparent colocalization between 7 nAChRs and postsynaptic densities. Rather, PSD-95 immunoreactivity appeared to be adjacent to 7-contining profiles (Figure 8A). In total, the results with pre- and postsynaptic markers indicate that 7 nAChRs are likely to be expressed at the presynaptic terminals of bipolar and amacrine cells but are less likely to be expressed by the postsynaptic members at bipolar cell synapses.

View larger version (140K): [in this window] [in a new window]   Figure 8 Confocal images of transverse sections of rabbit retina at the level of the IPL. (A) Section immunolabeled with sc-1447 (green) and anti PSD-95 (red). Many 7-labeled sites showed close association (A) but not apparent colocalization (inserted panel, higher magnification). (B–D,E–G) Sections simultaneously labeled using anti-NMDAR1 and anti-PSD-95, and anti-GluR2/3 and anti-PSD-95, respectively. The majority of NMDAR1- and GluR2/3-labeled clusters colocalized in hot spots with PSD-95. Images in B–G were taken from rabbit retina fixed in 4% 1-ethyl-3-3(-dimethylaminipropil) carbodiimide in phosphate buffer (Fletcher et al. 2000). Bars: A = 3.5 µm (panel = 7 µm); B–G = 4 µm.

	Discussion

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7 nAChR Involvement in the Rod and Cone Pathways and in On–Off Circuitry
These results demonstrate, for the first time, that 7 nAChRs are expressed by classes of bipolar and amacrine cells in the INL, in addition to GCs in the mammalian retina. Expression of 7 nAChRs by bipolar and amacrine cells indicates that the activation of this subtype of nAChRs in rabbit retina might significantly influence the processing of visual information by upstream neurons, as well as affecting processing by GCs.

The 7-containing bipolar and GCs transmit signals from photoreceptors forward to the brain. The 7-expressing amacrine cells may modify forward transmission by releasing glycine or GABA in response to stimulation by ACh or choline. Our imunohistochemical data are consistent with results from extracellular recordings in rabbit retina that suggest the expression of functional 7 nAChRs by GCs and upstream cells as well (Reed et al. 2002; Strang et al. 2005a). Figure 9 provides a summary of the different cell types that were immunoreactive to antibodies against 7 nAChRs.

View larger version (19K): [in this window] [in a new window]   Figure 9 Diagram summarizing the 7-expressing cell types in the rabbit retina. Letters on the drawing correspond to the description to the right of the image.

Our data suggest that the dedicated rod pathway neurons, rod bipolar cells, and AII amacrine cells do not express 7 nAChRs, nor do the dopaminergic and nitrinergic amacrine cells that modulate the activity of the AII amacrine cells. In contrast, many cone bipolar cells exhibited 7 immunoreactivity, including calbindin-positive cone bipolar cells. Interestingly, the calbindin-positive cone bipolar cells can receive input from AII amacrine cells and transmit both rod- and cone-mediated signals to ON-type GCs (Strettoi et al. 1994; Massey and Mills 1996). These data suggest that the release of ACh affects cone signals directly but may also affect rod signals downstream of the dedicated rod pathway.

7 nAChR Involvement in ON and OFF Circuitry
To perceive contrast between light and shadow under photopic conditions, parallel sets of visual channels that process ON and OFF qualities are present in mammalian retina (reviewed by Kolb 2003). In the rabbit retina, there are six distinct types of ON cone bipolar cells that connect to ON GCs in sublamina b of the IPL and six distinct types of OFF cone bipolar cells that connect to OFF GCs in sublamina a of the IPL (McGillem and Dacheux 2001). In our study, numerous 7-positive cone bipolar cells were also labeled with anti-glycine antibodies, and they may comprise four or five common types of ON of cone bipolar cells (Vaney et al. 1998). At least two types of ON cone bipolar cells—calbindin/glycine positive and calbindin negative/glycine positive—exhibited 7 immunoreactivity. The third type of 7 immunoreactive cone bipolar cells, which displayed neither calbindin nor glycine immunoreactivity, remains to be identified. These results indicate that primarily the ON cone bipolar pathway is influenced by 7 nAChRs.

Amacrine cells comprise the second major class of retinal neurons in rabbit retina that displayed 7 immunoreactivity. Our finding that most of the 7-positive amacrine cells contained glycine and that a smaller subset of 7-positive amacrine cells were GABAergic strikingly resembled the expression patterns of 3ß2 subtype of nAChRs. Specifically, 3ß2-expressing glycinergic amacrine cells were found to make up the majority of cholinoceptive amacrine cells in rabbit retina (Dmitrieva et al. 2003). However, the patterns of 7 and 3ß2 immunoreactivity were not identical; some amacrine cells expressed both types of nAChRs (DAPI-3 glycinergic cells), whereas others, such as the putative "flag" B glycinergic cells, were positive for 7 alone.

Glycinergic amacrine cells are usually "small field" cells and make more synapses onto amacrine and ganglion than onto bipolar cells. Their processes can span several strata within the IPL, but they are more compact in the plane parallel to the vitreal surface (Pourcho and Goebel 1985; MacNeil and Masland 1998; Menger et al. 1998; Kolb 2003). Different types of ON and OFF GCs in rabbit retina have been shown to express functional glycine receptors that mediate direct inhibitory input onto the GCs (Rotolo and Dacheux 2003a,b). One might predict that the release of ACh and its activation of Bgt-insensitive and -sensitive nAChRs on glycinergic cells preferentially control the activity of lateral circuits that feedback to other amacrine cells rather than information transfer to bipolar or GCs. However, organization of these local circuits is not well enough understood to speculate further.

7-Immunoreactive cells in the GCL constituted the numerically smallest population of cholinoceptive neurons in rabbit retina. 7-Immunoreactive neurons in the GCL represented two distinct types: some GCL cells expressed 7 and 3ß2 nAChRs and others exhibited only 7 immunoreactivity. The apparent heterogeneity of nAChR expression by neurons in the GCL as judged by IHC studies is consistent with the observed pharmacological diversity of GCs in rabbit retina. Many physiologically identified GC types, including subsets of sustained OFF and sustained ON, transient OFF (Strang et al. 2005a), and some directionally selective ON–OFF cells may express ß2-containing and 7 nAChRs (Reed et al. 2002; Strang et al. 2005b). Responses to cholinergic agonists of other subsets of GCs were mediated solely by 7 nAChRs (Strang et al. 2005a). Our data, together with the physiological studies cited above, suggest that ACh can directly affect the responses of GCs through the activation of 7 nAChRs and/or ß2-containing nAChRs on the GCs themselves, as well as on the upstream cells.

Putative Role of 7 nAChRs in Visual Processing
In the central nervous system, nAChRs mediate fast synaptic transmission only on rare occasions. More commonly, they modulate the release of neurotransmitters such as glutamate and GABA (Radcliffe et al. 1999). Light and electron microscopic studies have shown a "mismatch" between the locations of ACh release sites and the location of corresponding high-affinity receptors, providing anatomic evidence for a modulatory role (Descarries et al. 1997; Herkenham 1999). We observed a similar "mismatch" in the rabbit retina. There are reports that cholinergic cells in rabbit retina make synaptic outputs only onto GCs and other cholinergic cells (Brandon 1987; Famiglietti 1991). However, other studies of Bgt labeling (Pourcho 1979; Yamada et al. 2003) showed that starburst amacrine cells make presynaptic contacts onto bipolar cell terminals. Our data show that in addition to GCs and cholinergic cells, numerous bipolar cells and amacrine cells exhibited 7 immunoreactivity. In light of the studies by Brandon (1987) and Famiglietti (1991), our data are consistent with the probability that the 7 nAChRs expressed by GCs reflect direct cholinergic transmission from starburst cells, whereas activation of 7 nAChRs on amacrine and bipolar cells may exert modulatory effects on GC responses.

In our studies, 7 immunoreactivity often coincided with "hot spots", varicosities on the dendrites of cholinergic amacrine cells. Ultrastructural studies suggest that the majority of these varicosities are postsynaptic to bipolar and amacrine cells or presynaptic to GC dendrites (Famiglietti 1991). Therefore, 7-immunoreactive varicosities on cholinergic cells may represent pre- or/and postsynaptic sites at contacts with GCs or other cholinergic cells. These ultrastructural studies are consistent with our double-labeling studies with the presynaptic markers kinesin II and synaptophysin, suggesting a presynaptic role for 7 nAChRs on starburst cells.

At glutamatergic synapses, 7 nicotinic receptors were strictly confined to presynaptic structures in the IPL. Localization of 7 nAChRs, which have high Ca²⁺ permeability, on presynaptic terminals of bipolar cells raises two possibilities. First, ACh might increase the spontaneous release of glutamate from the bipolar terminal through activation of presynaptic 7 receptors, thus affecting the responses of specific postsynaptic GCs or amacrine cells. Second, the activation of 7 receptors coincident with transmission of bipolar signals might enhance glutamate-driven responses of target ganglion and amacrine cells. Consistent with our data, the application of MLA, a partial antagonist for 7 nAChRs, decreased the maintained firing and light-evoked responses in some identified GCs in rabbit retina (Reed et al. 2002; Strang et al. 2005a), and these effects may be at least partially attributable to a modulation of upstream glutamate release.

Our finding of 7 nAChRs in close association with synaptophysin on the dendrites of glycinergic and some GABAergic cells close to synapses indicates that the release of glycine and GABA could be enhanced via the activation of presynaptic 7 receptors and cause either inhibition or disinhibition of other amacrine and GCs involved in the same local circuits. However, our findings do not rule out a postsynaptic localization for 7 nicotinic receptors. In the CA1 region of adult hippocampus, double labeling for Bgt and synaptophysin revealed abundant expression of both epitopes, often in close apposition, that represented labeling of 7 at both pre- and postsynaptic elements at the electron microscopic level (Fabian-Fine et al. 2001).

The lack of reliable postsynaptic markers for inhibitory synapses in the retina that are compatible with the sc-1447 protocol did not allow us to test for the postsynaptic localization of 7. However, some of the 7 nAChRs in our studies coincided with GABA_A receptors on the dendrites of DAPI-3 glycinergic cells. GABA_A receptors are frequently found at postsynaptic sites at synapses with other amacrine cells in the IPL of rabbit retina (Greferath et al. 1994). This suggests that clustered 7 nAChRs and GABA_A receptors might be present at postsynaptic elements as well.

Post- or perisynaptic 7 nAChRs may mediate an increase in [Ca²⁺]_i through influx directly through the 7 nAChR, the activation of voltage-sensitive Ca²⁺ channels, or by release from intracellular stores. This increase could be sufficient to influence the activity of calcium-dependent kinases and phosphatases, thus regulating the function of GABA_A receptors at GABAergic synapses (Berg and Conroy 2002).

Physiological experiments have demonstrated that selected GCs in rabbit retina (Strang et al. 2005a) including directionally selective GCs (Strang et al. 2005b) express both 7 and non-7 nAChRs. Furthermore, our results indicate that, in addition to GCs, some amacrine cells express at least two types of nAChRs. They localize close to each other on the dendrites but apparently occupy different spots on the plasma membrane. In brain, expression of two functional subtypes of nAChRs, 7 and 4ß2, on the same neuron has been demonstrated by CA1 GABAergic interneurons in rat hippocampal slices (Alkondon et al. 1999). The mediation of fast (phasic) excitation of interneurons through the 7 subtype and slow (tonic) excitation of interneurons through 4ß2 results in two types of inhibition on target neurons. Similar to these findings, expression of both 7 and 3ß2 nAChRs on some inhibitory amacrine cells in rabbit retina raises the possibility of dual modulation of the release of glycine and/or GABA.

Thus, the apparent expression of 7 nAChRs by different retinal neurons at diverse subcellular locations suggests that they can serve many functions. Release of ACh may boost the responses of ON and ON–OFF types of GCs through the activation of postsynaptic 7 receptors on GCs or by enhancing ON bipolar cell signals via activation of presynaptic 7 nicotinic receptors. 7 nAChR activation on inhibitory amacrine cells that synapse onto other types of inhibitory amacrine cells likely would cause inhibition or disinhibition of multiple local circuits resulting in modulation of the responsiveness of selected GCs. Finally, activation of postsynaptic 7 nicotinic receptors at inhibitory synapses could modulate the activity of GABA_A receptors by triggering a cascade of calcium-dependent processes in cholinoceptive amacrine cells.

The possibility that cholinergic modulation of GCs via pathways involving bipolar cells has been previously suggested. Specifically, observations in chick (Vogel et al. 1977), mouse (Pourcho 1979), goldfish (Zucker and Yazulla 1982), and turtle (James and Klein 1985) all demonstrated Bgt binding associated with bipolar cells or amacrine–bipolar synapses. In addition, previous work from this laboratory (Keyser et al. 1993) showed Bgt-sensitive nAChR (8 subunits) antibody labeling of bipolar cells in the avian retina. Finally, data from non-human primate retina revealed that cholinergic cells synapse directly onto cone bipolar cell terminals, in addition to synapsing on amacrine and GCs (Yamada et al. 2003). In this respect, the observations we report here concerning bipolar cells are consistent in the context of a body of work that spans nearly 30 years. Details of the organization of cholinoceptive local circuits, the expression of additional nAChR subtypes in the retina, and their subcellular distribution remain issues for future investigation.

	Acknowledgments

 
This work was supported by NEI grants P30 EY-03039 and EY-07845.

	Footnotes

 
Received for publication October 13, 2006; accepted December 12, 2006

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

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