Electrophysiological, Pharmacological, and Molecular Evidence for α7-Nicotinic Acetylcholine Receptors in Rat Midbrain Dopamine Neurons

Abstract

Dopamine (DA) neurons located in the mammalian midbrain have been generally implicated in reward and drug reinforcement and more specifically in nicotine dependence. However, roles played by nicotinic acetylcholine receptors, including those composed of ␣7-subunits [␣7-nicotinic acetylcholine receptors (nAChRs)], in modulation of DA signaling and in nicotine dependence are not clearly understood. Although midbrain slice recording has been used previously to identify functional ␣7-nAChRs, these preparations are not optimally designed for extremely rapid and reproducible drug application, and rapidly desensitized, ␣7-nAChRmediated currents may have been underestimated or not detected. Here, we use patch-clamp, whole-cell current recordings from single neurons acutely dissociated from midbrain nuclei and having features of DA neurons to characterize acetylcholineinduced, inward currents that rapidly activate and desensitize, are mimicked by the ␣7-nAChR-selective agonist, choline, blocked by the ␣7-nAChR-selective antagonists, methyllycaconitine and ␣-bungarotoxin, and are similar to those of heterologously expressed, human ␣7-nAChRs. We also use reverse transcriptasepolymerase chain reaction, in situ hybridization, and immunocytochemical staining to demonstrate nAChR ␣7 subunit gene expression as message and protein in the rat substantia nigra pars compacta and ventral tegmental area. Expression of ␣7 subunit message and of ␣7-nAChR-mediated responses is developmentally regulated, with both being absent in samples taken from rats at postnatal day 7, but later becoming present and increasing over the next 2 weeks. Collectively, this electrophysiological, pharmacological, and molecular evidence indicates that nAChR ␣7 subunits and functional ␣7-nAChRs are expressed somatodendritically by midbrain DA neurons, where they may play important physiological roles and contribute to nicotine reinforcement and dependence.

Cannabinoid CB 2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice

“…We first examined the effects of the selective CB 2 R agonist JWH133 on DA neuronal firing using perforated patch-clamp recording in single dissociated VTA DA neurons. Identification of DA neurons was based on three criteria (30,31): electrophysiology [DA neurons exhibit low spontaneous firing rates (FRs; 1-3 Hz) with long action potential (AP) duration and a distinctive H-current], pharmacology [DA neuronal firing is inhibited by DA or by a D 2 receptor agonist (e.g., quinpirole)], and IHC staining (recorded neurons are TH-positive) (Fig. S6).…”

“…Individual group comparisons were carried out using the Student NewmanKeuls method. In addition, paired t tests were used to analyze some electrophysiological data, as described previously (30,31). FRs and burst firing were determined every 10 s, as described previously (57,58).…”

Nicotine Enhances the Excitability of Gaba Neurons in the Ventral Tegmental Area via Activation of Alpha 7 Nicotinic Receptors on Glutamate Terminals

“…Periods of drug exposure are specified in the text and figures or legends. When ACh or choline was used as a nAChR agonist, 1 μM atropine was routinely added to exclude actions mediated via endogenous muscarinic receptors, and this manipulation is known to have no clear effect on ACh-induced currents [33]. All experiments were performed at room temperature.…”

“…Perforated-patch whole-cell recording techniques were employed [33,34]. Compared to performing conventional whole-cell recordings, this approach was crucial for obtaining stable nAChR responses from dissociated VTA neurons, presumably due to minimal perturbation of the intracellular environment.…”

“…Single neurons were acutely dissociated from the VTA of 2-3-weekold mice (from Charles River), following procedures as previously described [33,34]. Briefly, mice were anesthetized with isoflurane, and brain tissue was rapidly removed and immersed in cold (2-4°C) artificial cerebrospinal fluid (ACSF), which contained (in mM): 124 NaCl, 5 KCl, 24 NaHCO 3 , 1.3 MgSO 4 , 1.2 KH 2 PO 4 , 2.4 CaCl 2 , and 10 glucose; pH 7.4.…”