No abstract

Nicotinic agonists elicit the release of dopamine from striatal synaptosomes by acting on presynaptic nicotinic acetylcholine receptors (nAChRs) on dopamine nerve terminals. Both alpha3beta2* and alpha4beta2 nAChR subtypes (but not alpha7* nAChRs) have been implicated. Here, we compared nAChR-evoked [(3)H]dopamine release from rat striatal synaptosome and slice preparations by using the nicotinic agonist anatoxin-a. In the more integral slice preparation, the concentration-response curve for anatoxin-a-evoked [(3)H]dopamine release was best fitted to a two-site model, giving EC(50) values of 241 nM and 5.1 microM, whereas only the higher-affinity component was observed in synaptosome preparations (EC(50) = 134 nM). Responses to a high concentration of anatoxin-a (25 microM) in slices (but not in synaptosomes) were partially blocked by ionotropic glutamate receptor antagonists (kynurenic acid, 6,7-dinitroquinoxaline-2,3-dione) and by alpha7*-selective nAChR antagonists (alpha-bungarotoxin, alpha-conotoxin-ImI, methyllycaconitine) in a nonadditive manner. In contrast, the alpha3beta2-selective nAChR antagonist alpha-conotoxin-MII partially inhibited [(3)H]dopamine release from both slice and synaptosome preparations, stimulated with both low (1 microM) and high (25 microM) concentrations of anatoxin-a. Antagonism by alpha-conotoxin-MII was additive with that of alpha7*-selective antagonists. These data support a model in which alpha7* nAChRs on striatal glutamate terminals elicit glutamate release, which in turn acts at ionotropic glutamate receptors on dopamine terminals to stimulate dopamine release. In addition, non-alpha7* nAChRs on dopamine terminals also stimulate dopamine release. These observations have implications for the complex cholinergic modulation of inputs onto the major efferent neurons of the striatum.

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α‐conotoxin‐MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)‐anatoxin‐a‐evoked release of [3H]dopamine from rat striatal synaptosomes and slices. α‐Conotoxin‐MII (112 nM) completely blocked acetylcholine‐evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα‐conotoxin‐MII. On perfused striatal synaptosomes and slices, α‐conotoxin‐MII dose‐dependently inhibited [3H]dopamine release evoked by 1 µM (±)‐anatoxin‐a with IC50 values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose‐response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α‐conotoxin‐MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2‐like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)‐anatoxin‐a‐evoked [3H]dopamine release by α‐conotoxin‐MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α‐conotoxin‐MII‐insensitive nAChR.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

Postsynaptic development at the neuromuscular junction depends on nicotinic transmission and secreted components from the presynaptic motor nerve terminal. Similarly, secreted components and synaptic activity are both thought to guide development of glutamatergic synapses in the CNS. Nicotinic synapses on chick ciliary neurons are structurally complex: a large presynaptic calyx engulfs the postsynaptic neuron and overlays a series of discrete mats of receptor-rich somatic spines tightly interwoven and folded against the soma. We used fluorescence imaging of alpha 7-containing nicotinic receptors and the spine constituent drebrin to monitor postsynaptic development. The results show that surgical disruption of the preganglionic input or removal of the ganglionic synaptic target tissue after synapses form in the ganglion does not disrupt the receptor-rich spine mats. Similarly, removal of the target tissue even prior to synapse formation in the ganglion does not prevent subsequent formation of the receptor clusters and associated spine constituents. Postsynaptic development is arrested, however, if normal innervation is prevented by ablating the preganglionic neurons prior to synapse formation. In this case the neurons express reduced levels of nicotinic receptors and cytoskeletal components and organize them only into early-stage clusters. Even low levels of residual innervation, however, can restore much of the normal postsynaptic receptor patterns. Chronic pharmacological blockade of cholinergic synaptic activity fails to replicate the effects of ablating the preganglionic nucleus. The results indicate that ciliary neurons are programmed to express postsynaptic components and can initiate clustering of alpha 7-containing receptors but need presynaptic guidance for maturation of the postsynaptic structure.