Dopamine control of social novelty preference is constrained by an interpeduncular-tegmentum circuit.

Animals are inherently motivated to explore social novelty cues over familiar ones, resulting in a novelty preference (NP), although the behavioral and circuit bases underlying NP are unclear. Combining calcium and neurotransmitter sensors with fiber photometry and optogenetics in mice, we find that mesolimbic dopamine (DA) neurotransmission is strongly and predominantly activated by social novelty controlling bout length of interaction during NP, a response significantly reduced by familiarity. In contrast, interpeduncular nucleus (IPN) GABAergic neurons that project to the lateral dorsal tegmentum (LDTg) were inhibited by social novelty but activated during terminations with familiar social stimuli. Inhibition of this pathway during NP increased interaction and bout length with familiar social stimuli, while activation reduced interaction and bout length with novel social stimuli via decreasing DA neurotransmission. These data indicate interest towards novel social stimuli is encoded by mesolimbic DA which is dynamically regulated by an IPN→LDTg circuit to control NP.

A neuronal coping mechanism linking stress-induced anxiety to motivation for reward.

Stress coping involves innate and active motivational behaviors that reduce anxiety under stressful situations. However, the neuronal bases directly linking stress, anxiety, and motivation are largely unknown. Here, we show that acute stressors activate mouse GABAergic neurons in the interpeduncular nucleus (IPN). Stress-coping behavior including self-grooming and reward behavior including sucrose consumption inherently reduced IPN GABAergic neuron activity. Optogenetic silencing of IPN GABAergic neuron activation during acute stress episodes mimicked coping strategies and alleviated anxiety-like behavior. In a mouse model of stress-enhanced motivation for sucrose seeking, photoinhibition of IPN GABAergic neurons reduced stress-induced motivation for sucrose, whereas photoactivation of IPN GABAergic neurons or excitatory inputs from medial habenula potentiated sucrose seeking. Single-cell sequencing, fiber photometry, and optogenetic experiments revealed that stress-activated IPN GABAergic neurons that drive motivated sucrose seeking express somatostatin. Together, these data suggest that stress induces innate behaviors and motivates reward seeking to oppose IPN neuronal activation as an anxiolytic stress-coping mechanism.

Viral Tracing Confirms Paranigral Ventral Tegmental Area Dopaminergic Inputs to the Interpeduncular Nucleus Where Dopamine Release Encodes Motivated Exploration.

Midbrain dopaminergic (DAergic) neurons of the ventral tegmental area (VTA) are engaged by rewarding stimuli and encode reward prediction error to update goal-directed learning. However, recent data indicate that VTA DAergic neurons are functionally heterogeneous with emerging roles in aversive signaling, salience, and novelty, based in part on anatomic location and projection, highlighting a need to functionally characterize the repertoire of VTA DAergic efferents in motivated behavior. Previous work identifying a mesointerpeduncular circuit consisting of VTA DAergic neurons projecting to the interpeduncular nucleus (IPN), a midbrain area implicated in aversion, anxiety-like behavior, and familiarity, has recently come into question. To verify the existence of this circuit, we combined presynaptic targeted and retrograde viral tracing in the dopamine transporter-Cre mouse line. Consistent with previous reports, synaptic tracing revealed that axon terminals from the VTA innervate the caudal IPN; whereas, retrograde tracing revealed DAergic VTA neurons, predominantly in the paranigral region, project to the nucleus accumbens shell, as well as the IPN. To test whether functional DAergic neurotransmission exists in the IPN, we expressed the genetically encoded DA sensor, dLight 1.2, in the IPN of C57BL/6J mice and measured IPN DA signals in vivo during social and anxiety-like behavior using fiber photometry. We observed an increase in IPN DA signal during social investigation of a novel but not familiar conspecific and during exploration of the anxiogenic open arms of the elevated plus maze. Together, these data confirm VTA DAergic neuron projections to the IPN and implicate this circuit in encoding motivated exploration.

Dynamic activity of interpeduncular nucleus GABAergic neurons controls expression of nicotine withdrawal in male mice.

A critical brain area implicated in nicotine dependence is the interpeduncular nucleus (IPN) located in the ventral midbrain and consisting primarily of GABAergic neurons. Previous studies indicate that IPN GABAergic neurons contribute to expression of somatic symptoms of nicotine withdrawal; however, whether IPN neurons are dynamically regulated during withdrawal in vivo and how this may contribute to both somatic and affective withdrawal behavior is unknown. To bridge this gap in knowledge, we expressed GCaMP in IPN GABAergic neurons and used in vivo fiber photometry to record changes in fluorescence, as a proxy for neuronal activity, in male mice during nicotine withdrawal. Mecamylamine-precipitated withdrawal significantly increased activity of IPN GABAergic neurons in nicotine-dependent, but not nicotine-naive mice. Analysis of GCaMP signals time-locked with somatic symptoms including grooming and scratching revealed reduced IPN GABAergic activity during these behaviors, specifically in mice undergoing withdrawal. In the elevated plus maze, used to measure anxiety-like behavior, an affective withdrawal symptom, IPN GABAergic neuron activity was increased during open-arm versus closed-arm exploration in nicotine-withdrawn, but not non-withdrawn mice. Optogenetic silencing IPN GABAergic neurons during withdrawal significantly reduced withdrawal-induced increases in somatic behavior and increased open-arm exploration. Together, our data indicate that IPN GABAergic neurons are dynamically regulated during nicotine withdrawal, leading to increased anxiety-like symptoms and somatic behavior, which inherently decrease IPN GABAergic neuron activity as a withdrawal-coping mechanism. These results provide a neuronal basis underlying the role of the IPN in the expression of somatic and affective behaviors of nicotine withdrawal.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons.

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.

Midbrain Dopamine Controls Anxiety-like Behavior by Engaging Unique Interpeduncular Nucleus Microcircuitry.

BACKGROUND: Dopamine (DA) is hypothesized to modulate anxiety-like behavior, although the precise role of DA in anxiety behaviors and the complete anxiety network in the brain have yet to be elucidated. Recent data indicate that dopaminergic projections from the ventral tegmental area (VTA) innervate the interpeduncular nucleus (IPN), but how the IPN responds to DA and what role this circuit plays in anxiety-like behavior are unknown. METHODS: We expressed a genetically encoded G protein-coupled receptor activation-based DA sensor in mouse midbrain to detect DA in IPN slices using fluorescence imaging combined with pharmacology. Next, we selectively inhibited or activated VTA→IPN DAergic inputs via optogenetics during anxiety-like behavior. We used a biophysical approach to characterize DA effects on neural IPN circuits. Site-directed pharmacology was used to test if DA receptors in the IPN can regulate anxiety-like behavior. RESULTS: DA was detected in mouse IPN slices. Silencing/activating VTA→IPN DAergic inputs oppositely modulated anxiety-like behavior. Two neuronal populations in the ventral IPN (vIPN) responded to DA via D1 receptors (D1Rs). vIPN neurons were controlled by a small population of D1R neurons in the caudal IPN that directly respond to VTA DAergic terminal stimulation and innervate the vIPN. IPN infusion of a D1R agonist and antagonist bidirectionally controlled anxiety-like behavior. CONCLUSIONS: VTA DA engages D1R-expressing neurons in the caudal IPN that innervate vIPN, thereby amplifying the VTA DA signal to modulate anxiety-like behavior. These data identify a DAergic circuit that mediates anxiety-like behavior through unique IPN microcircuitry.

Integrated miRNA-/mRNA-Seq of the Habenulo-Interpeduncular Circuit During Acute Nicotine Withdrawal.

Tobacco use is the leading preventable cause of mortality in the world. The limited number of smoking cessation aids currently available are minimally effective, highlighting the need for novel therapeutic interventions. We describe a genome-wide approach to identify potential candidates for such interventions. Next-generation sequencing was performed using RNA isolated from the habenulo-interpeduncular circuit of male mice withdrawn from chronic nicotine treatment. This circuit plays a central role in the nicotine withdrawal response. Differentially expressed miRNAs and mRNAs were validated using RT-qPCR. Many of the differentially expressed mRNAs are predicted targets of reciprocally expressed miRNAs. We illustrate the utility of the dataset by demonstrating that knockdown in the interpeduncular nucleus of a differentially expressed mRNA, that encoding profilin 2, is sufficient to induce anxiety-related behavior. Importantly, profilin 2 knockdown in the ventral tegmental area did not affect anxiety behavior. Our data reveal wide-spread changes in gene expression within the habenulo-interpeduncular circuit during nicotine withdrawal. This dataset should prove to be a valuable resource leading to the identification of substrates for the design of innovative smoking cessation aids.

Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function.

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 Pitx3IRES2-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.

A circuit-based mechanism underlying familiarity signaling and the preference for novelty.

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 transition 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. In mice, optogenetic silencing of IPN neurons increases salience of and interaction with familiar stimuli without affecting novelty responses, whereas photoactivation of the same neurons reduces exploration of novel stimuli mimicking familiarity. Bidirectional control of NP by the IPN depends on familiarity signals and novelty signals arising from excitatory habenula and dopaminergic ventral tegmentum 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.

Paternal nicotine exposure alters hepatic xenobiotic metabolism in offspring.

Paternal environmental conditions can influence phenotypes in future generations, but it is unclear whether offspring phenotypes represent specific responses to particular aspects of the paternal exposure history, or a generic response to paternal 'quality of life'. Here, we establish a paternal effect model based on nicotine exposure in mice, enabling pharmacological interrogation of the specificity of the offspring response. Paternal exposure to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring. This effect manifested as increased survival following injection of toxic levels of either nicotine or cocaine, accompanied by hepatic upregulation of xenobiotic processing genes, and enhanced drug clearance. Surprisingly, this protective effect could also be induced by a nicotinic receptor antagonist, suggesting that xenobiotic exposure, rather than nicotinic receptor signaling, is responsible for programming offspring drug resistance. Thus, paternal drug exposure induces a protective phenotype in offspring by enhancing metabolic tolerance to xenobiotics.

Anxiety and Nicotine Dependence: Emerging Role of the Habenulo-Interpeduncular Axis.

While innovative modern neuroscience approaches have aided in discerning brain circuitry underlying negative emotional behaviors including fear and anxiety responses, how these circuits are recruited in normal and pathological conditions remains poorly understood. Recently, genetic tools that selectively manipulate single neuronal populations have uncovered an understudied circuit, the medial habenula (mHb)-interpeduncular (IPN) axis, that modulates basal negative emotional responses. Interestingly, the mHb-IPN pathway also represents an essential circuit that signals heightened anxiety induced by nicotine withdrawal. Insights into how this circuit interconnects with regions more classically associated with anxiety, and how chronic nicotine exposure induces neuroadaptations resulting in an anxiogenic state, may thereby provide novel strategies and molecular targets for therapies that facilitate smoking cessation, as well as for anxiety relief.

Habenula cholinergic neurons regulate anxiety during nicotine withdrawal via nicotinic acetylcholine receptors.

Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4ß2 or α6ß2, but not α3ß4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics.

Functional Upregulation of α4* Nicotinic Acetylcholine Receptors in VTA GABAergic Neurons Increases Sensitivity to Nicotine Reward.

Chronic nicotine exposure increases sensitivity to nicotine reward during a withdrawal period, which may facilitate relapse in abstinent smokers, yet the molecular neuroadaptation(s) that contribute to this phenomenon are unknown. Interestingly, chronic nicotine use induces functional upregulation of nicotinic acetylcholine receptors (nAChRs) in the mesocorticolimbic reward pathway potentially linking upregulation to increased drug sensitivity. In the ventral tegmental area (VTA), functional upregulation of nAChRs containing the α4 subunit (α4* nAChRs) is restricted to GABAergic neurons. To test the hypothesis that increased functional expression of α4* nAChRs in these neurons modulates nicotine reward behaviors, we engineered a Cre recombinase-dependent gene expression system to selectively express α4 nAChR subunits harboring a "gain-of-function" mutation [a leucine mutated to a serine residue at the 9' position (Leu9'Ser)] in VTA GABAergic neurons of adult mice. In mice expressing Leu9'Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2(VTA):Leu9'Ser mice), subreward threshold doses of nicotine were sufficient to selectively activate VTA GABAergic neurons and elicit acute hypolocomotion, with subsequent nicotine exposures eliciting tolerance to this effect, compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2(VTA):Leu9'Ser mice at low nicotine doses that failed to condition control animals. Together, these data indicate that functional upregulation of α4* nAChRs in VTA GABAergic neurons confers increased sensitivity to nicotine reward and points to nAChR subtypes specifically expressed in GABAergic VTA neurons as molecular targets for smoking cessation therapeutics.

Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety during nicotine withdrawal.

Increased anxiety is a prominent 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 signalling, 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 mesointerpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knockdown of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signalling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

miRNAome analysis of the mammalian neuronal nicotinic acetylcholine receptor gene family.

Nicotine binds to and activates a family of ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine exposure alters the expression of various nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that microRNAs (miRNAs) may be involved in nAChR regulation. Using bioinformatics, miRNA library screening, site-directed mutagenesis, and gene expression analysis, we have identified a limited number of miRNAs that functionally interact with the 3'-untranslated regions (3' UTRs) of mammalian neuronal nAChR subunit genes. In silico analyses revealed specific, evolutionarily conserved sites within the 3' UTRs through which the miRNAs regulate gene expression. Mutating these sites disrupted miRNA regulation confirming the in silico predictions. In addition, the miRNAs that target nAChR 3' UTRs are expressed in mouse brain and are regulated by chronic nicotine exposure. Furthermore, we show that expression of one of these miRNAs, miR-542-3p, is modulated by nicotine within the mesocorticolimbic reward pathway. Importantly, overexpression of miR-542-3p led to a decrease in the protein levels of its target, the nAChR ß2 subunit. Bioinformatic analysis suggests that a number of the miRNAs play a general role in regulating cholinergic signaling. Our results provide evidence for a novel mode of nicotine-mediated regulation of the mammalian nAChR gene family.

Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms.

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 channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the ß4 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-naive mice and activated nonsomatostatin 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.

Nicotinic acetylcholine receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons.

Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse.

Nicotinic acetylcholine receptors containing the α4 subunit modulate alcohol reward.

BACKGROUND: Nicotine and alcohol are the two most co-abused drugs in the world, suggesting a common mechanism of action might underlie their rewarding properties. Although nicotine elicits reward by activating ventral tegmental area dopaminergic (DAergic) neurons via high-affinity neuronal nicotinic acetylcholine receptors (nAChRs), the mechanism by which alcohol activates these neurons is unclear. METHODS: Because most high-affinity nAChRs expressed in ventral tegmental area DAergic neurons contain the α4 subunit, we measured ethanol-induced activation of DAergic neurons in midbrain slices from two complementary mouse models, an α4 knock-out (KO) mouse line and a knock-in line (Leu9'Ala) expressing α4 subunit-containing nAChRs hypersensitive to agonist compared with wild-type (WT). Activation of DAergic neurons by ethanol was analyzed with both biophysical and immunohistochemical approaches in midbrain slices. The ability of alcohol to condition a place preference in each mouse model was also measured. RESULTS: At intoxicating concentrations, ethanol activation of DAergic neurons was significantly reduced in α4 KO mice compared with WT. Conversely, in Leu9'Ala mice, DAergic neurons were activated by low ethanol concentrations that did not increase activity of WT neurons. In addition, alcohol potentiated the response to ACh in DAergic neurons, an effect reduced in α4 KO mice. Rewarding alcohol doses failed to condition a place preference in α4 KO mice, paralleling alcohol effects on DAergic neuron activity, whereas a sub-rewarding alcohol dose was sufficient to condition a place preference in Leu9'Ala mice. CONCLUSIONS: Together, these data indicate that nAChRs containing the α4 subunit modulate alcohol reward.

Nicotine persistently activates ventral tegmental area dopaminergic neurons via nicotinic acetylcholine receptors containing α4 and α6 subunits.

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 α4ß2 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 α-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the α6 subunit. α-conotoxin MII[H9A;L15A] also significantly reduced the long-lasting 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 α4 or α6 nAChR subunits. Conversely, selective activation of nAChRs containing the α4 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 α-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing α4 and α6 subunits.