Characterization of nicotinic acetylcholine receptor-mediated [³H]-dopamine release from rat cortex and striatum

Abstract

The objective of this study was to use a new high throughput method to compare nicotinic acetylcholine receptor (nAChR)-mediated [³H]-dopamine (DA) release from slices of rat striatum and cortex. (−)Nicotine, (−)-cytisine, 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and (±)-epibatidine evoked release of striatal [³H]-DA with pEC₅₀ values of 6.7, 8.25, 5.11, and 9.08, respectively. The same agonists evoked release of cortical [³H]-DA with pEC₅₀ values of 6.98, 8.06, 5.58, and 9.59. Relative to (−)-nicotine, (−)-cytisine was a partial agonist in both tissues. In contrast, the maximal response evoked by DMPP differed between the two tissues. The rank order of potency for antagonists to block DA release was the same (mecamylamine (Mec)>dihydro-β-erythroidine (DHβE)>hexamethonium (Hex)>d-tubocurarine (d-TC)); however, the pIC₅₀ values varied between the two regions. Whereas Mec potently antagonized (−)-nicotine-evoked DA release similarly from striatum and cortex, with pIC₅₀ values of 6.07 and 6.53 respectively, the values obtained for DHβE, d-TC and Hex differed. Additionally, the present study was able to distinguish exocytotic vesicular-mediated from transporter-mediated DA release, by altering temperature of the incubation and exclusion of calcium. Assays carried out under these conditions indicate that approximately 60% of nicotine-evoked cortical DA release was likely mediated through the DA transporter. In contrast, under the same conditions only 15%–20% of striatal release appeared to be transporter-mediated. We conclude that the relative contributions of the mechanisms by which (−)-nicotine evokes DA release differ between striatum and cortex. In addition, the data suggest that the subtypes of nAChRs involved in regulating [³H]-DA release may be somewhat different in the two tissues.

Introduction

Previous studies have reported the ability of (−)-nicotine, as well as other nicotinic acetylcholine receptor (nAChR) agonists, to evoke release of several neurotransmitters, including acetylcholine, norepinephrine (NE), serotonin, and dopamine (DA), from various brain regions (MacDermott et al., 1999). Functional nAChRs, existing as hetero-oligomers comprising an α (α2–α6) with a β (β2–β4) subunit, or homo-oligomers comprising α7, α8, or α9 subunits, have been identified throughout the central nervous system (for reviews, see Lindstrom et al., 1996, Boyd, 1997). Since various subunit combinations can form functional receptors with varied physiological and pharmacological profiles, the potential exists for extensive diversity in the function of nAChRs to regulate neurotransmitter release. Indeed, previous studies have demonstrated that the nAChR subtypes which control [³H]-DA release from striatum differ from those that mediate [³H]-norepinephrine release from the hippocampus (Clarke and Reuben, 1996, Sacaan et al., 1995). However to our knowledge, no single study has characterized nAChR-mediated release of the same neurotransmitter in two different brain regions using a tissue slice preparation. Whiteaker and coworkers have examined (−)-nicotine-evoked DA release from striatal and frontal cortex synaptosomes (Whiteaker et al., 1995).

The present study characterized nAChR-mediated [³H]-DA release from striatum and cortex, using a novel method. Results obtained using striatum were compared with previously reported results using different methodology. To our knowledge, aside from one study using rat synaptosomes (Whiteaker et al., 1995), very little work has been published characterizing nAChR-mediated [³H]-DA release from slices of cortex. The ability of several nAChR agonists and antagonists was utilized to evaluate which nAChR subtypes mediate DA release in cortex and striatum. Additional studies were performed to characterize the mechanisms of (−)-nicotine-evoked DA release in striatum and cortex. The regulation of synaptic DA levels has multiple components, including control of vesicular release and alterations of DA transporter activity. There are two primary mechanisms for neurotransmitter release: exocytotic release of vesicular contents and release via the transporter. We evaluated (−)-nicotine-evoked striatal and cortical [³H]-DA release under assay conditions which discriminate between these two mechanisms (Lendvai et al., 1996, Vizi, 1998).