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

Abstract

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 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; Bmax = 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, appears 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-diphenylacetoxy-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) and one of low affinity (KD = 57.5 nM)/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(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneous binding of [3H]4‐DAMP to muscarinic cholinergic sites in the rat brain: Evidence from membrane binding and autoradiographic studies

“…[3H]4-DAMP muscarinic receptor autoradiography Coronal sections (20 pm thick) of rat brain were prepared as described previously (Quirion, 1985(Quirion, , 1987Quirion et al, 1989b). Slide-mounted sections of rat brain were stored at -70°C for at least 48 h prior to use.…”

“…Autoradiography of L3H1pirenzepine binding sites in the rat brain was carried out as described previously (Quirion and Boksa, 1986;Quirion et al, 1989b;Regenold et al, 1989). Briefly, slide-mounted sections were first pre-incubated in Krebs buffer (see above) for 15 min at 22°C.…”

“…The MI subtype of muscarinic receptor can be directly and selectively labeled using I3H1pirenzepine as ligand (Araujo et al, 1988(Araujo et al, ,1990Quirion et al, 1989b;Watson et al, 1982). Similarly, selective ligands for M,-muscarinic receptors include L3H1ACh, in the presence of nicotine, (Araujo et al, 1988;Gurwitz et al, 1985;Kellar et al, 1985;Quirion et al, 1989b;Schwartz, 19861, [3Hloxotremorine-M (Spencer et al, 1986;Gillard et al, 1987), [,HI(-)QNB in the presence of pirenzepine (Mash and Potter, 1986) and [,HIAF-DX 116 (Araujo et al, 1989Lapchak et al, 1989a, b;Regenold et al, 1989;Wang et al, 1987a). Using in vitro receptor autoradiographic techniques and selective ligands, a clear distinction between the distribution of M, and M, muscarinic receptors in the mammalian brain has been possible (Cortes et al, 1987;Mash and Potter, 1986;Quirion et al, 1989b;Regenold et al, 1989;Spencer et al, 1986).…”

No involvement of nicotinic receptors in the facilitation of acetylcholine outflow in mouse cortex in the presence of neostigmine and atropine

“…In the present investigation, there was no blocking effect of the facilitation seen after neostigmine and atropine with a variety of nicotinic antagonists used at concentrations capable of, or exceeding those, which block neuronal nicotinic effects in other tissues: mecamylamine ( Alkondon & Albuquerque, 1990 ), α‐bungarotoxin ( Alkondon & Albuquerque, 1995 ), methyllycaconitine ( Khan et al ., 1994 ; Alkondon & Albuquerque, 1995 ), α‐cobratoxin ( Alkondon & Albuquerque, 1990 ), and tubocurarine ( Araujo et al ., 1988 ; Lapchak et al ., 1989 ). Together these data suggest that nicotinic receptors are not involved in the facilitatory effect of neostigmine or acetylcholine seen after muscarinic blockade.…”

“…Their activation depolarizes cell bodies ( Mike, 1994 ) and nerve terminals ( Clarke, 1993 ) which results in transmitter release from a wide variety of neural types. As far as acetylcholine release is concerned, nicotinic agonists induce the release of acetylcholine from guinea‐pig myenteric plexus ( Briggs & Cooper, 1982 ; Mike, 1994 ), guinea‐pig ileum ( Soejima et al ., 1993 ; Dietrich & Kilbinger, 1995 ), guinea‐pig cortex ( Beani et al ., 1985 ), rat hippocampus and cortex ( Araujo et al ., 1988 ), rat cerebellum ( Lapchak et al ., 1989 ) and rat cortical minces ( Meyer et al ., 1987 ). Although less well established, nicotinic agonists facilitate the action‐potential evoked release of acetylcholine from rat hippocampus ( Araujo et al ., 1988 ), guinea‐pig cortex ( Beani et al ., 1985 ), rat cortex ( Loiacono & Mitchelson, 1990 ; Marchi & Raiteri, 1996 ), mouse brain synaptosomes ( Rowell & Winkler, 1984 ) and rat phrenic nerve ( Wessler et al ., 1986 ).…”