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Nicotinic Acetylcholine Receptor Subtypes Expression during Rat Retina Development and Their Regulation by Visual Experience

CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology, and Center of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy (M.M., S.V., L.R., F.C., C.G.); Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, Modena, Italy (M.Z., G.L.); Consiglio Nazionale delle Ricerche, Institute of Chemistry of Molecular Recognition, Milan, Italy (R.L.); and Scuola Normale Superiore, Pisa, Italy (A.V.)

Received October 29, 2003; accepted March 19, 2004

	Abstract

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By acting through retinal nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in the development of both the retina and central visual pathways. Ligand binding and immunoprecipitation studies with subunit-specific antibodies showed that the expression of Bungarotoxin (Bgtx) and high-affinity epibatidine (Epi) receptors is regulated developmentally and increases until postnatal day 21 (P21). The increase in Epi receptors is caused by a selective increase in the subtypes containing the 2, 4, 6, 2, and 3 subunits. Immunopurification studies revealed three major populations of Epi receptors on P21: 6^* receptors (26%), which contain the 632, 6432, and 63/232 subtypes; 4(non-6)^* receptors (60%), which contain the 242 and 42 subtypes; and (non-4/non-6)^* receptors (14%), which contain the 22/4 and 32/4 subtypes. These three populations can be pharmacologically discriminated using conotoxin MII, which binds the 6^* population with high affinity. In situ hybridization showed that the transcripts for all of the subunits are heterogeneously distributed throughout retinal neurons at P21, with 3, 6, and 3 transcripts preferentially concentrated in the ganglion cell layer, 5 in the inner nuclear layer, and 4 and 2 distributed rather homogeneously. To investigate whether nAChR expression is affected by visual experience, we also studied dark-reared P21 rats. Visual deprivation had no effect on the expression of Bgtx receptors or the developmentally regulated Epi receptors containing the 2, 6, and/or 3 subunits but significantly increased the expression of the Epi receptors containing the 4 and 2 subunits. Overall, this study demonstrates that the retina is the rat neural region that expresses the widest array of nAChR subtypes. These receptors have a specific distribution, and their expression is finely regulated during development and by visual experience.

In adult vertebrate retina, ACh released by the starburst amacrine cells activates a rich array of nAChRs. In situ hybridization and immunolocalization studies, together with Northern blot analyses, have shown that the retina expresses almost all of the nicotinic subunits present in homomeric and heteromeric receptors (Feller, 2002). In particular, 6 and 3 subunits are expressed in a restricted number of neuronal populations, which include catecholaminergic nuclei and visual pathways of the mammalian central nervous system (Le Novère et al., 1996; Champtiaux et al., 2002; Cui et al., 2003).

Biochemical and pharmacological studies using nicotinic ligands and subunit-specific polyclonal antibodies (Abs) have identified the presence of three Bgtx-binding subtypes in chick retina, the homomeric 7 and 8 and the heteromeric 7-8 subtype (Keyser et al., 1993; Gotti et al., 1994, 1997b). Most of the heteromeric [³H]Epi receptors in chick retina contain the 4 subunit (associated with the 4, 6, and/or 3 subunits) on postnatal day 1 (P1), and both chick Bgtx and heteromeric Epi receptors are developmentally regulated (Vailati et al., 1999, 2000; Barabino et al., 2001). With use of adult rabbit retina, Keyser et al. (2000) have shown that many of the heteromeric receptors contain the 2 structural subunit, which is partially associated with the 3 subunit.

Before phototransduction, spontaneous bursting activity in the developing vertebrate retina (also known as retinal waves) influences the size and complexity of retinal ganglion cell (RGC) dendrites and refines the connections between retinal axons and their thalamic targets (Wong, 1999; Feller, 2002). The role of nAChRs in this activity has been shown clearly by the use of nicotinic antagonists and knockout (KO) mice. Bansal et al. (2000) have shown that the retinal waves present between embryonic day 16 and birth are blocked by nonselective nicotinic antagonists, whereas between P0 and P11, they are blocked by conotoxin MII (CntxMII) a nicotinic antagonist believed to be specific for the 32^* and 62^* receptors (Cartier et al., 1996, Champtiaux et al., 2002, 2003); between P11 and P14, they are blocked by antagonists of the glutamatergic receptors. Bansal et al. (2000) also showed that mice lacking the 3 or 2 nicotinic subunits have retinal waves with altered spatiotemporal properties.

Further studies have shown that mice lacking the 2 structural subunit (but not those lacking the 4 subunit) have retinofugal projections to the dorsolateral geniculate nucleus and superior colliculus that do not segregate into eye-specific areas, an altered functional organization in the dorsolateral geniculate nucleus, an expanded binocular subfield of the primary cortex, and decreased visual acuity at cortical level (Rossi et al., 2001; Muir-Robinson et al., 2002, Grubb et al., 2003). Taken together, the results of all of these studies indicate that nAChRs containing the 2 subunit are essential for the anatomical and functional development of the visual system in rodents. However, the exact nature of the nAChR subtypes expressed during rat development and adulthood is still unclear.

The aims of the present study were threefold. First, we wished to identify the pattern of nAChR subunit expression during postnatal rat development until adulthood using a combination of ligand binding and immunoprecipitation techniques. Second, we sought to establish the subunit composition of the retina nAChR subtypes, characterize their pharmacological profile, and localize them at cell level by using in situ hybridization techniques on P21 (when nAChRs first reach adult density and subunit expression pattern). Finally, we investigated whether visual experience affects the expression of retinal nAChRs on P21.

	Materials and Methods

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Animals and Materials. Male pathogen-free Sprague-Dawley rats (Harlan-Nossan, Milan, Italy) were used on P1, P5, P10, P21, P52, or P84. They were kept under standardized temperature, humidity, and lighting conditions (lights on at 8.00 AM and off at 8.00 PM) and had free access to water and food. In the dark-rearing experiments, the rats were kept in total darkness from birth to P21 with free access to food and water and were anesthetized in the dark with chloral hydrate before killing.

All of the animal experimentation was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The protease inhibitors, nonradioactive Epi, nicotinic ligands, and Triton X-100 were purchased from Sigma Chemical (St. Louis, MO); the CnBr-activated Sepharose-4BCL, ¹²⁵I-protein A, and ¹²⁵I-Bgtx were from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK); (±)[³H]Epi (specific activity = 50-66 Ci/mmol) and ¹²⁵I-Epi (specific activity = 2200 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA); and the reagents for gel electrophoresis were from Bio-Rad (Hercules, CA). CntxMII was synthesized as described previously (Cartier et al., 1996).

Antibody Production and Characterization. The sequences of polyclonal Abs against the 2, 3, 4, 5, 6, 2, 3, and 4 peptides, which were raised and characterized as described previously (Del Signore et al., 2002; Zoli et al., 2002; Champtiaux et al., 2003), are shown in Table 1. Two different peptides were chosen for almost all of the subunits of the heteromeric receptors: one located in the cytoplasmic loop between M3 and M4 (CYT), which is the most divergent region of the subunits, and the other at the COOH terminal (COOH). Only one Ab was produced for the 2 subunit because the COOH peptide is almost identical with the 4 COOH. For the 6 subunit we used two Abs directed against two separate CYT peptides. The antibodies raised against the peptides were purified on an affinity column made by coupling the corresponding peptide to cyanogen bromide-activated Sepharose-4B according to the manufacturer's instructions.

The specificity and immunoprecipitation capacity of most antibodies has been reported previously (Zoli et al., 2002; Champtiaux et al., 2003). In addition, we tested the immunoprecipitation capacity of the anti-3 Abs on eye membrane extracts obtained from wild-type and 3 KO animals (Cui et al., 2003). The Abs directed against the CYT and COOH 3 peptides both immunoprecipitated 36% of the [³H]Epi receptors in the extracts obtained from the wild-type animals but did not immunoprecipitate the [³H]Epi receptors in those obtained from the KO animals. The anti-2 Ab specificity and immunoprecipitation capacity were tested on cell extracts obtained from human embryonic kidney cells transfected with the 24 human subunits (a generous gift from Dr. E. Sher, Eli Lilly & Co. Ltd, Basingstoke, Hampshire, UK). The anti-2 and 4 Abs, respectively, immunoprecipitated 95 ± 2% and 92 ± 3% of the [³H]Epi-labeled receptors (mean ± S.E.M. of three independent experiments), whereas no specific immunoprecipitation was determined using the Abs directed against the other subunits.

Preparation of Membranes and 2% Triton X-100 Extracts from Eyes and Retinas. The eyes were dissected, immediately frozen in liquid nitrogen, and stored at -80°C for later use. No difference in the binding of the fresh and frozen tissues was observed.

In every experiment, the eyes were homogenized separately in an excess of 50 mM sodium phosphate, pH 7.4, 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride for 2 min in an UltraTurrax homogenizer (IKA Labortecnik, Staufen, Germany). The homogenates were then diluted and centrifuged for 1.5 h at 60,000g. The retinas were dissected from frozen eyes, separately homogenized using a Potter homogenizer (Sartorius, Goettingen, Germany), and processed as described for the whole eyes.

The procedures of homogenization, dilution, and centrifugation of the eyes as whole or isolated retinas were performed twice, after which the pellets were collected; rapidly rinsed with 50 mM Tris HCl, pH 7, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, and 2 mM phenylmethylsulfonyl fluoride; and then resuspended in the same buffer containing a mixture of 20 µg/ml of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100 at a final concentration of 2% was added to the washed membranes, which were extracted for 2 h at 4°C.

The extracts from whole eyes or isolated retinas were then centrifuged for 1.5 h at 60,000g and recovered, and an aliquot of the resultant supernatants was collected for protein measurement using the BCA protein assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.

Binding Assay. 2-, 4-, and 8-containing receptors bind [³H]Epi with picomolar affinity, and 7 receptors bind Epi with nanomolar affinity (Gerzanich et al., 1995). To ensure that the 7 subtype did not contribute to [³H]Epi binding, the binding tissue extract and immunoprecipitation experiments were performed in the presence of 2 µM Bgtx, which specifically binds to the 7 subtype and prevents Epi from binding to the subtypes containing this subunit.

The Triton X-100 extracts of retina at different ages were preincubated with 2 µM Bgtx for 3 h and then labeled with 2 nM [³H]Epi. Tissue extract binding was performed using DE52 ion-exchange resin (Whatman, Maidstone, UK) as described previously (Vailati et al., 1999).

Immunoprecipitation of [³H]Epibatidine-Labeled Receptors by Anti-Subunit-Specific Antibodies. The extracts obtained from the eyes at different ages or from dissected retinas preincubated with 2 µM Bgtx, and labeled with 2 nM [³H]Epi, were incubated overnight with a saturating concentration of affinity purified IgG (20-30 µg; Sigma). The immunoprecipitation was recovered by incubating the samples with beads containing bound anti-rabbit goat IgG (Technogenetics, Trebbano, S.N., Milan, Italy). The level of Ab immunoprecipitation was expressed as the percentage of [³H]Epi-labeled receptors immunoprecipitated by the antibodies (taking the amount present in the Triton X-100 extract solution before immunoprecipitation as 100%) or as femtomole of immunoprecipitated receptors per eye or as femtomole of immunoprecipitated receptors per milligram of protein.

Receptor Subtype Immunopurification and Analysis. The extracts prepared from P21 rat eyes were incubated three times with 5 ml of Sepharose-4B with bound anti-6 Abs (column A) to remove the 6 subunit-containing receptors (6^* population). This 6^* population was eluted from column A by means of incubation with the 6 peptide and was then further incubated with the anti-4 Abs (column B) to remove the 6 receptors that also contain the 4 subunit (see Results). The 6 receptors bound to column B were eluted by competition with the 4 peptide, and those that did not bind the anti-4 Abs remained in the flow-through of the column and were analyzed.

The flow-through of column A (i.e., the retina extract devoid of 6-containing receptors) was incubated twice with 5 ml of Sepharose-4B with bound anti-2 (column C) or anti-4 Abs (column D). The bound receptors were eluted with 0.2 M glycine, pH 2.2, or by means of competition with 100 µM of the corresponding 2 or 4 peptides used for Ab production. The subunit content of the purified receptors was determined by immunoprecipitation using the purified subtypes eluted with the peptides labeled with 2 nM [³H]Epi and the subunit-specific Abs.

Gel Electrophoresis and Western Blotting. SDS-polyacrylamide gel electrophoresis was performed as described previously (Vailati et al., 1999) using 9% acrylamide. The proteins were electrophoretically transferred to nitrocellulose and subsequently probed with affinity-purified antipeptide antibodies. The bound antibodies were detected by means of ¹²⁵I-protein A.

Pharmacological Experiments on Immunoimmobilized Subtypes. The affinity-purified anti-6 or anti-2 Abs were bound to microwells (MaxiSorp; Nalge Nunc International, Naperville, IL) by incubating overnight at 4°C at a concentration of 10 µg/ml in 50 mM phosphate buffer, pH 7.5. On the following day, the wells were washed to remove the excess of unbound Abs and then incubated overnight at 4°C with 200 µl of 2% Triton X-100 eye membrane extract containing 20 to 40 fmol ¹²⁵I-Epi binding sites, which was prepared by sequentially immunodepleting or not the 6-containing receptors. After incubation, the wells were washed, and the presence of immobilized receptors revealed by means of ¹²⁵I-Epi binding. Binding techniques for immunoimmobilized subtypes and data analysis were as described in Vailati et al. (1999).

In Situ Hybridization. After the analysis of mRNA secondary structure using GCG sequence analysis software version 7.1 (Accelrys, San Diego, CA), oligodeoxynucleotide sequences were chosen in unique regions of the rat nAChR subunit mRNAs. The probe characteristics and specificity controls are reported elsewhere (Zoli et al., 1995; Le Novère et al., 1996). Specificity controls were performed on retina sections and included the demonstration that 1) two or more probes for each mRNA give identical labeling pattern; 2) the labeling disappears when labeled probes are incubated with 50x excess of unlabeled probe; and 3) probes with the same base composition but different sequence do not give the specific labeling pattern. The oligonucleotide probes were labeled at the 3' end using ³⁵S-dATP (Amersham) and terminal deoxynucleotidyl transferase (Roche Diagnostics, Indianapolis, IN) following the specifications of the manufacturer to a specific activity of 100 to 300 KBcq/pmol. The labeled probes were separated from unincorporated ³⁵S-dATP using G50 spin columns (Pharmacia, Peapack, NJ), precipitated in ethanol, and resuspended in distilled water containing 50 mM dithiothreitol.

P21 rat eyes were dissected out, immersed in 4% paraformaldehyde in phosphate-buffered saline overnight, embedded in gelatin, and frozen using crushed dry ice. The eyes were cut at the cryostat (20-µm thick sections) thaw mounted on gelatin-coated slides, and stored at -80°C for 1 to 3 days. The procedure was carried out according to Zoli et al. (1995). Probes were applied at a concentration of 2000 to 3000 Bcq/30 µl/section (corresponding to approximately 15 fmol/section). The slides were exposed for 14 days to ³H Hyperfilm (Amersham) and then to a photographic emulsion (Ilford, Cheshire, United Kingdom) for 2 to 3 months.

Grain counting was performed in the different retinal layers by means of an automatic image analyser (KS300) as described by Zoli et al. (1992) and Pedrazzi et al. (1998). For each labeling, four retinas were analyzed.

Statistical Analysis. Statistical analysis of the expression of [³H]Epi and ¹²⁵I-Bgtx receptors as well as the subunit content of the expressed [³H]Epi receptors was carried out by one-way analysis of variance (ANOVA), followed by post-hoc Dunnett test. Comparison of nAChR subunit mRNA labeling in different retinal layers was performed by one-way ANOVA followed by Bonferroni test for multiple comparisons.

	Results

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nAChR Expression during Postnatal Retina Development
[³H]Epibatidine-Binding Receptors. To investigate nAChR expression during postnatal retina development and aging, we performed binding studies using membranes prepared from whole eyes and isolated retinas obtained from the animals on P1, P5, P10, P21, P52, and P84.

We and others (Britto et al., 1992; Keyser et al., 1993; Gerzanich et al., 1995; Gotti et al., 1997b; Barabino et al., 2001) have shown previously that chick retina expresses a high level of Bgtx binding receptors that also bind [³H]Epi receptors with nanomolar affinity. To avoid the contribution of these receptors to [³H]Epi binding, we preincubated the tissue extracts with 2 µM Bgtx and thus only measured the binding of [³H]Epi to Bgtx-insensitive nAChRs.

The expression of [³H]Epi receptors was calculated in femtomole of bound [³H]Epi receptors per eye (mean values ± S.E.M. of four to five experiments) and femtomole of [³H]Epi receptors per milligram of retina protein (mean values ± S.E.M. of three to four experiments). The results are shown in Table 2 and Fig. 1, A and B. When expressed as femtomole per eye, the number of receptors increased almost linearly from P1 to P21 (from 11.1 to 73.6 fmol/eye) and then remained constant from P21 to P84 (71.6 fmol/eye on P52 and 73.0 fmol/eye on P84).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Developmental changes in 125I-Bgtx () and [3H]Epi () binding receptors expressed in whole eye membranes (A) and isolated retinas (B). The eyes or isolated retinas were dissected from the animals at the indicated times and frozen, and the membrane homogenates were prepared as described under Materials and Methods. At each indicated postnatal day, total binding was performed using 2 nM [3H]Epi or 10 nM 125I-Bgtx and subtracted from the aspecific binding performed in parallel using 2 nM [3H]Epi or 10 nM 125I-Bgtx and 100 nM cold Epi or 1 µM unlabeled Bgtx. For total and aspecific [3H]Epi binding, the eye membranes were always incubated with 2 µM cold Bgtx. The reported values are expressed as femtomoles of specific labeled 125I-Bgtx and [3H]Epi receptor per eye (A) or femtomoles per milligram of retina protein (B) and are the mean values ± S.E.M. of three to five experiments performed in triplicate (unless shown, the S.E.M. is in the range of the symbol). Statistical analysis was carried out by one-way ANOVA followed by post hoc Dunnett's test; #, P < 0.05 versus P1 mean value.

When expressed as femtomole per milligram of retina protein, the number of receptors did not change significantly from P1 to P5, sharply increased from P5 to P21, and then slightly decreased, being always significantly greater than the P1 values from P10 to adulthood (Fig. 1B and Table 2).

¹²⁵I-Bungarotoxin Binding Receptors. The results expressed as femtomole of ¹²⁵I-Bgtx receptors per eye (mean values ± S.E.M. of four experiments) are shown in Fig. 1A. As in the case of [³H]Epi, the number increased almost linearly from P1 (16.9 fmol/eye) to P21 (36.6 fmol/eye) and then remained constant until P84 (36.7 fmol/eye). The level was almost constant at each developmental time when expressed as femtomole per milligram of retina protein (Fig. 1B and Table 2).

Subunit Content of Retinal [³H]Epibatidine Receptors. The expression pattern of nAChR subunits during retina postnatal development was determined by quantitative immunoprecipitation experiments using subunit-specific antibodies and [³H]Epi-labeled receptors to quantify the relative contribution of each nicotinic subunit to [³H]Epi binding at each developmental stage. For each subunit (except 2), we used polyclonal Abs directed against two separate peptides. The results, expressed as femtomoles of immunoprecipitated receptors per milligram of retina protein, are the mean values of three to five separate experiments for each subunit at each developmental time (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Immunoprecipitation analysis of the subunit content of the [3H]Epi receptors expressed in retinas. Triton X-100 (2%) extracts were obtained from membranes prepared from retinas dissected from the animals on P1, P5, P10, P21, P52, and P59, preincubated with 2 µM Bgtx, and then labeled with 2 nM [3H]Epi. Immunoprecipitation was carried out as described under Materials and Methods using saturating concentrations (20-30 µg) of antisubunit Abs. Two Abs were used for each subunit (except for 2): one directed against a CYT peptide, and the other directed against a COOH peptide. In each experiment, the amount immunoprecipitated by each antibody was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG. The results are expressed as femtomoles of labeled [3H]Epi receptor per milligram of retina protein and are the mean values ± S.E.M. of three to four experiments performed in duplicate (unless shown, the S.E.M. is in the range of the symbol). Statistical analysis was carried out by one-way ANOVA followed by post hoc Dunnett's test; #, P < 0.05 versus P1 mean value.

By quantifying the number of receptors immunoprecipitated by the specific Ab as the percentage of the total number of [³H]Epi receptors, we also identified the major retinal subtypes present at each developmental time. P1 retina mainly contained the 4 (53.2 ± 4.3%) and 2 subunits (65.8 ± 4.2%), but there were also receptors containing the 3 (23.7 ± 5.0%), 6 (10.6 ± 2.4%), 3 (17.5 ± 4.4%), and 4 (22.7 ± 3.6%) subunits. From P1 to P21, there was an increase in the expression of the receptors containing the 2, 4, 6, 2, and 3 subunits and a decrease in the expression of those containing the 3 and 4 subunits.

By P21, the levels of the 2, 4, 6, 2, and 3 subunits expressed as femtomole per milligram of retina protein had increased by 33, 3.2, 7.3, 4.2, and 5.8 times over their P1 levels, respectively; the level of 3 and 5 increased 1.3 and 2.5 times, respectively, whereas the level of 4 subunit was 0.7 times lower (Fig. 2).

By P21 in addition to 2 (88.6 ± 7.3%) and 4 (56.2 ± 3.5%), which remain the major subunits in the retina, the 2 (23.0 ± 2.9%), 6 (26.3 ± 2.3%), and 3 (35.2 ± 3,8%) subunits were also well represented, whereas the 3 (9.2 ± 0.8%) and 4 (6.3 ± 1.2%) subunits were present in a small minority of Epi receptors.

Purification and Subunit Composition of the Major [³H]Epibatidine Binding Subtypes Expressed on P21. We performed immunopurification experiments using subunit-specific antibodies to establish the subunit assembly of the heteromeric nAChR subtypes in the retina on P21. For these experiments, we only used extracts obtained from whole eyes because preliminary ligand binding and immunoprecipitation found that, although the specific activity in the isolated retinas was higher (201.8 ± 17.2) than that found in eye membranes (94 ± 7 fmol/mg of protein), both the specific activity of whole-eye extracts (233 ± 24 fmol/mg of protein) and the percentage of subunit-specific immunoprecipitation of [³H]Epi receptors were very similar in whole eye and isolated retina. This indicates that the nAChRs present in the eyes as a whole and labeled by [³H]Epi are only present in the retina, because no binding is detected in the eye membranes deprived of retina.

6-Containing Receptors. The immunoprecipitation experiments showed that almost all of the receptors in the eluate of column A (see Materials and Methods) contained the 2 subunit (90.7 ± 2.4%) and a large majority contained the 3 subunit (71.4 ± 3.0%), whereas the 2, 3, 4, 5, and 4 subunits were present in 18.0 ± 3.0%, 12.9 ± 3.0%, 42.5 ± 3.4%, 3.0 ± 2.0%, and 7.0 ± 4.1% of the receptors, respectively (Fig. 3A). These receptors were defined as the 6^* population.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Immunoprecipitation analysis of the subunit content of the 6* receptors. The extracts prepared from P21 rat eyes were incubated with an affinity column with bound anti-6 Abs (see Materials and Methods)to bind the 6* population, which was eluted from the resin by means of incubation with the 6 peptide, labeled with 2 nM [3H]Epi, and then immunoprecipitated by the indicated subunit-specific Abs (A). The 6* population was further subfractioned by incubation with an affinity resin with bound anti 4 Abs. B and C show the results of the analyses of the eluate (46 receptors) (B) and the flow-through of the column (non-46* receptors) (C) labeled with 2 nM [3H]Epi and immunoprecipitated by the indicated subunit-specific Abs. Immunoprecipitation was carried out as described under Materials and Methods using saturating concentrations (20-30 µg) of antisubunit Abs. The amount immunoprecipitated by each antibody was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG, and the results obtained with each Ab are expressed as the percentage of total [3H]Epi binding present in the solution before immunoprecipitation. Each data point is the mean ± S.E.M. of three determinations performed in triplicate.

To investigate whether this population can be further subdivided, we subfractioned it by incubating with Sepharose beads with bound anti-4 Abs (column B) and then recovering by competition with the 4 peptide. The subfractioned 6^* receptors were analyzed by immunoprecipitation and found to contain the 6 (92.1 ± 0.9%), 4 (78.6 ± 0.6%), 3 (61.5 ± 5.1%), and 2 (92.9 ± 2.9%) subunits (Fig. 3B). As a further control, we also checked the subunit composition of the flow-through of column B and, as shown in Fig. 3C, found that it contained 6^* receptors (82.2 ± 2%) that were devoid of the 4 subunit but contained the 2 (81.2 ± 4.2%), 3 (53.9 ± 5%), 2 (12.8 ± 0.4%), and/or 3 (24.7 ± 1.0%) subunits. These subfractionation and immunoprecipitation studies showed that the rat retina receptor subtypes containing the 6 subunit are 6432, 62/332, and 632.

(Non-6)-Containing Receptors. After being immunodepleted of the 6-containing receptors, the membrane extract was incubated twice with Sepharose beads with bound anti-2 Abs (column C). We defined these 2-containing receptors as the (non-6)2^* receptor population. The 2 subunit in these receptors was associated with the 4 (71.7 ± 4.4%) and 2 subunits (25.2 ± 5.5%) (Fig. 4A).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Immunoprecipitation analysis of the (non-6)2* and 4(non-6)* populations. The extract devoid of the 6-containing receptors was incubated with resin with bound anti-2 Abs (A) or anti-4 Abs (B). The bound receptor populations were eluted by the corresponding peptides, labeled with 2 nM [3H]Epi, and immunoprecipitated by the indicated subunit-specific Abs. The results are expressed as described in Fig. 3.

To identify whether the 4 and 2 subunits coexist in the same subtype, we performed additional experiments in which the 6-depleted extract was directly incubated with Sepharose beads with bound anti-4 Abs (column D). The bound receptors eluted by competition with the 4 peptide were analyzed by immunoprecipitation experiments and showed that 90 ± 1.8% of them contained the 2 subunits, 87 ± 5.2% the 4 subunits, and 22 ± 0.6% the 2 subunit. These were therefore defined the 4(non-6)^* receptor population (Fig. 4B).

In the 4(non-6)^* population, coimmunoprecipitation of the 2 and 4 subunits clearly indicated the presence of a subtype containing the 2, 4, and 2 subunits, and a subtype containing the 4 and 2 subunits. We found that 14.1 ± 1.5% of these receptors also contain the 3 subunit, thus indicating that this subunit is present in a minority of retinal receptors without being associated with the 6 subunit.

Moreover, binding and immunoprecipitation analysis of the flow-through of the anti-4 affinity column (column D) revealed the presence of [³H]Epi receptors that contained the 2 and 3 subunits associated with the 2 (77.8 ± 1.6%) and/or 4 (22.1 ± 2.0%) subunits. This population represented 14% of the total number of [³H]Epi receptors and was defined as the (non-4/non-6)^* population, i.e., one or more subtypes containing the 2 (40.0 ± 3.0% %), 3 (31.4 ± 2.0%), 2 (77.8 ± 3.1%), and/or 4 (22.1 ± 2.0%) subunits but not the 4 and 6 subunits.

Distribution of nAChR Subunit mRNAs in the Rat Retina at P21. We performed an in situ hybridization study of the distribution of heteromeric nAChR subunit mRNAs in P21 rat retina. The probes for all of the heteromeric subunit mRNAs (except 4) gave a positive signal in the retina, each with a specific layer distribution.

Analysis of the in situ hybridization preparations showed that rat retina contains relatively high levels of 3, 5, 6, and 3 mRNAs, moderate levels of 2 and 4 mRNAs, and relatively low levels of 2 mRNA; no specific signal could be detected for 4 mRNA. Besides differences in the overall intensity of the labeling, the distribution of the different subunit transcripts was heterogeneous (Fig. 5) and subunit-specific.

View larger version (149K): [in this window] [in a new window]   Fig. 5. Distribution of nAChR subunit mRNAs in rat retina at P21. Dark-field microphotographs of emulsion autoradiograms showing 2, 3, 4, 5, 6, 2, 3, and 4 nAChR subunit mRNAs in the retina of P21 rat. To compare these images with the grain counting results shown in Fig. 6, it needs to be remembered that the quantitation was corrected for background subtraction and specific probe activity. In the images shown here, the relative specific activity was 2 = 0.16, 3 = 0.50, 4 = 0.18, 5 = 0.42, 6 = 1.00, 2 = 0.25, 3 = 0.29, and 4 = 0.38.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Density of nAChR subunit mRNAs in the retina of P21 rats. Density of photographic emulsion grains/retinal layer of 2, 3, 4, 5, 6, 2, and 3 nAChR subunit mRNA labeling in the retina of P21 rat. For methodological details, see Materials and Methods. Mean values ± S.E.M. (n = 4).

Western Blot Analysis of the 6^* and (Non-6)2^* Receptor Populations. The immunopurification analysis described above identified two major populations of heteromeric receptors (those containing and those not containing 6). The subunit composition of the P21 6^* and (non-6)2^* receptor populations was analyzed by Western blotting using the same subunit-specific Abs as those used for the immunoprecipitation experiments. The results confirmed that the 2, 4, and 2 subunits were present in both populations, but the 6 subunit was only present in the 6^* population (Fig. 7). Although present at much higher levels in the 6^* population, the 3 subunit was also present in very small amounts in the (non-6)2^* receptor population. The anti-2 Ab (lanes 1 and 7) recognized a major peptide with a molecular mass of 60 ± 1 kDa in both populations; the anti-3 Ab (lanes 2 and 8) faintly recognized a peptide of 51 ± 1 kDa; the anti-4 (lanes 3 and 9) recognized a single band of 68 kDa in both populations, and the anti-2 Ab (lanes 5 and 11) recognized a single band of 52 kDa in both populations. The anti-6 Ab recognized a single band of 57 kDa (lanes 4 and 10) only in the 6^* receptor population, and the anti-3 Ab (lanes 6 and 12) recognized three peptides of 58, 50, and 40 kDa mainly in the 6^* receptor population. In agreement with the immunoprecipitation results, the anti-5 did not recognize any band in either receptor population (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Western blot analysis of P21 affinity-purified 6* and (non-6)2* nAChRs. The receptors were prepared as described under Materials and Methods. The eluted receptors were concentrated and separated on 9% acrylamide SDS gels, electrotransferred to nitrocellulose, and then probed with 5 to 10 µg/ml of the indicated Abs. The bound Abs were revealed by means of 125I-protein A. The molecular mass markers (top to bottom) are 97, 66, and 45 kDa.

Pharmacological Characterization of the 6^* and (Non-6)2^* Receptor Populations. We pharmacologically characterized the nicotinic subtypes in rat retina by performing binding experiments on the immunoimmobilized subtypes as described under Materials and Methods. Because the equilibrium binding assays revealed no significant differences in the affinity for [³H]Epi of the 6^* and (non-6)2^* receptor populations [apparent K_d values of, respectively, 10.2 (CV 34%) and 8.7 pM (CV 25%)], competition binding studies were performed using a number of nicotinic ligands. No significant differences were detected for the agonists acetylcholine, nicotine, cytosine, and DMPP or the antagonists dihydro--erythroidine and D-tubocurarine (Table 4), but significant differences were observed for CntxMII, which showed a statistically significant better fit for a two-site model with a high- (K_i = 1.1 nM) and low-affinity site (K_i > 10 µM) when tested on the 6^* nAChRs (Fig. 8 and Table 4). We only determined a single low-affinity site for the (non-6)2^* receptors, with K_i > 10 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Inhibition of 125I-Epi binding to native immunoimmobilized 6*() and (non-6)2* () by CntxMII. The curves were obtained by fitting three to four separate competition experiments using the LIGAND program.

Dark-Rearing Slightly Affects the Number and Subunit Composition of Rat Retina [³H]Epibatidine Receptors, but Does Not Affect ¹²⁵I-Bungarotoxin Receptors. Activity-dependent synaptic plasticity is a fundamental feature of the vertebrate central nervous system. To determine whether retinal nAChRs are regulated by visual activity, the expression of ¹²⁵I-Bgtx and [³H]Epi nicotinic retinal receptors on P21 and the subunit composition of the³H-Epi receptors in dark-reared (DR) rats and rats raised in a diurnal light/dark cycle (LR) were compared.

Radioactive ligand binding studies of the membranes obtained from the eyes of the DR and LR animals showed that the levels of ¹²⁵I-Bgtx receptors were not significantly different (mean ± S.E.M. of five experiments, DR = 34.4 ± 2.0 fmol/mg of membrane protein, LR = 31.4 ± 2.2 fmol/mg of membrane protein) (Fig. 9A). The DR animals however, had approximately 30% more [³H]Epi receptors (110.2 ± 7.2 and 86.4 ± 5.0 fmol/mg of membrane protein, P < 0.05) (Fig. 9B) than the LR animals.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Effects of dark rearing on nAChR expression. 125I-Bgtx binding (A) and [3H]Epi (B) in eye membranes obtained from LR and DR animals on P21. C, immunoprecipitation of nAChR subunits in 2% Triton X-100 extracts of eye membranes obtained from P21 LR and DR animals. Each value represents the mean ± S.E.M. of three separate experiments. , P < 0.05 (Student's t test).

Given the considerable increase in [³H]Epi receptors containing the 2, 4, 6, 2, and 3 subunits on P21, the levels of the subunits contained in 2% Triton X-100 eye extracts from the LR and DR animals were determined. The increase in the number of receptors in the DR rats was statistically significant and associated with an increase in the number of receptors containing the 4 and 2 subunits but not of those containing the 2, 3, 5, 6, 2, and 3 subunits (Fig. 9C).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The various nicotinic effects of ACh during retina development may be mediated by the different receptor subtypes that may have a different pattern of signaling (Dmitrieva et al., 2001; Zhou, 2001). Identifying the nAChR subtypes involved in ACh retinal action is thus important for understanding retina circuitry and developing pharmacological tools that modulate specific retinal functions.

In this molecular and pharmacological study, we identified the nAChR subtypes expressed in rat retina and studied their expression at different postnatal developmental stages. Our main findings were that 1) the numbers of Bgtx and Epi nAChRs increased from P1 to P21, when they reached adult levels, with the increase in Epi receptors being caused by selective increases in the receptors containing the 2, 4, 6, 2, and 3 subunits; 2) immunopurification experiments showed that P21 retina contains a relatively wide array of different heteromeric nAChR subtypes, and in situ hybridization experiments showed that nAChR subunit mRNAs have highly heterogeneous distribution patterns throughout the retinal layers; and 3) although visual experience does not markedly alter the developmental expression of nAChRs, there was a selective increase in the expression of the 42 subtype in the retina of DR animals.

Because our findings concerning retinal subtype expression and their subunit assembly are derived from ligand binding experiments and the immunoprecipitation of [³H]Epi-labeled receptors with subunit-specific Abs, they critically depend on Ab specificity and efficiency. These parameters were carefully checked by means of previously described immunoprecipitation experiments using tissues obtained from KO animals and purified receptors (Zoli et al., 2002; Champtiaux et al., 2003) but still require previously discussed caveats (Zoli et al., 2002).

In agreement with the results of previous in situ hybridization studies (Zoli et al., 1995) and in accordance with the hypothesis that nAChRs play an important role during the pre- and perinatal retinal development, we found high concentrations of both Epi and Bgtx receptors at birth. However, although the number of both classes of receptors increased during the postnatal period, there were more Epi than Bgtx receptors. Taken from the current hypothesis that homomeric Bgtx-sensitive receptors have five ligand binding sites per receptor and that heteromeric receptors have only two (Le Novère and Changeux, 1995; Corringer et al., 2000), Epi receptors are more expressed than Bgtx receptors at P1 and become largely predominant during postnatal development and adulthood.

As observed in adult rabbit retina (Keyser et al., 2000), we found that the large majority of heteromeric receptors in developing and adult rat retina contain the 2 subunit, although at birth, approximately 20% of receptors were 4^* nAChRs. The amount of 4^* nAChR then remained constant whereas the amount of 2^* nAChRs increased markedly so that by P21, the large majority of retinal receptors contained the 2 subunit. The prevalence of 2^* receptors seems to be mammal-specific because we have previously shown that the 4 subunit is the major postnatally expressed structural subunit in chick retina after a developmental shift from 2to 4 during embryonic development (Vailati et al., 1999, 2003). Besides 2 and 4, which are constantly the most concentrated subunits of the heteromeric receptors, during postnatal development, there is a clear change in the concentration of the other subunits. In the early phase of retinal development (P1), 24% of the receptors contain the 3 and 23% the 4 subunits, but there is a selective increase in the expression of receptors containing the 2, 6, 4, 2, and 3 subunits by P5, which reaches a peak by P21.

Previous pharmacological and KO animal studies have shown that mouse retinal waves depend on heteromeric nAChRs; in particular, 2 KO animals have no nAChR-mediated waves between P0 and P8 (Bansal et al., 2000). This can now be interpreted as a consequence of the fact that the large majority (>80%) of heteromeric nAChRs contain the 2 subunit at all postnatal developmental stages, and therefore, very few nAChRs are left to mediate retinal waves after 2 subunit deletion.

The contribution of 3-containing receptors changes during development: on P1, they represent a substantial fraction of heteromeric nAChRs (24%) and functionally participate in retinal wave activity (Bansal et al., 2000), but although their number (expressed as femtomole per milligram of protein) remains almost constant during development, their relative contribution to the total number of Epi receptors markedly decreases. On the other hand, the receptors containing the 6 subunit are highly up-regulated during development, and their number increases 7.3 times between P1 and P21, when they become largely predominant over 3-containing receptors. Because the amino acid sequence of the 3 subunit is very close to that of the 6 subunit (70% identity), it is likely that most of the retinal receptors in adult vertebrates identified previously as containing 3 on the basis of immunolocalization and immunopurification experiments may actually have contained 6 rather than 3 subunits (Whiting et al., 1991; Keyser et al., 2000).

Another important finding is the considerable developmental increase in the number of 2-containing receptors (more than 33-fold from P1 to P21), which account for 23% of all heteromeric receptors by P21. In addition, 2 mRNA distribution in the retina is unique among nAChR subunits because 2 mRNA labeling was only detected in the GCL and external INL, thus suggesting that 2-containing nAChRs may be expressed selectively in a subpopulation of RGCs. 2-Containing receptors in the retina may play a particular role in the functional and anatomical development of visual systems, as indirectly suggested by the recent finding that 2 KO mice have an altered anatomical and functional visual development, whereas 4 or 6 KO animals do not (Rossi et al., 2001; Champtiaux et al., 2002). It is therefore possible that principal subunits other than 6 and 4 constitute the 2^* nAChRs, which are necessary for the normal development of the visual system and/or that subunit heterogeneity plays a role in the functional compensation of the missing subunits in KO animals.

The immunopurification studies isolated three populations of P21 heteromeric retinal nAChRs: the 6^* receptor population (approximately 26% of total Epi binding), the 4(non-6)^* receptor population (approximately 60% of the receptors), and the (non-4/non-6)^* receptor population (around 14% of receptors). All three populations are heterogeneous, but immunoprecipitation and further immunopurification of the purified receptors allowed for the identification of the subunit composition of most of the different subtypes. A summary of the identified subtypes and their relative percentages of all of the Epi receptors present in rat retina are shown in Table 5.