Nigrostriatal dopaminergic neurons express many nicotinic acetylcholine receptor (nAChR) subunits capable of forming multiple nAChR subtypes. These subtypes are expressed differentially along the neuron and presumably mediate diverse responses. beta3 subunit mRNA has restricted expression but is abundant in the substantia nigra and ventral tegmental areas. To investigate the potential role(s) of nicotinic receptors containing the beta3 subunit in dopaminergic tracts, we generated mice with a null mutation in the beta3 gene. We were thereby able to identify a population of beta3-dependent alpha-conotoxin MII-binding nAChRs that modulate striatal dopamine release. Changes were also observed in locomotor activity and prepulse inhibition of acoustic startle, behaviors that are controlled, in part, by nigrostriatal and mesolimbic dopaminergic activity, respectively, suggesting that beta3-containing nAChRs modulate these behaviors.

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Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors.

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed ␣ 4 ␤ 2 nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested. Neuronal nicotinic acetylcholine receptors (nAChRs)1 in mammalian brain compose a family of proteins encoded by 11 genes (␣ 2-7 , ␣ 9 -10 , and ␤ 2-4 ) that assemble into pentameric ligandgated ion channels (1-4). Although all combinations of subunits that form functional nicotinic receptors can bind nicotine, ␣ 4 -and ␤ 2 -containing receptors form the high affinity nicotine binding site (5-9) and are therefore thought to be the primary nAChR subtype affected by the relatively low nicotine concentrations (100 -500 nM) found in the blood of smokers (10).Chronic exposure of ␣4␤2 receptors to nicotine, as occurs in smokers, results initially in receptor desensitization, followed by subsequent up-regulation of high affinity nicotine binding sites (11). Upon nicotine withdrawal, the increased number of nAChRs recover from desensitization, resulting in excess activity in the nAChR system. It has been proposed that this cycle of nicotine-induced receptor up-regulation may contribute to the negative symptoms associated with nicotine withdrawal, resulting in continued tobacco consumption and, eventually, nicotine dependence (12).The mechanisms and cellular machinery required for nicotine-induced up-regulation remain unknown. However, a large body of research supports a consensus on several relevant points. First, up-regulation is observed in the brains of human smokers (13) as well as in chronically nicotine-treated animal models (8,14) and in cultured cells heterologously expressing nAChRs (15-18), suggesting that up-regulation requires basic, conserved cellular processes. It has been convincingly shown that increased nicotine binding reflects an increase in receptor number rather than receptor affinity (15, 19 -21), implying that up-regulation involves receptor protein dynamics rather th...

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