Genetic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco addiction and lung cancer, but underlying mechanisms are unknown. Here, we report dramatically increased nicotine consumption in mice with null mutation in Chrna5. This effect was `rescued' in knockout mice by re-expressing α5 subunits in medial habenula (MHb), and recapitulated in rats through α5 subunit knockdown in MHb. Remarkably, α5 subunit knockdown in MHb did not alter the rewarding effects of nicotine but abolished the inhibitory effects of higher nicotine doses on brain reward systems. The MHb extends projections almost exclusively to the interpeduncular nucleus (IPN). We found diminished IPN activation in response to nicotine in α5 knockout mice and disruption of IPN signaling increased nicotine intake in rats. Our findings suggest that nicotine activates the habenulo-interpeduncular pathway through α5-containing nAChRs, triggering an inhibitory motivational signal that acts to limit nicotine intake.

The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

Pharmacological evaluation of nicotine-stimulated dopamine release from striatum has yielded data consistent with activation of a single population of nicotinic acetylcholine receptors (nAChR). However, discovery that ␣-conotoxin MII (␣-CtxMII) partially inhibits the response indicates that two classes of presynaptic nAChRs mediate dopamine release. We have investigated the pharmacology and subunit composition of these two classes of nAChR. Inhibition of nicotine-stimulated dopamine release from mouse striatal synaptosomes by ␣-CtxMII occurs within minutes; recovery is slow. The IC 50 is 1 to 3 nM. ␣-CtxMII-sensitive and -resistant components have significant differences in pharmacology. The five agonists tested were more potent at activating the ␣-CtxMII-sensitive nAChRs; indeed, this receptor is the highest affinity functional nAChR found, so far, in mouse brain. In addition, cytisine was more efficacious at the ␣-CtxMII-sensitive sites. Methyllycaconitine was 9-fold more potent at inhibiting the ␣-CtxMII-sensitive sites, whereas dihydro-␤-erythroidine was a 7-fold more potent inhibitor of the ␣-CtxMII-resistant response. Both the transient and persistent phases of nicotine-stimulated dopamine release were partially inhibited by ␣-CtxMII with equal potency. The subunit composition of functional nAChRs, was assessed in mice with null mutations for individual nAChR subunits. The ␤2 subunit is an absolute requirement for both classes. In contrast, deletion of ␤4 or ␣7 subunits had no effect. The ␣-CtxMIIsensitive response requires ␤3 and is partially dependent upon ␣4 subunits, probably ␣6␤3␤2 and ␣4␣6␤3␤2, whereas the ␣-CtxMII-resistant release requires ␣4 and is partially dependent upon ␣5 subunits, probably ␣4␤2 and ␣4␣5␤2.Nicotinic cholinergic receptors (nAChRs) are expressed both peripherally and centrally. Many, if not most, brain nAChRs are located presynaptically, where they serve to modulate neurotransmitter release (Wonnacott, 1997;Nishi et al., 2000). Activation of presynaptic nAChRs may elicit neurotransmitter release in the absence of a propagated signal (Vizi and Lendvai, 1999), and may increase action-potential induced transmitter release and, consequently, enhance synaptic fidelity (McGehee et al., 1995;Gray et al., 1996).Neuronal nAChR subunits form pentameric complexes, closely resembling the nAChR found at the neuromuscular junction (Leonard and Bertrand, 2001). Some of these receptors are homomeric (␣7), but most are heteromeric. Subunit composition of heteromeric nAChRs expressed in Xenopus laevis oocytes or transfected cell lines has profound effects on agonist potency and efficacy as well as sensitivity to antagonists (Luetje and Patrick, 1991;Harvey and Luetje, 1996;

We apply the Bayesian framework to assess the presence of a correlation between two quantities. To do so, we estimate the probability distribution of the parameter of interest, ρ, characterizing the strength of the correlation. We provide an implementation of these ideas and concepts using python programming language and the pyMC module in a very short (∼ 130 lines of code, heavily commented) and user-friendly program.We used this tool to assess the presence and properties of the correlation between planetary surface gravity and stellar host activity level as measured by the log(R HK ) indicator. The results of the Bayesian analysis are qualitatively similar to those obtained via p-value analysis, and support the presence of a correlation in the data. The results are more robust in their derivation and more informative, revealing interesting features such as asymmetric posterior distributions or markedly different credible intervals, and allowing for a deeper exploration.We encourage the reader interested in this kind of problem to apply our code to his/her own scientific problems. The full understanding of what the Bayesian framework is can only be gained through the insight that comes by handling priors, assessing the convergence of Monte Carlo runs, and a multitude of other practical problems. We hope to contribute so that Bayesian analysis becomes a tool in the toolkit of researchers, and they understand by experience its advantages and limitations.

Summary α6-containing (α6*) nicotinic ACh receptors (nAChRs) are selectively expressed in dopamine (DA) neurons and participate in cholinergic transmission. We generated and studied mice with gain-of-function α6* nAChRs, which isolate and amplify cholinergic control of DA transmission. In contrast to gene knockouts or pharmacological blockers, which show necessity, we show that activating α6* nAChRs and DA neurons is sufficient to cause locomotor hyperactivity. α6L9’S mice are hyperactive in their home cage and fail to habituate to a novel environment. Selective activation of α6* nAChRs with low doses of nicotine, by stimulating DA but not GABA neurons, exaggerates these phenotypes and produces a hyperdopaminergic state in vivo. Experiments with additional nicotinic drugs show that altering agonist efficacy at α6* provides fine-tuning of DA release and locomotor responses. α6*-specific agonists or antagonists may, by targeting endogenous cholinergic mechanisms, provide a new method for manipulating DA transmission in Parkinson’s disease, nicotine dependence, or attention deficit hyperactivity disorder.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose ␣4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased ␣4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change ␣4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional ␣4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases ␣4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated ␣4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional ␣4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo-interpeduncular (Hb-IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (ϩ/ϩ) and ␤2 knock-out (Ϫ/Ϫ) mice to establish that the Hb and IPn contain significant ␤2* and ␤4* populations of nAChR receptors (each of which is heterogeneous). The ␤4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (␣2␤2*, ␣4␤3␤2*, ␣3␤3␤4*, ␣6␤3␤4*). Our studies on IPn synaptosomes obtained from ϩ/ϩ and ␣2, ␣4, ␣5, ␣6, ␣7, ␤2, ␤3, and ␤4 Ϫ/Ϫ mice show that only the ␣3␤4 and ␣3␤3␤4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in ␤3 Ϫ/Ϫ mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and ␣3␤4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the ␤3 subunit may be important for transporting the ␣3␤4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb-IPn system and identify ␣3␤4 and ␣3␤3␤4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.