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A comparison of the effects of nicotine on dopamine and non-dopamine neurons in the rat ventral tegmental area: an in vitro electrophysiological study

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Abstract

Increased neurotransmission within the mesolimbic dopamine system is considered an essential component for the rewarding and dependence producing properties of nicotine. Nicotinic acetylcholine receptors on dopamine containing neurons in the ventral tegmental area are thought to be a prime target for nicotine’s stimulatory effects. However, there is no evidence regarding the actions of nicotine on ventral tegmental GABAergic interneurons which play an important modulatory role in mesolimbic dopamine neuronal excitability. In the present study we sought to characterize the effects of nicotine on the activity of both dopamine and non-dopamine neurons in the juvenile rat ventral tegmentum. Extracellular recording techniques in rat brain slices and two methods of drug perfusion were used. Nicotine was found to markedly increase the firing rate of both groups, although the dopamine neuronal response pattern was considerably different and more vigorous than that in the non-dopamine neurons. The nicotine-induced excitations were also reversed by mecamylamine. Furthermore, desensitization to nicotine’s stimulatory effects occurred in both neuronal populations, although non-dopamine neurons appeared to desensitize to a greater degree. In fact, the desensitization accompanying sequential uninterrupted applications of nicotine appears to occur at concentrations below that described to produce receptor activation. The low nM concentrations of nicotine used in the present study are comparable to plasma levels of nicotine found after smoking a cigarette or even with passive inhalation of tobacco smoke. Thus, the present results not only confirm that nicotine stimulates the firing rate of ventral tegmental area dopamine neurons, but also that GABAergic neurons may be an important target for nicotine’s central nervous system effects. The less robust response in the non-dopamine presumptive GABAergic population and their more pronounced desensitization could lead to disinhibition of dopamine neurons thereby facilitating a more sustained increase in the response of mesolimbic dopamine neurons to nicotine.

Introduction

Tobacco-smoking has been recognized as the leading cause of morbidity and mortality in the United States [45]. Psychopharmacological studies further demonstrated that nicotine could serve as a primary positive reinforcer and that nicotine addiction is a crucial component in chronic tobacco use [43]. Although nicotine produces diverse actions in the central nervous system (CNS) 1, 47, substantial evidence links nicotine-induced activation of nicotinic acetylcholine receptors (nAChR) in the brain with the behavioral reward and arousal processes that form the neurobiological underpinnings for tobacco dependence [35]. However, the precise mechanism(s) by which nicotine elicits these effects remains to be determined.

Mesolimbic dopamine pathways have been implicated in the reinforcing properties of the major drugs of abuse [23]. The neurons comprising this system are located in the midbrain ventral tegmental area (VTA), and they innervate the limbic-related structures in forebrain such as the nucleus accumbens, prefrontal cortex, tuberculum olfactorium, and nucleus interstitial striae terminalis pars dorsalis. The VTA also receives afferent input from many transmitter systems, but contains two principal cell types: tyrosine-hydroxylase containing neurons whose axon terminals release dopamine in mesolimbic structures, and non-tyrosine hydroxlase containing neurons, the majority of which are γ-aminobutyric acid (GABA)ergic and function as local interneurons to control the activity of the principal dopamine neurons 19, 20, 30. Recent work has confirmed that the VTA also contains a subset of GABA neurons which also send axon projections to cortical areas 42, 46.

In addition to other drugs of abuse, midbrain VTA dopamine neurons have been implicated in mediating nicotine’s reinforcing effects [28]. The depletion of mesolimbic dopamine by 6-hydroxydopamine injections into the rat VTA was found to markedly reduce nicotine self-administration. The microinfusion of the nicotinic receptor antagonist dihydro-β-erythroidine (DH-β-E) into the VTA, but not into the nucleus accumbens, decreased the self-administration of nicotine, whereas the muscarinic antagonist atropine had no effect [10]. Thus, it has been suggested that nicotine acts primarily on nAChRs within the VTA to initiate processes which are essential for the behaviorally activating and reinforcing properties of the drug [28]. Moreover, nicotine can produce these effects at blood concentrations within the range found in cigarette smokers 9, 10, 17, 33.

Autoradiographic mapping techniques have revealed that the potential targets for nicotine in the mesolimbic system include nAChRs located on cell bodies in VTA and terminals in the nucleus accumbens [8]. Findings from functional studies in in vitro preparations are also consistent with this proposal, as nicotine has been demonstrated to alter dopamine uptake [18], its release 32, 37 and metabolism [44]. However, the locomotor stimulant effect and the self-administration of nicotine are accompanied by, and appear to be dependent upon, activation of mesolimbic dopamine neurons 7, 14, 27. Of the few electrophysiological studies that have assessed the effects of nicotine on midbrain dopamine neurons, nicotine was found to increase cell firing rates, an effect which desensitized after continual or repeated exposure to the drug 5, 16, 31. However, there is no evidence available as to the actions of nicotine on non-dopamine neurons in the VTA, or whether these neurons are also susceptible to desensitization.

Therefore, the aim of the present study was to compare the response of both VTA dopamine and non-dopamine neurons over a range of low to high concentrations of nicotine, and to determine whether these two neuronal populations exhibit a differential degree of desensitization.

Section snippets

Reagents

(-)-Nicotine hydrogen tartrate (nicotine) salt was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Mecamylamine hydrochloride was purchased from Research Biochemicals, Inc. (Natick, MA, USA).

Preparation of the midbrain slices

Male Sprague–Dawley rats (110–120 g) were deeply anaesthetized with ether and decapitated. The brain was quickly removed and immediately immersed in ice-cold (2–6°C) artificial cerebrospinal fluid (ACSF). The ACSF contained (in mM) NaCl, 124; KCl, 2.5; KH2PO4, 1.25; MgSO4 · 7H2O, 2.4, CaCl2 · 2H2, O

Effects of continuous nicotine perfusion on dopamine and non-dopamine neurons

In the first set of experiments, the continuous application of escalating concentrations of nicotine (0.02–12.5 μM) produced a concentration dependent increase in the firing rate of dopamine neurons (n = 8) (Fig. 1a). An examination of the dose-response curve indicates that nicotine’s stimulatory effects become evident at approximately 0.1 μM, reaching a maximum of +114 ± 23% at 12.5 μM (n = 8) (Fig. 1c). The graphic method of calculation yielded an EC50 of 0.25 μM. The nicotine-induced

Discussion

The present results show that in a rat midbrain slice preparation VTA neurons, identified as dopamine or non-dopamine by electrophysiological characteristics, are excited upon exposure to nicotine. Furthermore, this effect occurred over a range of concentrations that have been shown to support nicotine self-administration in animals 9, 10, 33. Also, these concentrations are likely to be achieved in smokers where brain levels of nicotine have been found to be several fold higher than blood

Acknowledgements

This work was supported by the Arizona Disease Control Research Commission.

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