Summary Background Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes. Results Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channel rhodopsin elicited physical withdrawal symptoms in both nicotine-naïve and chronic nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blocking IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naïve mice and activated non-somatostatin neurons in the IPN. Conclusions Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Increased anxiety is a predominant withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here, we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signaling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a meso-interpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knock-down of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signaling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

Recently, the smoking cessation therapeutic varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to reduce alcohol consumption. However, the mechanism and nAChR subtype(s) involved are unknown. Here we demonstrate that varenicline and alcohol exposure, either alone or in combination, selectively activates dopaminergic (DAergic) neurons within the posterior, but not the anterior, ventral tegmental area (VTA). To gain insight into which nAChR subtypes may be involved in the response to alcohol, we analyzed nAChR subunit gene expression in posterior VTA DAergic neurons. Ethanol-activated DAergic neurons expressed higher levels of ␣4, ␣6, and ␤3 subunit genes compared with nonactivated neurons. To examine the role of nicotinic receptors containing the ␣4 subunit (␣4* nAChRs) in varenicline-induced reduction of alcohol consumption, we examined the effect of the drug in two complementary mouse models, a knock-out line that does not express the ␣4 subunit (␣4 KO) and another line that expresses ␣4* nAChRs hypersensitive to agonist (Leu9ЈAla). While varenicline (0.1-0.3 mg/kg, i.p.) reduced 2% and 20% alcohol consumption in wild-type (WT) mice, the drug did not significantly reduce consumption in ␣4 KO animals. Conversely, low doses of varenicline (0.0125-0.05 mg/kg, i.p.) that had little effect in WT mice dramatically reduced ethanol intake in Leu9ЈAla mice. Infusion of varenicline into the posterior, but not the anterior VTA was sufficient to reduce alcohol consumption. Together, our data indicate that activation of ␣4* nAChRs is necessary and sufficient for varenicline reduction of alcohol consumption.

Rationale-Recent reports describe a restricted access ethanol consumption paradigm where C57Bl/6J mice drink until intoxicated. Termed "drinking in the dark" (DID), this paradigm has been used as a model of binge drinking. Although neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated in alcohol drinking in rats pre-trained to self-administer ethanol, their role in binge-like ethanol consumption is unknown.Objectives-To determine if nAChRs are involved in binge drinking as measured by the DID assay in C57Bl/6J mice.Methods-Adult male C57Bl/6J mice were injected i.p. with nicotinic receptor antagonists including mecamylamine, hexamethonium, dihydro-β erythroidine, and methyllycaconitine. Immediately following injection mice were presented with 20 % ethanol for 2 hours in the DID assay to measure ethanol consumption. Nicotinic agonists including cytisine and nicotine were also evaluated. The effects of mecamylamine and nicotine on ethanol-induced dopaminergic neuronal activation in the VTA were evaluated via immunohistochemistry.Results-Mecamylamine dose dependently reduced ethanol consumption; whereas the peripheral antagonist hexamethonium had no significant effect. Nicotinic agonists cytisine and nicotine reduced ethanol consumption. None of the effective nicotinic receptor drugs reduced sucrose drinking. Mecamylamine blocked ethanol activation of dopaminergic neurons while nicotine alone activated them without additional activation by ethanol.Conclusions-Neuronal nAChRs are involved in ethanol consumption in the DID paradigm. The effects of mecamylamine, nicotine, and cytisine on ethanol intake appear to be specific because they do not reduce sucrose drinking. Mecamylamine reduces alcohol consumption by blocking activation of dopaminergic neurons; whereas nicotinic agonists may activate the same reward pathway as alcohol.

Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brain's mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity ␣4␤2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by ␣-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the ␣6 subunit. ␣-conotoxin MII[H9A;L15A] also significantly reduced the longlasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either ␣4 or ␣6 nAChR subunits. Conversely, selective activation of nAChRs containing the ␣4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to ␣-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing ␣4 and ␣6 subunits.

Nicotine activation of nicotinic acetylcholine receptors (nAChRs) within the dopaminergic (DAergic) neuron-rich ventral tegmental area (VTA) is necessary and sufficient for nicotine reinforcement. Here we show that rewarding doses of nicotine activated VTA DAergic neurons in a region-selective manner, preferentially activating neurons in the posterior VTA (pVTA) but not the anterior VTA (aVTA) or tail VTA (tVTA). Nicotine (1µM) directly activated pVTA DAergic neurons in adult mouse midbrain slices but had little effect on DAergic neurons within the aVTA. Quantification of nAChR subunit gene expression revealed that pVTA DAergic neurons expressed higher levels of α4, α6, and β3 transcripts, compared to aVTA DAergic neurons. Activation of nAChRs containing the α4 subunit (α4* nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: Nicotine failed to activate pVTA DAergic neurons in α4 knock-out animals; in contrast pVTA α4* nAChRs were selectively activated by nicotine, in mutant mice expressing agonist hypersensitive α4* nAChRs (Leu9’Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by α4* nAChRs and were significantly larger in pVTA neurons compared to aVTA neurons. Infusion of an α6* nAChR antagonist into the VTA blocked activation of pVTA DAergic neurons in WT mice and Leu9’Ala mice at nicotine doses that only activate the mutant receptor indicating that α4 and α6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA via α4α6* nAChRs. These receptors represent novel targets for smoking cessation therapies.

Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here, we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3 IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.

Novelty preference (NP) is an evolutionarily conserved, essential survival mechanism often dysregulated in neuropsychiatric disorders. NP is mediated by a motivational dopamine signal that increases in response to novel stimuli thereby driving exploration. However, the mechanism by which once novel stimuli transitions to familiar stimuli is unknown. Here we describe a neuroanatomical substrate for familiarity signaling, the interpeduncular nucleus (IPN) of the midbrain, which is activated as novel stimuli become familiar with multiple exposures. Optogenetic silencing of IPN neurons increases salience of and interaction with familiar stimuli without affecting novelty responses; whereas, photo-activation of the same neurons reduces exploration of novel stimuli mimicking familiarity. Bi-directional control of NP by the IPN depends on familiarity- and novelty-signals arising from excitatory habenula and dopaminergic ventral tegmental area inputs, which activate and reduce IPN activity, respectively. These results demonstrate that familiarity signals through unique IPN circuitry that opposes novelty seeking to control NP.