Characterization of nicotinic acetylcholine receptor-mediated [3H]-dopamine release from rat cortex and striatum
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).
Section snippets
Methods
Male Sprague Dawley rats (200–400 g) (Harlan, Madison, WI) were housed four per cage, and food and water were available ad libitum. Rats were allowed to acclimate to housing conditions 4 days after arrival. Animals were treated in accordance with the IUPAC guidelines.
Effect of nicotinic acetylcholine receptor (nAChR) agonists
Initial studies compared the ability of four nAChR agonists to evoke [3H]-DA release from rat striatal slices (Fig. 1). All agonists tested evoked a concentration-dependent increase in [3H]-DA release, with a rank order potency of (±)-epibatidine (EB)>cytisine>nicotine>1,1-dimethyl-4-phenyl piperazinium (DMPP). (−)-Cytisine (Fig. 1C) and (±)-EB (Fig. 1B) were 40- and 240-fold more potent than (−)-nicotine respectively, while DMPP (Fig. 1D) was the least potent agonist tested. The maximal
Discussion
The present study introduces a new methodology for the study of [3H]-DA release from a suspension of tissue slices. In place of the laborious commonly used superfusion method, a 96-well format has been developed. Using this assay, a number of compounds, over a wide range of concentrations, can be screened in the same time frame as one superfusion assay.
nAChR-mediated DA release in two regions of rat brain was characterized. Results using various nAChR agonists and antagonists suggest that the
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