Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

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Abstract
The present study shows that N-[3H]meth-ylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD= 5.0 nM; BmdK= 8.2 fmol/ mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD= 4.8 nM BmaK= 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]-pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116. ap-pears to bind to two classes of binding sites, i.e., a high-affinity (KD= 3 nM)/low-capacity (BMax= 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD= 28.4 nM) and higher capacity (Bmax= 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenyIacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD= 0.5 nM)/low capacity (Bmax= 19.5 fmol/mg of protein) nd one of low affinity (KD= 57.5 nA/)/high capacity (Bmax 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10−7M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and te-trodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-β-erythroidine, d-tubocurarine, and k-bungarotoxin, but was insensitive to either atropine or α-bungarotoxin. Tests of the effects of muscarinic drugs, such as oxotremorine (nonselective agonist), atropine (nonselective antagonist), and AF-DX 116 (M2 selective antagonist), on spontaneous and evoked acetylcholine release revealed the absence of muscarinic autoreceptor-mediated regulation of acetylcholine release from cerebellar brain slices. These results support the hypothesis that in the rat cerebellum some nicotinic sites, as labeled by [3H]MCC. are localized to cholinergic terminals where they mediate positive feedback control of acetylcholine release.