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Developmental Expression of Heteromeric Nicotinic Receptor Subtypes in Chick Retina

Consiglio Nazionale delle Ricerche (CNR), Institute of Neuroscience, Cellular and Molecular Pharmacology Section, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy (S.V., M.M., F.C., C.G.); CNR Center of Hormone Chemistry, Milan, Italy (R.L.); and Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, University of Milan, Italy (G.E.R.)

Received January 13, 2003; accepted February 12, 2003

	Abstract

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Acting through nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in retinal development and the formation of retinal connections to target tissues, but very little is known about the nAChR subtypes expressed in vertebrate retina during neuronal development. We used immunoprecipitation and [³H]epibatidine binding to study the expression of chick retina -bungarotoxin–insensitive heteromeric nAChRs during development and adulthood, and found that it is strictly developmentally regulated, reaching a peak on postnatal day 1. The increase in [³H]epibatidine receptors is caused mainly by an increase in the receptors containing the 2, 6, 3, and 4 subunits. The contribution of subunits to [³H]epibatidine receptors significantly changes during development: the 2 subunit is contained in the majority (84%) of receptors on embryonic day (E) 7 but in only 32% on postnatal day (P) 1, whereas the 4-containing receptors increase from 22% to 78% during the same period. Using a sequential immunodepletion procedure, we purified the 2- and 4-containing subtypes and found that they coassemble with 4 and/or 3 on E11, and also with the 2, 6, and 3 on P1. After the immunodepletion of 6-containing receptors, the 2- and 4-containing receptors have a very similar pharmacological profile on P1. Parallel immunoprecipitation experiments in other brain areas showed that the developmentally regulated receptors in optic lobe are those containing the 2, 5, and 2 subunits and those containing the 4 and 2 subunits, whereas the receptors in forebrain-cerebellum contain the 4 and 2 subunits with or without the 5 subunit. These results indicate that there is an increase in receptor heterogeneity and complexity in chick retina during development that is also maintained in adulthood.

However, the role of nAChRs in dendritic remodeling and the spontaneous activity of RGCs seems to be species-specific and developmentally regulated (Wong et al., 1998, 2000). In the chick, pharmacological experiments with nicotinic antagonists have shown that nicotinic cholinergic transmission is important in driving dendritic filopodia motility and spontaneous activity early in retinal development, but less so as development continues (Wong, 1999; Sernagor et al., 2000; Wong et al., 2000; Wong and Wong, 2001). The influence of ACh on in vivo retinal development probably depends on the nAChR subtype expressed at each stage, but it is not yet clear how many subtypes are expressed or which subtypes are the most important.

Chick neuronal nAChRs are cationic channels whose opening is physiologically controlled by the ACh neurotransmitter. They form a heterogeneous family of pentameric oligomers made up of combinations of subunits encoded by at least 12 different genes. Although there are many subtypes consisting of different subunits, based on their phylogenetic, functional, and pharmacological properties (Le Novère and Changeux, 1995; Corringer et al., 2000), two main classes have been identified: the -bungarotoxin (Bgtx)-sensitive receptors made of 7, 8, or 9 subunits, which can form homomeric or heteromeric receptors, and the Bgtx-insensitive receptors made of 2–6 and 2–4 subunits, which form heteromeric receptors. In heteromeric receptors, more than one type of or subunit can participate in the formation of the receptor pentamer, thus increasing the number of possible receptor subtypes with different pharmacological and functional properties (Lindstrom, 2000).

Previous nicotinic ligands binding and immunolocalization studies have shown that chick retina expresses both classes of nAChRs, which are localized on amacrine, displaced amacrine, ganglion, and bipolar retinal cells (Betz, 1981; Whiting et al., 1991; Britto et al., 1992, 1994; Anand et al., 1993; Keyser et al., 1993; Hamassaki-Britto et al., 1994). Moreover, biochemical and pharmacological studies have identified the presence of three Bgtx binding subtypes in chick retina, the homomeric 7 and 8 subtypes and the heteromeric 7–8 subtype, all of which have a developmentally regulated expression (Keyser et al., 1993; Gotti et al., 1994, 1997). In situ hybridization and immunolocalization studies, together with Northern blot analyses, have shown that chick retina contains almost all of the nicotinic subunits present in heteromeric receptors (Matter et al., 1990; Whiting et al., 1991; Britto et al., 1992, 1994; Hamassaki-Britto et al., 1994; Hernandez et al., 1995; Fucile et al., 1998). In particular, there is a selective expression of the 6 and 3 subunits, which are only present in catecholaminergic nuclei and retina in the mammalian central nervous system (Le Novère et al., 1996). In previous biochemical, immunological, and pharmacological studies, we have shown that most of the Bgtx-insensitive [³H]Epi receptors in chick retina contain the 4 subunit (associated with the 4, 6, and/or 3 subunits) on postnatal day (P) 1 (Vailati et al., 1999, 2000; Barabino et al., 2001), but nothing is known about their developmental expression.

We used ligand binding and immunoprecipitation experiments to study the expression of the high-affinity [³H]Epi binding receptors in chick retina and the optic tectum (its target tissue), using the forebrain-cerebellum tissue as a further control. Because we found a developmental change in the retinal receptors containing the 2 and 4 subunits, we also immunopurified the subtypes containing these subunits on embryonic day (E) 11 and P1 and studied their subunit coassembly and pharmacology.

	Materials and Methods

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Materials. The protease inhibitors, the nonradioactive Epi, nicotinic ligands, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO); the CnBr-activated Sepharose 4BCL [PDB] and ¹²⁵I-protein A from Amersham Biosciences UK, Ltd. (Buckinghamshire, UK); (±)-[³H]Epi from PerkinElmer Life Sciences (Boston, MA); and the reagents for gel electrophoresis from Bio-Rad Laboratories (Hercules, CA).

Antibody Production and Characterization. The polyclonal antibodies (Abs) against the 2, 3, 4, 5, 6, 2, 3, and 4 peptides were raised and characterized as described previously (Vailati et al., 1999, 2000; Balestra et al., 2000; Barabino et al., 2001). Two different peptides were chosen for all the subunits: one located in the cytoplasmic loop between M3 and M4, which is the most divergent region of the subunits, and the other located at the COOH-terminal (COOH). 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 of the antibodies has been previously reported (Vailati et al., 1999, 2000; Balestra et al., 2000; Barabino et al., 2001), and additional experiments on BOSC 23 cell lines transfected with different nAChR subunits are reported under Results.

Preparation of Retina, Chick Optic Lobe, and Forebrain-Cerebellum Triton X-100 Extracts. The embryos, 1-day-old animals, and adult animals were of the Gallus gallus strain and obtained from a local hatchery. The embryos were kept in the dark, whereas the chicks were kept under natural lighting conditions. The retina, optic lobe, and forebrain-cerebellum samples were dissected from in ovo chickens on E7, E11, E14, and E18 and from chickens on P1, P35, and P59, immediately frozen in liquid nitrogen, and stored at –80°C for later use. No differences in the binding properties of the fresh and frozen tissues were observed. In every experiment, the three types of tissue were separately homogenized 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. The homogenates were then diluted and centrifuged for 1.5 h at 60,000g.

This procedure of homogenization, dilution, and centrifugation was performed twice, after which the pellets were collected, rapidly rinsed with 50 mM sodium phosphate, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride, and then resuspended in the same buffer containing a mixture of 10 µg/ml 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 were then centrifuged for 1.5 h at 60,000g, recovered, and an aliquot of the resultant supernatants was collected for protein measurement using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard.

Binding Assay. The (±)-[³H]Epi with a specific activity of 66.6 Ci/mmol was purchased from PerkinElmer Life Sciences; the nonradioactive Epi was purchased from Sigma/RBI (Natick, MA). Because 2-, 4-, and 8-containing receptors bind [³H]Epi with picomolar affinity and 7 receptors bind it with nanomolar affinity (Gerzanich et al., 1995), the binding tissue extract and immunoprecipitation experiments were performed in the presence of 2 µM Bgtx, which specifically binds to the 7 and 8 subtypes and blocks [³H]Epi binding, to ensure that the 7 and 8 subtypes did not contribute to [³H]Epi binding

The Triton X-100 extracts of retina, optic lobe, and forebrain-cerebellum 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]Epi-Labeled Receptors by Antisubunit-Specific Abs. The extracts obtained from the three tissues at different ages, 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). The immunoprecipitation was recovered by incubating the samples with beads containing bound anti-rabbit goat IgG (Technogenetics, 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 femtomoles of immunoprecipitated receptors per milligram of protein.

Receptor Subtype Immunopurification and Analysis. The extracts prepared from E11 and P1 chick retina were incubated twice with 5 ml of Sepharose-4B and bound anti-2 Abs to remove the 2 receptors and then twice with 5 ml of Sepharose-4B with bound anti-4 Abs; 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 peptide 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 chick 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 2% Triton X-100 extract of P1 chick retina was immunodepleted of 6-containing receptors by passing it over a column of Sepharose-4B with bound anti-6 Abs.

The affinity-purified anti-2 or anti-4 Abs were bound to micro-wells (Maxi-Sorp; Nalge Nunc International, Naperville, IL) by means of overnight incubation 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 retina membrane extract containing 50 to 100 fmol of [³H]Epi binding sites, which was prepared by sequentially immunodepleting it of the 6-containing receptors. After incubation, the wells were washed and the presence of immobilized receptors revealed by means of [³H]Epi binding. The binding techniques for immunoimmobilized subtypes and the data analysis were the same as those described previously (Vailati et al., 1999).

Expression of nAChR Subunits in BOSC 23 Cells. Transient transfections of the nAChR subunits were carried out in the retroviral packaging cell line BOSC 23, as described previously (Ragozzino et al., 1997). The cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT). The subunit cDNAs were added in equivalent amounts (8 µg each per 100-mm dish). Between 8 and 12 h after transfection, the cells were washed twice and fed again with Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells were collected in ice-cold phosphate-buffered saline (Invitrogen) 36 to 48 h after transfection and stored at –70°C.

	Results

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Characterization of the Subunit Specificity of the Antibodies. We have previously purified 6-containing receptors from chick retina and 252 and 42 subtypes from the optic lobe on P1 and have shown their subunit composition by means of quantitative immunoprecipitation (see Table 1) and Western blotting. The 2, 5, and 2 Abs had respective immunoprecipitation capacities of 51, 66, and 80% on the 252 subtype. The Abs directed against the 6 or 4 subunits both had an immunoprecipitation capacity of almost 90% on the 6-purified subtype, whereas the anti-3 and anti-3 Abs only immunoprecipitated 42 and 51% of these receptors. This incomplete immunoprecipitation by anti-3 and anti-3 Abs may have been caused by the limited presence of the 3 and 3 subunits in the 64 receptors or by reduced immunoprecipitation efficiency. To answer this question, we transiently transfected the BOSC cells with the 32 or 432 or 44 chick subunits and measured the [³H]Epi-labeled receptors immunoprecipitated in 2% Triton X-100 extract by the same antibodies as those used to immunoprecipitate the native receptors (see Table 1). The studies of transfected cells confirmed the subunit specificity of the Abs and showed that the anti-3, anti-4, anti-2, and anti-4 Abs had an immunoprecipitation capacity ranging from 80 to 97%, whereas the anti-3 Ab (although they are subunit-specific, because they do not recognize the other subunits) recognized only 45% of the [³H]Epi receptors in the cells transfected with 432 subunits. This may be because the anti-3 Ab has a relatively low capacity or because the 3 subunit is associated with 4 and 2 in only 45% of the receptors in this cell line. On the basis of these results and the previous immunoprecipitation study, we can only conclude that the 3 Ab has an immunoprecipitation capacity of at least 45%.

[³H]Epibatidine-Binding Receptors in Chick Central Nervous System during Development. We and others (Gerzanich et al., 1995; Barabino et al., 2001) have previously shown that chick retina expresses a high level of Bgtx-binding receptors and that these receptors 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 nicotinic receptors. We have previously shown the presence of [³H]Epi-labeled receptors in chick retina on P1 (Vailati et al., 1999; Barabino et al., 2001). To investigate their expression during embryonic development and the aging process, we performed binding studies on 2% Triton X-100 retina extracts obtained from chicks on E7, E11, E14, E18, P1, P35, and P59 and compared the expression of the [³H]Epi receptors with that of the receptors present in the optic lobe and forebrain-cerebellum at the same ages (see Fig. 1). The receptor level (mean ± S.E.M. of three experiments) was similar in retina, optic lobe, and forebrain-cerebellum on E7 (41.4 ± 2.9, 46.9 ± 3.2, and 46.9 ± 3.2 fmol/mg of protein, respectively) and E11 (103.2 ± 5.6, 86.9 ± 2.1, and 78.3 ± 2.7 fmol/mg of protein) but subsequently increased much more in the retina and optic lobe (246.6 ± 21.4 and 224.7 ± 2.9 fmol/mg of protein on P1) than in the forebrain-cerebellum (77.5 ± 2 fmol/mg of protein on P1). After birth, the receptor level gradually decreased in every tissue (170.6 ± 33.4 and 149 ± 20.9 fmol/mg of protein on P35 and on P59 in retina; 161.6 ± 17.6 and 140.3 ± 14.9 fmol/mg of protein in optic lobe; and 46.5 ± 5.7 and 46.1 ± 3.4 fmol/mg of protein in forebrain-cerebellum).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Developmental changes in [3H]Epi-binding receptors expressed in the retina, optic lobe, and forebrain-cerebellum. The tissues were dissected from the embryos at the indicated times and frozen. Triton X-100 (2%) extracts were prepared as described under Materials and Methods, preincubated with 2 µM Bgtx, and then labeled with 2 nM [3H]Epi. The reported values are expressed as femtomoles of labeled [3H]Epi receptor per milligram of protein and are the mean values ± S.E.M. of three to four experiments performed in triplicate.

Subunit Content of the [³H]Epi Receptors in Retina. To identify whether different subtypes are expressed during embryonic development, at birth and afterward, we performed 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: one located in the cytoplasmic loop and the other in the COOH-terminal region. The reported values are the mean values obtained in three separate experiments for each subunit. On E7, the large majority of retinal receptors contained the 4 and 2 subunits. By E11 (together with the receptors containing the 4 and 2 subunits), there was an increase in the receptors containing the 3 and/or 3 and/or 4 subunits; by E14, there was an increase in the expression of the 6 and/or 2 subunits (see Fig. 2). By P1, the 6, 3, 2, 4, and 3 subunits had increased 70, 9, 26, 16, and 9 times, respectively, over their levels on E7, whereas the increase in the 4, 5, and 2 subunits was much more limited (respectively 3.1, 4.6, and 2.5 times) (see Fig. 3)

View larger version (26K): [in this window] [in a new window]   Fig. 2. Immunoprecipitation analysis of the subunit content of the [3H]Epi receptors expressed in the retina, optic lobe, and forebrain-cerebellum. Triton X-100 (2%) extracts were prepared from tissues dissected from the animals on E7, E11, E14, E18, P1, P35, 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 2): one directed against a subunit cytoplasmic peptide and the other 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 protein and are the mean values ± S.E.M. of three experiments performed in duplicate (unless shown, the S.E.M. is in the range of the symbol).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Developmental changes in the expression of nicotinic receptor subunits in 2% Triton X-100 extract of retina. The receptors preincubated with 2 µM Bgtx and labeled with [3H]Epi were immunoprecipitated at the indicated times as described in the legend to Fig. 2. The reported values are the -fold increases in immunoprecipitation for each subunit at each developmental time normalized to the E7 value expressed as femtomoles per milligram of protein.

Subunit Content of the [³H]Epi Receptors in Optic Lobe and Forebrain-Cerebellum. In parallel experiments, we checked the expression of the subunit receptors present in the optic lobe and found that the 4-containing receptors were already highly expressed on E7 and had increased 2- to 3-fold by P59. The level of the 2-containing receptors was similar to that of the 4-containing receptors on E7 but then increased much more (from 30.57 ± 5.3 on E7 to 188.1 ± 3 fmol/mg of protein on P1). As described previously (Balestra et al., 2000), there was a selective increase in the 2 and 5 subunits from E11 to P1 (from 2 ± 0.4 to 57.5 ± 2.5 fmol/mg of protein for the 2 subunit and from 1.2 ± 1 to 62 ± 1 fmol/mg of protein for the 5), and their level of expression was still high on P35 and P59.

The immunoprecipitation studies of the forebrain-cerebellum only detected the presence of a considerable level of 4 and 2 subunits on E7 (21.5 ± 1 and 30.5 ± 5 fmol/mg of protein) and these levels increased by approximately 2 to 3 times between E7 and P1. We also detected a developmental increase in the 5-containing receptor (from 2.4 to 13 fmol/mg of protein) and, to a much lesser extent, the 2-, 3-, and 4-containing receptors, but we never detected the presence of any 6- or 3-containing receptors at any time.

Change in the Expression of the 2- and 4-Containing Receptors in Retina. The above results show that there is a change in the expression of the subunits during development and adulthood. As shown in Fig. 2, the level of 2-containing receptors was higher than that of 4-containing receptors on E7, increased until E14, remained constant until P1, and then slightly decreased. The level of the 4-containing receptors increased almost linearly from E7 to P1 and then decreased (although it remained higher than that of the 2-containing receptors). This suggests that the contribution of the subunits to the expressed subtypes changes during development. On E7, 84% of the [³H]Epi receptors contained the 2 subunit and only 22% the 4 subunit; by P1, however, 78% of the receptors contained the 4 and only 32% the 2 subunit (Fig. 4). To identify the subunits coassembled with the 2 or 4 subunits during early and late embryonic development, we immunopurified the receptors containing the 2 and 4 subunits from 2% Triton X-100 extracts (there was too little material available on E7) on E11 and P1.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Changes in 2/4 subunit expression in chick retina. The level of expression of the 2- and 4-containing receptors in extracts obtained from animals at the indicated times is expressed as the percentage of [3H]Epi-labeled receptors present in the solution before immunoprecipitation. The reported values are the mean ± S.E.M. of three to four separate experiments.

We first immunopurified the 2-containing receptors by passing the extracts obtained on E11 or P1 over a column with bound anti-2 Abs, and then the flowthroughs of these columns (devoid of the 2-containing receptors) were passed over a column with bound anti-4 Abs. The 2- or 4-bound receptors were recovered by competition with the 2 or 4 peptides, labeled with 2 nM [³H]Epi, and then immunoprecipitated by means of subunit-specific Abs. The results of the quantitative immunoprecipitation studies of the purified receptors are shown in Fig. 5, A and B. On E11, the and subunits in the 2-containing receptors were 3 (12.2 ± 0.8%), 4 (55 ± 5%), 3 (28 ± 2%), and 4 (6.7 ± 1.1%), whereas the other subunits were almost absent. On P1, the subunits in the 2-containing receptors were more heterogeneous, with 2, 3, 4, 5, 6, 3, and 4 being present in 28 ± 1.7, 11 ± 1.4, 23 ± 4, 3.6 ± 2, 19.3 ± 2.4, 30 ± 1.7, and 18.7 ± 4% of receptors, respectively (Fig. 5A).

View larger version (24K): [in this window] [in a new window]   Fig. 5. Immunoprecipitation of purified 2- and 4-containing receptors. The subtypes were purified from tissues obtained from animals on E11 or P1 as described under Materials and Methods. After extensive dialysis to remove the 4 or 2 peptides used to elute the receptors from the affinity columns, the receptors were labeled with 2 nM [3H]Epi and immunoprecipitated using saturating concentrations (20–30 µg) of the indicated Abs. The results are expressed as percentages of the [3H]Epi-labeled receptors, taking the amount of receptor present in the solution before immunoprecipitation as 100%. The percentage of immunoprecipitation was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG. The values are the mean ± S.E.M. of three determinations.

The purified 4-containing receptors mainly contained the 3 (72 ± 5%), 4 (22.5 ± 0.9%), and 6 (11 ± 1%) subunits on E11, whereas their subunit composition on P1 was much more complex, with the anti-2, anti-3, anti-4, anti-5, anti-6, and anti-3 subunit-specific Abs, respectively, immunoprecipitating 14.7 ± 0.9, 35 ± 2.6, 40 ± 2.6, 5 ± 1, 27 ± 0.9, and 19 ± 4% (Fig. 5B) of the receptors. The subunit compositions of the 2- and 4-containing receptors on P1 were also analyzed on Western blots using the same subunit-specific Abs as those used for the immunoprecipitation and previously tested on Western blots of purified chick subtypes (Vailati et al., 1999, 2000; Balestra et al., 2000; Barabino et al., 2001).

The results confirmed that the 2, 3, 4, and 6 subunits coassemble with the 2 and 4 subunits (Fig. 6). The anti-2 Ab recognized a major peptide with a molecular mass of 60 ± 1 kDa on 2- and 4-purified subtypes (lanes 1 and 9); the anti-3 Ab recognized a major band of 56 kDa and a lower band of 54 kDa (lanes 2 and 10); the anti-4 Ab recognized a major band of 68 kDa (lanes 3 and 11); and the anti-6 Ab a single band of 57 kDa (lanes 5 and 13). The anti-5 Ab (lanes 4 and 12) did not recognize any band, thus indicating that the expression of the receptors containing this subunit was too low (as also found by immunoprecipitation). The anti-2 Abs recognized a peptide with a molecular mass of 54 kDa on the 2- (lane 6) but not on the 4-containing receptors (lane 14), whereas the anti-3 (lanes 7 and 15) and anti-4 Abs (lanes 8 and 16) recognized peptides with molecular masses of 55 and 52 kDa. We also used immunoprecipitation experiments to test the purified E11 and P1 receptors for the presence of 7 or 8 subunits coassembled in the 2- or 4-containing receptors, but neither was detectable at either developmental time (Fig. 5).

View larger version (60K): [in this window] [in a new window]   Fig. 6. Western blot analysis of P1 affinity-purified 2- and 4-containing receptors. The receptors were prepared as described in the legend to Fig. 5. 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 2- and 4-Containing Receptors Present on P1. Most studies of the functional role of retinal nAChRs rely on pharmacological experiments performed using nicotinic drugs. To see whether the available nicotinic drugs are selective on the retina subtypes, we pharmacologically characterized the 2- and 4-containing receptors using nicotinic agonists and antagonists. Because we have previously shown (Vailati et al., 1999; Barabino et al., 2001) that the -conotoxin MII binds to 6-containing receptors with high affinity and selectivity, we performed the binding experiments on 2- and 4-containing receptors previously depleted of the 6-containing receptors. [³H]Epi binds to the 2- and 4-containing receptors with a high affinity; the K_d values calculated from four separate experiments were 27 pM (coefficient of variation, 20%) and 26 pM (coefficient of variation, 16%) for the 2- and 4-containing receptors, respectively.

The pharmacological profiles of the 2- and 4-containing receptors were characterized by testing the relative efficacy by which cholinergic agonists and antagonists inhibited the binding of 0.05 nM [³H]Epi at equilibrium. The K_i values of the inhibition curves obtained by simultaneously fitting the data of three to four different experiments are shown in Table 2, together with the K_i values of the same drugs for the 6-containing receptors (Vailati et al., 1999). We determined that the rank order of antagonist potency for 2- and 4-containing receptors was identical: dihydro--erythroidine > d-tubocurarine > MG624 > decamethonium > hexamethonium. The -conotoxin MII, which was the most potent drug (K_i, 66 nM) in competing for 6 receptors (Table 2), inhibited the binding of 2- and 4-containing receptors only at very high concentrations of >2 µM.

	Discussion

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ACh is synthesized and released in vertebrate retina by two populations of amacrine cells (one in the inner nuclear layer and the other in the ganglion cell layer) and can regulate many aspects of neuronal development by acting on nAChRs (reviewed in Feller, 2002). The various nicotinic effects of ACh may be mediated by the different receptor subtypes functionally expressed during development (Zhou, 2001), which can activate different signaling pathways (Dmitrieva et al., 2001) or have a different pattern of signaling because of the biophysical properties of the different receptor subtypes (Role and Berg, 1996).

In this molecular and pharmacological study, we identified the major nAChR subtypes expressed in chick retina and studied their expression at different stages of development. Our main findings were that 1) during embryonic development, there is an increase in [³H]Epi binding receptors because of a developmental increase that is particularly prominent for the receptors containing the 2, 6, 3, and 4 subunits; 2) there is a developmental change in subunit expression, with the large majority of receptors containing the 2 subunit early in development and two thirds containing the 4 subunit by P1; 3) the major subtype early in development is that containing the 42 subunits, but subtype expression becomes more heterogeneous by E11, and even more so at hatching; and 4) the 7 or 8 subunits never coassemble with heteromeric subunits in receptors containing the 2 or 4 subunits on E11 or P1.

Our conclusions concerning the subtypes expressed in retina and their subunit coassembly are based on the immunoprecipitation of [³H]Epi-labeled receptors using subunit-specific Abs, and thus critically depend on antibody specificity and efficiency, which were carefully checked in immunoprecipitation experiments on purified receptors and transfected cells.

On the basis of the current hypothesis that homomeric Bgtx-sensitive receptors have five ligand-binding sites per receptor and heteromeric receptors have only two (Le Novère and Changeux, 1995; Corringer et al., 2000), the most abundant class of nAChRs expressed throughout embryonic retinal development are the Bgtx receptors containing the 7 and/or 8 subunits (Gotti et al., 1994). However, heteromeric receptors binding [³H]Epi are also highly expressed during embryonic development and their number increases 6-fold between E7 and P1. The level and temporal expression of retinal [³H]Epi receptors are very similar to those of the optic lobe receptors, which suggests a common receptor regulation in the visual pathway that is not present in other regions (the increase in nAChRs in the forebrain-cerebellum is much more limited and peaks between E14 and E18).

The receptors expressed in the early phase of retinal development (E7) are those containing the 42 subunits, and their expression is qualitatively and quantitatively very similar to that of the subtypes present in the optic lobe and forebrain-cerebellum. By E11, there is an increase in the expression of the 3, 3, and 4 subunits, and affinity purification of the 2- and 4-containing receptors at this age shows that the 3 and 4 subunits are present in both, but 4 is associated mainly with the 2 subunit and 3 with the 4 subunit; the 3 subunit is present in a similar fraction of both types of receptors. After E14, there is a considerable increase in the number of receptors containing the 6 and 2 subunits that reaches a peak by P1, when both the 2- and 4-containing subtypes are heterogeneously associated with the 2, 3, 4, and 6 subunits. The results of our immunoprecipitation studies are consistent with those of previous Northern blot analyses showing an increase in 3, 6, and 3 mRNAs from the early to late stages of embryonic development and then a decrease in adult animals (Matter et al., 1990; Whiting et al., 1991; Fucile et al., 1998).

The heterogeneity in the subunit composition of the 2- and 4-containing receptors at P1 is not revealed by their pharmacological profile, in that both have a very similar affinity and rank order of potency for the tested nicotinic agonists and antagonists. Unlike the heterologously expressed 2- or 4-containing rat subtypes (Parker et al., 1998), the presence of more than one type of and subunit/receptor may change the pharmacology of native chick retinal subtypes. Another possibility is that the affinities of different subtypes to nicotinic drugs are so close that they cannot be discriminated by binding.

Moreover, our pharmacological experiments showed that the 2- and 4-containing receptors devoid of the 6-containing receptors have a low affinity for the -conotoxin MII toxin, thus confirming that the 6 subunit is the crucial subunit conferring high affinity for this toxin in chick subtypes, as previously shown (Vailati et al., 1999; Barabino et al., 2001), and also recently demonstrated in 6 KO mice by Champtiaux et al. (2002). -Conotoxin MII is therefore the only available tool capable of discriminating some of the nAChR subtypes in the chick retina.

Another important finding of this study is that there is a developmental increase in the number of 2-containing retinal receptors, reach a peak on P1 and decrease only slightly in adulthood. These receptors represent roughly 18% of the heteromeric receptors at P1 and are associated with the 2 and/or 4 subunits but never with the 6 subunit. The presence of the 2 subunit in retina may be important in the functional and anatomical development of visual systems, as also 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 subunits other than 6 and 4 are important for the development of the visual system and/or that subunit heterogeneity plays a role in the functional compensation of subunits in KO animals.

We found that the major subunit expressed in chick retina on P1 and in adulthood is the 4 subunit, whereas Keyser et al. (2000) found that the large majority of heteromeric receptors in adult rabbit retina contain the 2 subunit and we have found the same preponderance throughout postnatal development and adulthood in rat retina (C. Gotti, M. Moretti, S. Vailati, unpublished results). These results, together with the previous demonstration of the specific expression of the homomeric 8 subtype in retina (Keyser et al., 1993; Gotti et al., 1994), demonstrate the species-specific expression of nAChR subtypes in retina.

Comparison of nAChR expression in the retina and optic lobe shows greater developmental expression of 2-containing receptors in the latter, in which the 2 subunit is mainly assembled with the 2 and 5 subunits in the 252 subtype and with the 4 subunit in the 42 subtype. The expression of the 2 and 5 subunits remains high on P35 and P59, thus suggesting that this subtype is also selectively expressed in the optic lobe in adult animals. Interestingly, the increase in 2 subunit expression that we detected at protein level has been previously observed at the mRNA level by Matter et al. (1990), who found that it is absent in eyeless animals and appears when the optic nerve axons are invading the optic tectum and making retino-tectal synapses (peaking at E12).

Unlike Fucile et al. (1998), whose Northern blot studies failed to detect 6 mRNA in the optic tectum at any age, we found that there was a small increase in 6-containing receptors after E14. It is possible that 6-containing receptors are made in the retina and then transported to the tectum starting from E12. Our studies of chick forebrain-cerebellum showed that the largest developmental increase involved the 4, 2, and 5 subunits, thus indicating a developmental increase in receptors containing the 4, 2, and the 452 subunits. These results are consistent with the earlier findings of Conroy and Berg (1998) in chick brain.

Although we do not know the physiological role of all of the subtypes described here, our results provide a more defined picture of the heteromeric nAChR subtypes expressed in retina during embryonic development, upon hatching, and in adult ages. On the basis of these findings and those of previous studies of Bgtx-sensitive receptors (Keyser et al., 1993; Gotti et al., 1994, 1997), we can conclude that the heteromeric receptors containing the 3 and/or 4 subunits with the 2 subunit, and the homomeric receptors containing the 7 subunit, are expressed long before there is any evidence of synaptic connections and at later stages of embryonic development, whereas the receptors containing the 2, 6, 3, and 4 subunits, and the Bgtx receptors containing the 8 subunit, are present (and may thus play a role) only late in development, when complex functional circuits have been established in the retina itself and the retinal projections to subcortical structures.

	Acknowledgements

 
We thank Dr. Benedetta Barabino for the generous gift of the transfected cells, Dr. J. M. McIntosh for the generous gift of -conotoxin MII, Dr. Michele Zoli for critically reading the manuscript, and Kevin Smart for help with the manuscript.

	Footnotes

 
This work was supported in part by grants to Francesco Clementi from the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (grant MM05152538), the Italian Ministry of Health (grant ICS 030.3/RA 0048), and the European Research Training Network (grant HPRN-CT-2002-00258).

ABBREVIATIONS: nAChR, neuronal nicotinic acetylcholine receptor; ACh, acetylcholine; RGC, retinal ganglion cell; KO, knock-out; Bgtx, -bungarotoxin; Epi, epibatidine; E, embryonic day; P, postnatal day; Abs, polyclonal antibodies; COOH, subunit COOH peptide; MG624, N,N,N,-triethyl-2-(4-trans-stilbenoxy)ethylammoniam iodide.

Address correspondence to: Dr. Cecilia Gotti, CNR, Institute of Neuroscience, Section of Cellular and Molecular Pharmacology, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy. E-mail: c.gotti{at}csfic.mi.cnr.it

	References

Top Abstract Materials and Methods Results Discussion References

 
Anand R, Peng X, Ballesta JJ, and Lindstrom J (1993) Pharmacological characterization of -bungarotoxin-sensitive acetylcholine receptors immunoisolated from chick retina: contrasting properties of 7 and 8 subunit-containing subtypes. Mol Pharmacol 44: 1046–1050.[Abstract]

Balestra B, Vailati S, Moretti M, Hanke W, Clementi F, and Gotti C (2000) Chick optic lobe contains a developmentally regulated 252 nicotinic receptor subtype. Mol Pharmacol 58: 300–311.[Abstract/Free Full Text]

Bansal A, Singer JH, Hwang BJ, Xu W, Beaudet A, and Feller MB (2000) Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina. J Neurosci 20: 7672–7681.[Abstract/Free Full Text]

Barabino B, Vailati S, Moretti M, McIntosh MJ, Longhi R, Clementi F, and Gotti C (2001) An 44 nicotinic receptor subtype is present in chick retina. Identification, characterization and pharmacological comparison with the transfected 44 and 64 subtypes. Mol Pharmacol 59: 1410–1417.[Abstract/Free Full Text]

Betz H (1981) Characterisation of the -bungarotoxin receptor in chick-embryo retina. Eur J Biochem 117: 131–139.[Medline]

Britto LRG, Hamassaki-Britto DE, Ferro ES, Keyser KT, Karten HJ, and Lindstrom JM (1992) Neurons of the chick brain and retina expressing both -bungarotoxin-sensitive and -bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis. Brain Res 590: 193–200.[CrossRef][Medline]

Britto LRG, Torrao AS, Hamassaki-Britto DE, Mpodozis J, Keyser KT, Lindstrom JM, and Karten HJ (1994) Effects of retinal lesion upon the distribution of nicotinic acetylcholine receptor subunits in the chick visual system. J Comp Neurol 350: 473–484.[CrossRef][Medline]

Champtiaux N, Han ZY, Bessis A, Rossi FM, Zoli M, Marubio L, McIntosh JM, and Changeux JP (2002) Distribution and pharmacology of alpha6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J Neurosci 22: 1208–1217.[Abstract/Free Full Text]

Conroy WG and Berg DK (1998) Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the 4, 2 and 5 gene products. Mol Pharmacol 53: 392–401.[Abstract/Free Full Text]

Corringer PJ, Le Novère N, and Changeux JP (2000) Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40: 431–458.[CrossRef][Medline]

Daw NW, Brunken WJ, and Parkinson D (1989) The function of synaptic transmitters in the retina. Annu Rev Neurosci 12: 205–225.[CrossRef][Medline]

Dmitrieva NA, Lindstrom JM, and Keyser KT (2001) The relationship between GABA-containing cells and the cholinergic circuitry in the rabbit retina. Visual Neurosci 18: 93–100.[CrossRef][Medline]

Feller MF (2002) The role of nAChR-mediated spontaneous retinal activity in visual system development. J Neurobiol 53: 556–567.[CrossRef][Medline]

Fucile S, Matter J-M, Erkman L, Ragozzino D, Barabino B, Grassi F, Alemà S, Ballivet M, and Eusebi F (1998) The neuronal 6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells. Eur J Neurosci 10: 172–178.[CrossRef][Medline]

Gerzanich V, Peng X, Wang F, Wells G, Anand R, Fletcher S, and Lindstrom J (1995) Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors. Mol Pharmacol 48: 774–782.[Abstract]

Gotti C, Moretti M, Longhi R, Briscini L, Balestra B, and Clementi F (1994) Expression of -Bungarotoxin receptor subtypes in chick central nervous system during development. J Recept Res 14: 335–346.[Medline]

Gotti C, Moretti M, Maggi R, Longhi R, Hanke W, Klinke N, and Clementi F (1997) 7 and 8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties. Eur J Neurosci 9: 1201–1211.[CrossRef][Medline]

Hamassaki-Britto DE, Gardino PF, Hokoc JN, Keyser KT, Karten HJ, Lindstrom JM, and Britto LRG (1994) Differential development of alpha-bungarotoxin-sensitive and alpha-bungaro-insensitive nicotinic acetylcholine receptors in the chick retina. J Comp Neurol 347: 161–170.[CrossRef][Medline]

Hernandez M-C, Erkman L, Matter-Sadzinski L, Roztocil T, Ballivet M, and Matter J-M (1995) Characterization of the nicotinic acetylcholine receptor 3 gene. J Biol Chem 270: 3224–3233.[Abstract/Free Full Text]

Keyser KT, Britto RG, Schoepfer R, Whiting P, Cooper J, Conroy W, Brozozowska-Prechtl A, Karten HJ, and Lindstrom J (1993) Three subtypes of -Bungarotoxin-sensitive nicotinic acetylcholine receptors are expressed in chick retina. J Neurosci 13: 442–454.[Abstract]

Keyser KT, Macneil MA, Dmitrieva N, Wang F, Masland RH, and Lindstrom JM (2000) Amacrine, ganglion and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors. Visual Neurosci 17: 743–752.[CrossRef][Medline]

Le Novère N and Changeux JP (1995) Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J Mol Evol 40: 155–172.[CrossRef][Medline]

Le Novère N, Zoli M, and Changeux J-P (1996) Neuronal nicotinic receptor 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. Eur J Neurosci 8: 2428–2439.[CrossRef][Medline]

Lindstrom J (2000) The Structure of NAChRs, in Neuronal Nicotinic Receptors, Handbook of Experimental Pharmacology (Clementi F, Fornasari D, and Gotti C eds) vol 144, Springer-Verlag, Berlin.

Lipton SA (1988) Spontaneous release of acetylcholine affects the physiological nicotinic responses of rat retinal ganglion cells in culture. J Neurosci 8: 3857–3868.[Abstract]

Matter J-M, Matter-Sadzinski L, and Ballivet M (1990) Expression of neuronal nicotinic acetylcholine receptor genes in the developing chick visual system. EMBO (Eur Mol Biol Organ) J 9: 1021–1026.[Medline]

Muir-Robinson G, Hwang BJ, and Feller MB (2002) Retinogeniculate axons undergo eye-specific segregation in the absence of eye-specific layers. J Neurosci 22: 5259–5264.[Abstract/Free Full Text]

Parker MJ, Beck A, and Luetje CW (1998) Neuronal nicotinic receptor 2 and 4 subunits confer large differences in agonist binding affinity. Mol Pharmacol 54: 1132–1139.[Abstract/Free Full Text]

Ragozzino D, Fucile S, Giovanelli A, Grassi F, Mileo A, Ballivet M, Alemà S, and Eusebi F (1997) Functional properties of neuronal nicotinic acetylcholine receptor channels expressed in transfected human cells. Eur J Neurosci 9: 480–488.[CrossRef][Medline]

Role LW and Berg KD (1996) Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16: 1077–1085.[CrossRef][Medline]

Rossi FM, Pizzorusso T, Porciatti V, Marubio LM, Maffei L, and Changeux JP (2001) Requirement of the nicotinic acetylcholine receptor 2 subunit for the anatomical and functional development of the visual system. Proc Natl Acad Sci USA 98: 6453–6458.[Abstract/Free Full Text]

Sernagor E, Eglen SJ, and O'Donovan MJ (2000) Differential effects of acetylcholine and glutamate blockade on the spatiotemporal dynamics of retinal waves. J Neurosci 20: RC56(1–6).[Abstract/Free Full Text]

Vailati S, Hanke W, Bejan A, Barabino B, Longhi R, Balestra B, Moretti M, Clementi F, and Gotti C (1999) Functional 6-containing nicotinic receptors are present in chick retina. Mol Pharmacol 56: 11–19.[Abstract/Free Full Text]

Vailati S, Moretti M, Balestra B, McIntosh M, Clementi F, and Gotti C (2000) 3 subunit is present in different nicotinic receptor subtypes in chick retina. Eur J Pharmacol 393: 23–30.[CrossRef][Medline]

Whiting PJ, Schoepher R, Conroy WG, Gore MJ, Keyser KT, Shimasaki S, Esch F, and Lindstrom JM (1991) Expression of nicotinic acetylcholine receptor subtypes in brain and retina. Brain Res 10: 61–70.

Wong RO (1999) Retinal waves and visual system development. Annu Rev Neurosci 22: 29–47.[CrossRef][Medline]

Wong WT, Myhr K, Miller ED, and Wong RO (2000) Developmental changes in the neurotransmitter regulation of correlated spontaneous retinal activity. J Neurosci 20: 351–360.[Abstract/Free Full Text]

Wong WT, Sanes JR, and Wong RO (1998) Developmentally regulated spontaneous activity in the embryonic chick retina. J Neurosci 18: 8839–8852.[Abstract/Free Full Text]

Wong WT and Wong RO (2001) Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat Neurosci 4: 351–352.[CrossRef][Medline]

Zhou ZJ (2001) The function of the cholinergic system in the developing mammalian retina. Prog Brain Res 131: 599–613.[Medline]

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