Nicotinic acetylcholine receptors formed from combinations of ␣3, ␤2, ␤4, and ␣5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human ␣3, ␤2, ␤4, and ␣5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of ␣3␤2, ␣3␤4, ␣3␤2␣5, and ␣3␤4␣5 subunits but not for other combinations of one, two, or three of these subunits. ␣5 subunits assembled inside the oocyte with ␤2 but not with ␣3 subunits or other ␣5 subunits. ␣5 subunits coassembled very efficiently with ␣3␤2 or ␣3␤4 combinations. The presence of ␣5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also ␣3 and ␤2 or ␣3 and ␤4 subunits. The presence of ␣5 subunits increased the rate of desensitization of both receptors containing also ␣3 and ␤2 or ␣3 and ␤4 subunits. In the case of receptors containing ␣3 and ␤4 subunits, the addition of ␣5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing ␣3 and ␤2 subunits, the addition of ␣5 subunits reduced the EC 50 for acetylcholine from 28 to 0.5 M and the EC 50 for nicotine from 6.8 to 1.9 M, while increasing the efficacy of nicotine from 50% on ␣3␤2 receptors to 100% on ␣3␤2␣5 receptors. Both ␣3␤2 and ␣3␤2␣5 receptors expressed in oocytes sedimented at the same 11 S value as native ␣3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma ␣3, ␤2, and ␣5 subunits were co-assembled, and 56% of the receptor subtypes containing ␣3 subunits also contained ␤2 subunits. The ␤2 subunitcontaining receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from ␣3 and ␤2 or ␣3, ␤2, and ␣5 subunits rather than the low affinity exhibited by receptors formed from ␣3 and ␤4 or ␣3, ␤4, and ␣5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing ␣3 subunits in SH-SY5Y cells. The affinities of ␣3␤2 receptors expressed in oocytes were similar to the affinities of native ␣3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium. Nicotinic acetylcholine receptors (AChRs)1 are members of a gene superfamily of homologous ligand-gated ion channels which include receptors for glycine, ␥-aminobutyric acid, and serotonin (1). There are three branches of the AChR gene family (2-5). The best characterized are muscle and electric organ AChRs which consist of a pentameric array of homologous subunits oriented around a central ion channel like barrel staves. The order of these subunits around the channel is ␣1␥␣1␦␤1 in the fetal form and ␣1⑀␣1␦␤1 in the adult form (6). The two ligand binding sites in each AChR are thought to be formed at the interfaces between ␣1 and ␥, ␦, or ⑀ subunits (6). One group of neuronal AChRs which is capable of f...

Human nicotinic acetylcholine receptor (AChR) subtypes ␣3␤2, ␣3␤2␣5, ␣3␤4, and ␣3␤4␣5 were stably expressed in cells derived from the human embryonic kidney cell line 293. ␣3␤4 AChRs were found in prominent 2-m patches on the cell surface, whereas most ␣3␤2 AChRs were more diffusely distributed. The functional properties of the ␣3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on ␣3␤4 AChRs and nearly a full agonist on ␣3␤2␣5 AChRs. Chronic exposure of cells expressing ␣3␤2 AChRs or ␣3␤2␣5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of ␣3␤4 or ␣3␤4␣5 AChRs, i.e. the up-regulation of ␣3 AChRs depended on the presence of ␤2 but not ␤4 subunits in the AChRs. This was also found to be true of ␣3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, ␣3␤2 AChRs were expressed at much lower levels than ␣3␤4 AChRs, but in the presence of nicotine, the amount of ␣3␤2 AChRs exceeded that of ␣3␤4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated ␣3␤2 AChRs were functional. The nicotinic antagonists curare and dihydro-␤-erythroidine also up-regulated ␣3␤2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of ␣3␤2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of ␣3␤2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

A recent report described the isolation of cDNA clones encoding alpha 7 and alpha 8 subunits of alpha-bungarotoxin-sensitive nicotinic ACh receptors (alpha BgtAChRs) from chick brain and demonstrated that they were related to, but distinct from, the alpha subunits of nicotinic ACh receptors (nAChRs) from muscles and neurons. Monoclonal antibodies against the two alpha BgtAChR subunits were used to demonstrate that at least two subtypes are present in embryonic day 18 chicken brain. The predominant brain subtype contains alpha 7 subunits, while a minor subtype contains both alpha 7 and alpha 8 subunits. Both subtypes may also contain other subunits. Here we report the results of immune precipitation studies and immunohistochemical studies of alpha BgtAChRs in the chick retina. In addition to the two subtypes found in brain, a new alpha BgtAChR subtype that contains alpha 8 subunits, but not alpha 7 subunits, was identified and was found to be the major subtype in chick retina. This subtype has a lower affinity for alpha-bungarotoxin (alpha Bgt) than does the subtype containing only alpha 7 subunits. Small amounts of this alpha 8 subtype were also detected in brain by labeling with higher concentrations of 125I-alpha Bgt than had been used previously. The subtype containing only alpha 7 subunits comprised 14% of the alpha BgtAChRs in hatchling chick retina. The subtype containing alpha 8 subunits (but no alpha 7 subunits) accounted for 69%, and the alpha 7 alpha 8 subtype accounted for 17%. Amacrine, bipolar, and ganglion cells displayed alpha 8 subunit immunoreactivity, and a complex pattern of labeling was evident in both the inner and outer plexiform layers. In contrast, only amacrine and ganglion cells exhibited alpha 7 subunit immunoreactivity, and the pattern of alpha 7 subunit labeling in the inner plexiform layer differed from that of alpha 8 subunit labeling. These disparities suggest that the alpha BgtAChR subunits are differentially expressed by different populations of retinal neurons. In addition, the distribution of alpha BgtAChR subunit immunoreactivity was found to differ from that of alpha-Bgt-insensitive nAChR subunits.

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Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three beta and eight alpha subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain beta2 subunits, some of which are in combination with alpha3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 microm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 microm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.

The effects of intraocular injections of ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of choline uptake, on the rabbit retina were assessed electrophysiologically, pharmacologically, anatomically, and behaviorally. Survival times from 1 day to 30 days were investigated. After 24 h, the shortest time tested, the directional selectivity of On-Off responding ganglion cells having the characteristic morphology of On-Off directionally selective directionally selective (DS) ganglion cells, as revealed by intracellular dye injection, was significantly reduced, both by an apparent decrease of preferred direction responses and an increase in responses to null-direction movement. No toxin-mediated changes in the dendritic trees of these cells were noted. Cells in AF64A-affected retinas having the DS morphology did not respond significantly to GABAergic or cholinergic agents such as picrotoxin and eserine, but did respond to nicotine. Recordings from a small random sample of other ganglion cell classes in the same retinas yielded no obvious changes in response properties. The direct effects on starburst (cholinergic) amacrine cells, which were identified by intraocular injection of the fluorescent dye DAPI with the AF64A, were investigated by intracellular injections of Lucifer yellow, and by immunohistochemical staining with antibodies to choline acetyltransferase (ChAT). Although starburst amacrine cell somas survived the AF64A treatment for at least several days, the dendrites could not be visualized by fluorescent dye injection in affected retinas due to dye leakage of the injected fluorescent dye from either the soma or proximal dendritic region. ChAT staining revealed a sequence in which ChAT-positive cells were undetectable first in the inner nuclear layer, and then in the ganglion cell layer. Cholinergic amacrine cells in the central retina were also affected before those in the periphery. The electrophysiological changes observed typically preceded the loss of ChAT activity. Behavioral tests for optokinetic nystagmus responses also revealed a lack of such responses in the affected eyes.

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.