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The hippocampus is essential for spatial learning and memory. To assess learning we used contextual fear conditioning (cFC), where animals learn to associate a place with aversive events like foot-shocks. Candidate memory mechanisms for cFC are long-term potentiation and long-term depression, but there is little direct evidence of them operating in the hippocampus in vivo following cFC. Also, little is known about the behavioral state changes induced by cFC. To address these issues, we recorded local field potentials in freely behaving mice by stimulating in the left dorsal CA1 region and recording in the right dorsal CA1 region. Synaptic strength in the commissural pathway was monitored by measuring field excitatory postsynaptic potentials (fEPSPs) before and after cFC. After cFC, the commissural pathway’s synaptic strength was potentiated. Although recordings occurred during the wake phase of the light/dark cycle, the mice slept more in the post-conditioning period than in the pre-conditioning period. Relative to awake periods, in non-rapid eye movement (NREM) sleep the fEPSPs were larger in both pre- and post-conditioning periods. We also found a significant negative correlation between the animal’s speed and fEPSP size. Therefore, to avoid confounds in the fEFSP potentiation estimates, we controlled for speed-related and sleep-related fEPSP changes and still found that cFC induced long-term potentiation, but no significant long-term depression. Synaptic strength changes were not found in the control group that simply explored the fear-conditioning chamber, indicating that exploration of the novel place did not produce the measurable effects caused by cFC. These results show that following cFC, the CA1 commissural pathway is potentiated, likely contributing to the functional integration of the left and right hippocampi in fear memory consolidation. In addition, the cFC paradigm produces significant changes in an animal’s behavioral state, which are observable as proximal changes in sleep patterns.

Disruption of neuronal chloride ion (Cl(−)) homeostasis has been linked to several pathological conditions, including substance use disorder, yet targeted pharmacotherapies are lacking. In this study, we explored the potential of serotonin 2A receptor (5-HT(2A)R) agonism to reduce alcohol consumption in male wildtype C57Bl/6J mice and to ameliorate alcohol-induced inhibitory plasticity in the midbrain. We found that administration of the putative 5-HT(2A)R agonist TCB-2 attenuated alcohol consumption and preference but did not alter water or saccharin consumption. We hypothesized that the selective behavioral effects of TCB-2 on alcohol drinking were due, at least in part, to effects of the agonist on ventral tegmental area (VTA) neurocircuitry. Alcohol consumption impairs Cl(−) transport in VTA GABA neurons, which acts as a molecular adaptation leading to increased alcohol self-administration. Using ex vivo electrophysiological recordings, we found that exposure to either intermittent volitional alcohol drinking or an acute alcohol injection diminished homeostatic Cl(−) transport in VTA GABA neurons. Critically, in vivo TCB-2 administration normalized Cl(−) transport in the VTA after alcohol exposure. Thus, we show a potent effect of alcohol consumption on VTA inhibitory circuity, in the form of dysregulated Cl(−) homeostasis, that is reversible with agonism of 5-HT(2A)Rs. Our results provide insight into the potential therapeutic action of 5-HT(2A)R agonists for alcohol abuse.

Aversive memories are important for survival, and dopaminergic signaling in the hippocampus has been implicated in aversive learning. However, the source and mode of action of hippocampal dopamine remain controversial. Here, we utilize anterograde and retrograde viral tracing methods to label midbrain dopaminergic projections to the dorsal hippocampus. We identify a population of midbrain dopaminergic neurons near the border of the substantia nigra pars compacta and the lateral ventral tegmental area that sends direct projections to the dorsal hippocampus. Using optogenetic manipulations and mutant mice to control dopamine transmission in the hippocampus, we show that midbrain dopamine potently modulates aversive memory formation during encoding of contextual fear. Moreover, we demonstrate that dopaminergic transmission in the dorsal CA1 is required for the acquisition of contextual fear memories, and that this acquisition is sustained in the absence of catecholamine release from noradrenergic terminals. Our findings identify a cluster of midbrain dopamine neurons that innervate the hippocampus and show that the midbrain dopamine neuromodulation in the dorsal hippocampus is sufficient to maintain aversive memory formation.

Stress is known to alter GABAergic signaling in the ventral tegmental area (VTA), and this inhibitory plasticity is associated with increased alcohol self-administration. In humans, serotonin 2A receptor (5-HT 2A R) agonists can treat stress- and alcohol-related disorders, but the neural substrates are ill-defined. Thus, we reasoned that 5-HT 2A R pharmacotherapies may ameliorate the stress-induced dysregulated inhibitory VTA circuitry that contributes to subsequent alcohol abuse. We found that acute stress exposure in mice compromised GABA-mediated inhibition of VTA GABA neurons corresponding with increased ethanol-induced GABAergic transmission. This stress-induced inhibitory plasticity was reversible by applying the 5-HT 2A R agonist TCB-2 ex vivo via functional enhancement of the potassium-chloride cotransporter KCC2. The signaling pathway linking 5-HT 2A R activation and normalization of KCC2 function was dependent on protein kinase C signaling and phosphorylation of KCC2 at serine 940 (S940), as mutation of S940 to alanine prevented restoration of chloride transport function by TCB-2. Through positive modulation of KCC2, TCB-2 also reduced elevated ethanol-induced GABAergic signaling after stress exposure that has previously been linked to increased ethanol consumption. Collectively, these findings provide mechanistic insights into the therapeutic action of 5-HT 2A R agonists at the neuronal and circuit levels of brain reward circuitry.

Although alcohol (i.e., ethanol) is a major drug of abuse, the acute functional effects of ethanol on the reward circuitry are not well defined in vivo. In freely moving rats, we examined the effect of intravenous ethanol administration on neuronal unit activity in the posterior ventral tegmental area (VTA), a central component of the mesolimbic reward system. VTA units were classified as putative dopamine (DA) neurons, fast-firing GABA neurons, and unidentified neurons based on a combination of electrophysiological properties and DA D2 receptor pharmacological responses. A gradual infusion of ethanol significantly altered the firing rate of DA neurons in a concentration-dependent manner. The majority of DA neurons were stimulated by ethanol and showed enhanced burst firing activity, but a minority was inhibited. Ethanol also increased the proportion of DA neurons that exhibited pacemaker-like firing patterns. In contrast, ethanol mediated a variety of effects in GABA and other unidentified neurons that were distinct from DA neurons, including a nonlinear increase in firing rate, delayed inhibition, and more biphasic activity. These results provide evidence of discrete electrophysiological effects of ethanol on DA neurons compared with other VTA cell types, suggesting a complex role of the VTA in alcohol-induced responses in freely moving animals.

Abstinence from chronic use of addictive drugs triggers an aversive withdrawal syndrome that compels relapse and deters abstinence. Many features of this syndrome are common across multiple drugs, involving both affective and physical symptoms. Some of the network signaling underlying withdrawal symptoms overlaps with activity that is associated with aversive mood states, including anxiety and depression. Given these shared features, it is not surprising that a particular circuit, the dorsal diencephalic conduction system, and the medial habenula (MHb) and interpeduncular nucleus (IPN), in particular, have been identified as critical to the emergence of aversive states that arise both as a result and, independently, of drug addiction. As the features of this circuit continue to be characterized, the MHb-IPN axis is emerging as a viable target for therapeutics to aid in the treatment of addiction to multiple drugs of abuse as well as mood-associated disorders. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Stress is known to alter GABAergic signaling in the ventral tegmental area (VTA), and this inhibitory plasticity is associated with increased alcohol self-administration. In humans, serotonin 2A receptor (5-HT(2A)R) agonists can treat stress- and alcohol-related disorders, but the neural substrates are ill-defined. Thus, we reasoned that 5-HT(2A)R pharmacotherapies may ameliorate the stress-induced dysregulated inhibitory VTA circuitry that contributes to subsequent alcohol abuse. We found that acute stress exposure in mice compromised GABA-mediated inhibition of VTA GABA neurons corresponding with increased ethanol-induced GABAergic transmission. This stress-induced inhibitory plasticity was reversible by applying the 5-HT(2A)R agonist TCB-2 ex vivo via functional enhancement of the potassium-chloride cotransporter KCC2. The signaling pathway linking 5-HT(2A)R activation and normalization of KCC2 function was dependent on protein kinase C signaling and phosphorylation of KCC2 at serine 940 (S940), as mutation of S940 to alanine prevented restoration of chloride transport function by TCB-2. Through positive modulation of KCC2, TCB-2 also reduced elevated ethanol-induced GABAergic signaling after stress exposure that has previously been linked to increased ethanol consumption. Collectively, these findings provide mechanistic insights into the therapeutic action of 5-HT(2A)R agonists at the neuronal and circuit levels of brain reward circuitry.

Nicotine use increases the risk for subsequent abuse of other addictive drugs, but the biological basis underlying this risk remains largely unknown. Interactions between nicotine and other drugs of abuse may arise from nicotine-induced neural adaptations in the mesolimbic dopamine (DA) system, a common pathway for the reinforcing effects of many addictive substances. Previous work identified nicotine-induced neuroadaptations that alter inhibitory transmission in the ventral tegmental area (VTA). Here, we test whether nicotine-induced dysregulation of GABAergic signaling within the VTA increases the vulnerability for benzodiazepine abuse that has been reported in smokers. We demonstrate in rats that nicotine exposure dysregulates diazepam-induced inhibition of VTA GABA neurons and increases diazepam consumption. In VTA GABA neurons, nicotine impaired KCC2-mediated chloride extrusion, depolarized the GABAAreversal potential, and shifted the pharmacological effect of diazepam on GABA neurons from inhibition toward excitation. In parallel, nicotine-related alterations in GABA signaling observedex vivowere associated with enhanced diazepam-induced inhibition of lateral VTA DA neuronsin vivo. Targeting KCC2 with the agonist CLP290 normalized diazepam-induced effects on VTA GABA transmission and reduced diazepam consumption following nicotine administration to the control level. Together, our results provide insights into midbrain circuit alterations resulting from nicotine exposure that contribute to the abuse of other drugs, such as benzodiazepines.

Alcohol use disorders (AUDs) continue to be a significant public health problem. Early life stress and adversity have long-lasting effects on a wide range of behaviors, including responses to drugs of abuse. Epidemiological evidence indicates that exposure to early life stress contributes to alcohol use disorders and, while it is known that stress and alcohol both act on overlapping mesolimbic circuitry, the cellular mechanisms underlying the relationship between stress and alcohol intake are not well understood. Previous work has demonstrated that acute stress increases ethanol intake mediated by changes in GABA signaling within the ventral tegmental area (VTA). Here we investigated if adolescent stress exposure might elicit long-term, persistent increases in ethanol self-administration associated with altered VTA GABA signaling. To this end, we exposed adolescent postnatal day (PND) 28 male rats to 14 days of chronic variable stress (CVS) and then examined operant ethanol self-administration begun at least 30 days later. We found that adolescent stress exposure resulted in significantly increased ethanol self-administration in adulthood. In contrast, adult (PND 82) male rats exposed to the same CVS protocol did not display increased ethanol self-administration that was begun 30 days later. Furthermore, we found that adolescent stress exposure resulted in enhancement of ethanol-induced GABA signaling onto VTA dopamine neurons and impairments in VTA GABA chloride homeostasis. The results indicate that adolescence is a period vulnerable to stress, which produces long-term changes in VTA GABA signaling associated with increased ethanol self-administration behavior.

Nicotine is a highly addictive drug found in tobacco that drives its continued use despite the harmful consequences. The initiation of nicotine abuse involves the mesolimbic dopamine system, which contributes to the rewarding sensory stimuli and associative learning processes in the beginning stages of addiction. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), which come in a diverse collection of subtypes. The nAChRs that contain the α4 and β2 subunits, often in combination with the α6 subunit, are particularly important for nicotine's ability to increase midbrain dopamine neuron firing rates and phasic burst firing. Chronic nicotine exposure results in numerous neuroadaptations, including the upregulation of particular nAChR subtypes associated with long-term desensitization of the receptors. When nicotine is no longer present, for example during attempts to quit smoking, a withdrawal syndrome develops. The expression of physical withdrawal symptoms depends mainly on the α2, α3, α5, and β4 nicotinic subunits in the epithalamic habenular complex and its target regions. Thus, nicotine affects diverse neural systems and an array of nAChR subtypes to mediate the overall addiction process. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Behavioral adaptations occur through remodeling of brain circuits, as arising, for instance, from experience-dependent synaptic plasticity. Drugs of abuse and aversive stimuli, such as stress, act on the mesocorticolimbic system, dysregulating adaptive mechanisms and leading to a variety of aberrant behaviors associated with neuropsychiatric disorders. Until recently, research in the field has commonly focused on experience-dependent synaptic plasticity at excitatory synapses. However, there is growing evidence that synaptic plasticity within inhibitory circuits is an important contributor to maladaptive behaviors. We speculate that restoring normal inhibitory synaptic transmission is a promising therapeutic target for correcting some of the circuit abnormalities underlying neuropsychiatric disorders.

Stress and tobacco smoking are risk factors for alcoholism, but the underlying neural mechanisms are not well understood. Although stress, nicotine, and alcohol have broad, individual effects in the brain, some of their actions converge onto the same mechanisms and circuits. Stress and nicotine augment alcohol-related behaviors, in part via modulation of alcohol-evoked neuronal plasticity and metaplasticity mechanisms. Stress modulates alcohol-evoked plasticity via the release of signaling molecules that influence synaptic transmission. Nicotine also activates some of the same signaling molecules, cells, and circuits, producing a convergence of both stress and nicotine onto common plasticity mechanisms that influence alcohol self-administration. We describe several forms of alcohol-induced plasticity, including classic Hebbian plasticity at glutamatergic synapses, and we highlight less appreciated forms, such as non-Hebbian and GABAergic synaptic plasticity. Risk factors such as stress and nicotine initiate lasting neural changes that modify subsequent alcohol-induced synaptic plasticity and increase the vulnerability to alcohol addiction.

SUMMARY Adolescent smoking is associated with pathological drinking later in life, but the biological basis for this vulnerability is unknown. To examine how adolescent nicotine exposure influences subsequent ethanol intake, nicotine was administered during adolescence or adulthood, and responses to alcohol were measured 1 month later. We found that adolescent, but not adult, nicotine exposure altered GABA signaling within the ventral tegmental area (VTA) and led to a long-lasting enhancement of alcohol self-administration. We detected depolarizing shifts in GABAA reversal potentials arising from impaired chloride extrusion in VTA GABA neurons. Alterations in GABA signaling were dependent on glucocorticoid receptor activation and were associated with attenuated dopaminergic neuron responses to alcohol in the lateral VTA. Importantly, enhancing chloride extrusion in adolescent nicotine-treated animals restored VTA GABA signaling and alcohol self-administration to control levels. Taken together, this work suggests that adolescent nicotine exposure increases the risk profile for increased alcohol drinking in adulthood., In Brief Thomas et al. show that nicotine treatments during adolescence, but not adulthood, cause a long-term increase in alcohol self-administration in adult rodents. Adolescent nicotine exposure shifts GABAA receptor signaling within the ventral tegmental area circuitry, thereby altering subsequent responses to alcohol.

Introduction: Virtual reality (VR) has been shown to be effective in eliciting responses to nicotine cues in cigarette smokers. The primary aim of this study was to investigate whether cigarette-deprived smokers would exhibit increased craving and changes in heart rate when viewing cigarette related cues as compared to non-smoking cues in a VR environment, and the secondary aim was to assess the extent to which self-assessed measures of withdrawal and dependence correlated with VR craving. Methods: Nicotine-dependent cigarette smokers were recruited for a 2 day study. On Day 1, participants smoked as usual and on Day 2 were deprived from smoking overnight. On both days, participants completed self-assessment questionnaires on withdrawal, craving, and nicotine-dependence. Participants completed a VR session during the cigarette deprivation condition only (Day 2). During this session, they were exposed to active smoking and placebo (non-smoking) cues. Results: The data show that self-reported levels of “craving” (p < .01) and “thinking about cigarettes” (p < .0001) were significantly greater after exposure to the active cues versus non-smoking cues. Significant increases in heart rate were found for 3 of 4 active cues when compared to non-smoking cues (p < .05). Finally, significant positive correlations were found between self-reported craving prior to the VR session and craving induced by active VR cues (p < .01). Conclusions: In this report, active VR cues elicited craving during cigarette deprivation. This is the first study to demonstrate that self-reported craving, withdrawal symptoms, and nicotine dependence severity predict cue-induced craving in the VR setting.

Although evidence suggests that DA modulates hippocampal function, the mechanisms underlying that dopaminergic modulation are largely unknown. Using perforated-patch electrophysiological techniques to maintain the intracellular milieu, we investigated how the activation of D1-type DA receptors regulates spike timing-dependent plasticity (STDP) of the medial perforant path (mPP) synapse onto dentate granule cells. When D1-type receptors were inhibited, a relatively mild STDP protocol induced LTP only within a very narrow timing window between presynaptic stimulation and postsynaptic response. The stimulus protocol produced timing-dependent LTP (tLTP) only when the presynaptic stimulation was followed 30 ms later by depolarization-induced postsynaptic action potentials. That is, the time between presynaptic stimulation and postsynaptic response was 30 ms (Δt= +30 ms). When D1-type receptors were activated, however, the same mild STDP protocol induced tLTP over a much broader timing window: tLTP was induced when −30 ms ≤ Δt≤ +30 ms. The result indicated that D1-type receptor activation enabled synaptic potentiation even when postsynaptic activity preceded presynaptic stimulation within this Δtrange. Results with null mice lacking the Kv4.2 potassium channel and with the potassium channel inhibitor, 4-aminopyridine, suggested that D1-type receptors enhanced tLTP induction by suppressing the transientIA-type K+current. Results obtained with antagonists and DA receptor knock-out mice indicated that endogenous activity of both D1 and D5 receptors modulated plasticity in the mPP. The DA D5 receptors appeared particularly important in regulating plasticity of the mPP onto the dentate granule cells.

Dopamine (DA) signaling in the central nervous system mediates the addictive capacities of multiple commonly abused substances, including cocaine, amphetamine, heroin and nicotine. The firing of DA neurons residing in the ventral tegmental area (VTA), and the release of DA by the projections of these neurons in the nucleus accumbens (NAc), is under tight control by cholinergic signaling mediated by nicotinic acetylcholine (ACh) receptors (nAChRs). The capacity for cholinergic signaling is dictated by the availability and activity of the presynaptic, high-affinity, choline transporter (CHT, SLC5A7) that acquires choline in an activity-dependent matter to sustain ACh synthesis. Here, we present evidence that a constitutive loss of CHT expression, mediated by genetic elimination of one copy of the Slc5a7 gene in mice (CHT+/-), leads to a significant reduction in basal extracellular DA levels in the NAc, as measured by in vivo microdialysis. Moreover, CHT heterozygosity results in blunted DA elevations following systemic nicotine or cocaine administration. These findings reinforce a critical role of ACh signaling capacity in both tonic and drug-modulated DA signaling and argue that genetically imposed reductions in CHT that lead to diminished DA signaling may lead to poor responses to reinforcing stimuli, possibly contributing to disorders linked to perturbed cholinergic signaling including depression and attention-deficit hyperactivity disorder (ADHD).

Epidemiological studies consistently find correlations between nicotine and alcohol use, yet the neural mechanisms underlying their interaction remain largely unknown. Nicotine and alcohol (i.e., ethanol) share many common molecular and cellular targets that provide potential substrates for nicotine–alcohol interactions. These targets for interaction often converge upon the mesocorticolimbic dopamine system, where the link to drug self-administration and reinforcement is well documented. Both nicotine and alcohol activate the mesocorticolimbic dopamine system, producing downstream dopamine signals that promote the drug reinforcement process. While nicotine primarily acts via nicotinic acetylcholine receptors, alcohol acts upon a wider range of receptors and molecular substrates. The complex pharmacological profile of these two drugs generates overlapping responses that ultimately intersect within the mesocorticolimbic dopamine system to promote drug use. Here we will examine overlapping targets between nicotine and alcohol and provide evidence for their interaction. Based on the existing literature, we will also propose some potential targets that have yet to be directly tested. Mechanistic studies that examine nicotine–alcohol interactions would ultimately improve our understanding of the factors that contribute to the associations between nicotine and alcohol use.

Recreational drug use leads to compulsive substance abuse in some individuals. Studies on animal models of drug addiction indicate that persistent long-term potentiation (LTP) of excitatory synaptic transmission onto ventral tegmental area (VTA) dopamine (DA) neurons is a critical component of sustained drug seeking. However, little is known about the mechanism regulating such long-lasting changes in synaptic strength. Previously, we identified that translational control by eIF2α phosphorylation (p-eIF2α) regulates cocaine-induced LTP in the VTA (Huang et al., 2016). Here we report that in mice with reduced p-eIF2α-mediated translation, cocaine induces persistent LTP in VTA DA neurons. Moreover, selectively inhibiting eIF2α-mediated translational control with a small molecule ISRIB, or knocking down oligophrenin-1—an mRNA whose translation is controlled by p-eIF2α—in the VTA also prolongs cocaine-induced LTP. This persistent LTP is mediated by the insertion of GluR2-lacking AMPARs. Collectively, our findings suggest that eIF2α-mediated translational control regulates the progression from transient to persistent cocaine-induced LTP. DOI: http://dx.doi.org/10.7554/eLife.17517.001

Physiological and behavioral evidence supports that dopamine (DA) receptor signaling influences hippocampal function. While several recent studies examined how DA influences CA1 plasticity and learning, there are fewer studies investigating the influence of DA signaling to the dentate gyrus. The dentate gyrus receives convergent cortical input through the perforant path fiber tracts and has been conceptualized to detect novelty in spatial memory tasks. To test whether DA-receptor activity influences novelty-detection, we used a novel object recognition (NOR) task where mice remember previously presented objects as an indication of learning. Although DA innervation arises from other sources and the main DA signaling may be from those sources, our molecular approaches verified that midbrain dopaminergic fibers also sparsely innervate the dentate gyrus. During the NOR task, wild-type mice spent significantly more time investigating novel objects rather than previously observed objects. Dentate granule cells in slices cut from those mice showed an increased AMPA/NMDA-receptor current ratio indicative of potentiated synaptic transmission. Post-training injection of a D1-like receptor antagonist not only effectively blocked the preference for the novel objects, but also prevented the increased AMPA/NMDA ratio. Consistent with that finding, neither NOR learning nor the increase in the AMPA/NMDA ratio were observed in DA-receptor KO mice under the same experimental conditions. The results indicate that DA-receptor signaling contributes to the successful completion of the NOR task and to the associated synaptic plasticity of the dentate gyrus that likely contributes to the learning.

Adolescents are especially prone to drug addiction, but the underlying biological basis of their increased vulnerability remains unknown. We reveal that translational control by phosphorylation of the translation initiation factor eIF2α (p-eIF2α) accounts for adolescent hypersensitivity to cocaine. In adolescent (but not adult) mice, a low dose of cocaine reduced p-eIF2α in the ventral tegmental area (VTA), potentiated synaptic inputs to VTA dopaminergic neurons, and induced drug-reinforced behavior. Like adolescents, adult mice with reduced p-eIF2α-mediated translational control were more susceptible to cocaine-induced synaptic potentiation and behavior. Conversely, like adults, adolescent mice with increased p-eIF2α became more resistant to cocaine's effects. Accordingly, metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD)—whose disruption is postulated to increase vulnerability to drug addiction—was impaired in both adolescent mice and adult mice with reduced p-eIF2α mediated translation. Thus, during addiction, cocaine hijacks translational control by p-eIF2α, initiating synaptic potentiation and addiction-related behaviors. These insights may hold promise for new treatments for addiction. DOI: http://dx.doi.org/10.7554/eLife.12052.001, eLife digest Drug addiction a is major mental health problem that presents a huge financial, social and legal burden worldwide. Adolescents are notoriously prone to drug abuse and addicts typically begin using drugs at a young age. However, an explanation for why young people are particularly vulnerable to the effects of addictive substances remains elusive. Addictive drugs change how the brain works, in particular by strengthening the connections (synapses) between brain cells (neurons) and making it easier for neurons to communicate with each other. Such strengthening of synaptic connections, which can be observed when the activity of the neurons is recorded with microelectrodes, relies on new proteins being made in the brain. Since adolescents have a greater capacity than adults to make new proteins, Huang et al. hypothesized that changes in synaptic strength might occur more easily in the brain of adolescents, explaining why they are more likely to become addicted to drugs than adults. A protein called eIF2α plays a key role in regulating the production of new proteins. Huang et al. discovered that reduced eIF2α activity accounts for why adolescents are particularly vulnerable to the synaptic and behavioral effects of cocaine. Giving adolescent mice a low dose of cocaine reduced the activity of eIF2α, caused an increase in the strength of synaptic connections in a part of the brain that processes pleasurable feelings, and promoted drug-reinforced behavior. This did not occur in adult mice. Reducing the activity of eIF2α using either genetics or pharmacological methods caused adult mice to become as vulnerable as adolescents to cocaine-induced changes in synaptic strength and addiction-related behavior. Conversely, increasing the activity of eIF2α made adolescent mice more resistant to cocaine’s effects; in other words, adolescents responded to cocaine more like adults. Huang et al. also found that other drugs of abuse, including alcohol, methamphetamine and nicotine, all reduce eIF2α activity, suggesting that eIF2α is a common target of different drugs of abuse. In a related study, Placzek et al. investigated the role of eIF2α in nicotine addiction in mice and humans. These findings raise several intriguing questions. How do cocaine and other drugs of abuse reduce eIF2α activity? Could variations in the activity of eIF2α or other components of the eIF2α pathway in the brain explain why some people are more likely to abuse drugs? Finally, could compounds that regulate the activity of eIF2α be useful for treating addiction? DOI: http://dx.doi.org/10.7554/eLife.12052.002

Adolescents are particularly vulnerable to nicotine, the principal addictive component driving tobacco smoking. In a companion study, we found that reduced activity of the translation initiation factor eIF2α underlies the hypersensitivity of adolescent mice to the effects of cocaine. Here we report that nicotine potentiates excitatory synaptic transmission in ventral tegmental area dopaminergic neurons more readily in adolescent mice compared to adults. Adult mice with genetic or pharmacological reduction in p-eIF2α-mediated translation are more susceptible to nicotine’s synaptic effects, like adolescents. When we investigated the influence of allelic variability of the Eif2s1 gene (encoding eIF2α) on reward-related neuronal responses in human smokers, we found that a single nucleotide polymorphism in the Eif2s1 gene modulates mesolimbic neuronal reward responses in human smokers. These findings suggest that p-eIF2α regulates synaptic actions of nicotine in both mice and humans, and that reduced p-eIF2α may enhance susceptibility to nicotine (and other drugs of abuse) during adolescence. DOI: http://dx.doi.org/10.7554/eLife.12056.001, eLife digest Nicotine addiction is a serious public health problem. People who start using nicotine during adolescence are more likely to become addicted to it during adulthood, but the reasons for this are not well understood. Nicotine causes long-lasting changes in the brain that are responsible for the feelings of pleasure and reward. In particular, nicotine strengthens the connections between neurons at structures called synapses and increases communication between reward-related neurons in key reward areas of the brain. This hijacking of the natural reward system requires new proteins to be made. However, the relationship between protein synthesis and adolescents being particularly vulnerable to nicotine addiction was not known. In a related study, Huang et al. found that the reduced activity of a protein called eIF2α, which controls the production of new proteins, accounts for why adolescents are more likely to become addicted to cocaine than adults. Thus, Placzek et al. wanted to know whether the same was true for nicotine and whether the proteins controlled by eIF2α are involved in the way human nicotine addicts experience reward. Placzek et al. found that adolescent mice are more susceptible than adult mice to the changes in synaptic strength that are caused by nicotine. This increased susceptibility results from reduced activity levels of the protein eIF2α. Reducing the activity of eIF2α in adult mice made their synapses as likely to change strength in response to nicotine as the synapses of adolescent mice. Placzek et al. also used a technique called functional magnetic resonance imaging and found that compared to non-smokers, the brain activity of human smokers was significantly reduced when given a natural reward. Further studies revealed a variation in the gene encoding the eIF2α protein that affects how smokers respond to a reward, suggesting that this variant is linked to the likelihood that a person will be addicted to nicotine. This work raises several important questions. In addition to regulating the initial adaptive changes induced in the brain by nicotine, does eIF2α activity affect compulsive nicotine use? If so, could targeting parts of the eIF2α pathway help treat nicotine addiction? Finally, further studies could explore whether the gene variant identified by Placzek et al. affects how users of other drugs (such as cocaine or alcohol) respond to natural rewards. DOI: http://dx.doi.org/10.7554/eLife.12056.002

In this study, we examined the relationship between the level of daily cigarette consumption and the startle response to affective and cigarette-related cues among treatment-seeking smokers. Before receiving any behavioral or pharmacological treatment, 136 smokers attended a baseline laboratory session, during which we recorded their reflexive eyeblink responses to acoustic startle probes while they were viewing pleasant, unpleasant, neutral, and cigarette-related pictures. We found that 1) cigarette-related and pleasant pictures similarly reduced the startle magnitude compared to neutral pictures; 2) the magnitude of startle modulation rendered by pleasant or unpleasant pictures did not differ among light, moderate, and heavy smokers; and 3) startle attenuation by cigarette-related pictures was greater in heavy smokers than in light smokers. These results suggest that similar to pleasant stimuli, cigarette-related cues are motivationally salient for smokers, and that this salience increases with nicotine dependence.

Altered functional interactions among midbrain dopamine (DA) neurons contribute to the reinforcing properties of environmental stimuli and addictive drugs. To examine correlations among DA neurons, acute nicotine was administrated to rats via an intraperitoneal catheter and unit activity was measured using multi-tetrode in vivo recordings. Nicotine administration enhanced the correlated activity of simultaneously recorded DA neurons from the ventral tegmental area (VTA). The strength of the correlations between DA neuron pairs, as measured by cross covariance among two spike trains, showed dynamic firing changes over time. Nicotine produced a gradual rise in firing rate and burst activity that reached a stable plateau approximately 20 min after the intraperitoneal nicotine infusion. Shortly after that time the cross correlations measured using 5-ms bins increased significantly above baseline. In addition, nicotine increased the firing rates of DA neurons in the posterior VTA more than in the anterior VTA. Unlike nicotine, eticlopride administration also boosted DA neuron firing activity but did not enhance synchronization, indicating that the cross correlations induced by nicotine were not due to a non-specific increase in firing rate. The overall results show that nicotine induces nearly synchronous firing by a subset of DA neurons, and those changes in correlative firing will enhance the DA signal that contributes to nicotine-induced behavioral reinforcement.

Nicotine is the principal addictive component that drives continued tobacco use despite users' knowledge of the harmful consequences. The initiation of addiction involves the mesocorticolimbic dopamine system, which contributes to the processing of rewarding sensory stimuli during the overall shaping of successful behaviors. Acting mainly through nicotinic receptors containing the α4 and β2 subunits, often in combination with the α6 subunit, nicotine increases the firing rate and the phasic bursts by midbrain dopamine neurons. Neuroadaptations arise during chronic exposure to nicotine, producing an altered brain condition that requires the continued presence of nicotine to be maintained. When nicotine is removed, a withdrawal syndrome develops. The expression of somatic withdrawal symptoms depends mainly on the α5, α2, and β4 (and likely α3) nicotinic subunits involving the epithalamic habenular complex and its targets. Thus, nicotine taps into diverse neural systems and an array of nicotinic acetylcholine receptor (nAChR) subtypes to influence reward, addiction, and withdrawal.

SummaryAddictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

Mesolimbic dopamine (DA) systems play a critical role in tobacco addiction driven by nicotine. Nicotine activates midbrain DA neurons and, consequently, elevates DA concentrations in targets, especially in the nucleus accumbens (NAc) of the ventral striatum. The route of drug administration influences the impact of addictive drugs. Here, we examine whether the nature of the administration alters DA neuron activity and DA concentrations in the NAc. Using unhabituated naïve freely moving rats, microdialysis measurements showed that nicotine administered via needle injection caused greater DA release in the NAc than the same dose administered via an implanted chronic cannula. After habituation to the needle injections, however, there was no significant difference in DA signaling between the needle and cannula routes of administration. Consistent with these microdialysis results after habituation, our in vivo tetrode unit recordings showed no significant difference in midbrain DA neuron activity in response to nicotine delivered by needle or cannula as long as predictive cues were avoided.

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the α3 and α4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with α3-containing nAChRs (α3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the α3β2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of α3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER andcis-Golgi compartments. Our investigations also showed that the α3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of α3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the α3 subunit and, consequently, the number of receptors at the cell surface.

Nicotinic acetylcholine receptors (nAChRs) are expressed throughout the hippocampus, and nicotinic signaling plays an important role in neuronal function. In the context of learning and memory related behaviors associated with hippocampal function, a potentially significant feature of nAChR activity is the impact it has on synaptic plasticity. Synaptic plasticity in hippocampal neurons has long been considered a contributing cellular mechanism of learning and memory. These same kinds of cellular mechanisms are a factor in the development of nicotine addiction. Nicotinic signaling has been demonstrated by in vitro studies to affect synaptic plasticity in hippocampal neurons via multiple steps, and the signaling has also been shown to evoke synaptic plasticity in vivo. This review focuses on the nAChRs subtypes that contribute to hippocampal synaptic plasticity at the cellular and circuit level. It also considers nicotinic influences over long-term changes in the hippocampus that may contribute to addiction.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and participate in a variety of physiological functions. Recent advances have revealed roles of nAChRs in the regulation of synaptic transmission and synaptic plasticity, particularly in the hippocampus and midbrain dopamine centers. In general, activation of nAChRs causes membrane depolarization and directly and indirectly increases the intracellular calcium concentration. Thus, when nAChRs are expressed on presynaptic membranes their activation generally increases the probability of neurotransmitter release. When expressed on postsynaptic membranes, nAChR-initiated calcium signals and depolarization activate intracellular signaling mechanisms and gene transcription. Together, the presynaptic and postsynaptic effects of nAChRs generate and facilitate the induction of long-term changes in synaptic transmission. The direction of hippocampal nAChR-mediated synaptic plasticity - either potentiation or depression - depends on the timing of nAChR activation relative to coincident presynaptic and postsynaptic electrical activity, and also depends on the location of cholinergic stimulation within the local network. Therapeutic activation of nAChRs may prove efficacious in the treatment of neuropathologies where synaptic transmission is compromised, as in Alzheimer's or Parkinson's disease.

Summary Stress is a well-known risk factor for subsequent alcohol abuse, but the neural mechanisms underlying interactions between stress and alcohol remain largely unknown. Addictive drug reinforcement and stress signaling involve common neural circuitry, including the mesolimbic dopamine system. We demonstrate in rodents that pre-exposure to stress attenuates alcohol-induced dopamine responses and increases alcohol self-administration. The blunted dopamine signaling resulted from ethanol-induced excitation of GABA neurons in the ventral tegmental area. Excitation of GABA neurons was mediated by GABA A receptor activation and involved stress-induced functional downregulation of the K + , Cl − cotransporter, KCC2. Blocking stress hormone receptors, enhancing KCC2 function, or preventing excitatory GABA signaling by alternative methods all prevented the attenuated alcohol-induced dopamine response and prevented the increased alcohol self-administration. These results demonstrate that stress alters the neural and behavioral responses to alcohol through a neuroendocrine signal that shifts inhibitory GABA transmission toward excitation.

Nicotine addiction drives tobacco use by one billion people worldwide, causing nearly six million deaths a year. Nicotine binds to nicotinic acetylcholine receptors that are normally activated by the endogenous neurotransmitter acetylcholine. The widespread expression of nicotinic receptors throughout the nervous system accounts for the diverse physiological effects triggered by nicotine. A crucial influence of nicotine is on the synaptic mechanisms underlying learning that contribute to the addiction process. Here, we focus on the acquisition phase of smoking addiction and review animal model studies on how nicotine modifies dopaminergic and cholinergic signaling in key nodes of the reinforcement circuitry: ventral tegmental area, nucleus accumbens (NAc), amygdala, and hippocampus. Capitalizing on mechanisms that subserve natural rewards, nicotine activates midbrain dopamine neurons directly and indirectly, and nicotine causes dopamine release in very broad target areas throughout the brain, including the NAc, amygdala, and hippocampus. In addition, nicotine orchestrates local changes within those target structures, alters the release of virtually all major neurotransmitters, and primes the nervous system to the influence of other addictive drugs. Hence, understanding how nicotine affects the circuitry for synaptic plasticity and learning may aid in developing reasoned therapies to treat nicotine addiction.

Studies in human populations consistently demonstrate an interaction between nicotine and ethanol use, each drug influencing the use of the other. Here we present data and review evidence from animal studies that nicotine influences operant self-administration of ethanol. The operant reinforcement paradigm has proven to be a behaviorally relevant and quantitative model for studying ethanol-seeking behavior. Exposure to nicotine can modify the reinforcing properties of ethanol during different phases of ethanol self-administration, including acquisition, maintenance, and reinstatement. Our data suggest that non-daily intermittent nicotine exposure can trigger a long-lasting increase in ethanol self-administration. The biological basis for interactions between nicotine and ethanol is not well understood but may involve the stress hormone systems and adaptations in the mesolimbic dopamine system. Future studies that combine operant self-administration with techniques for monitoring or manipulating in vivo neurophysiology may provide new insights into the neuronal mechanisms that link nicotine and alcohol use.

The hippocampus is a center for learning and memory that receives abundant cholinergic innervation and richly expresses nicotinic acetylcholine receptors (nAChRs). Nicotinic mechanisms acting on the hippocampus influence attention, learning, and memory. During Alzheimer's dementia, nAChRs and cholinergic innervation decline in the hippocampus. Using mouse hippocampal slices, we examined the potential diversity of nAChR influences at the Schaffer collateral synapse onto CA1 pyramidal neurons. When nAChR currents were elicited locally at those excitatory synapses, various outcomes were possible depending on the relationship between the nAChR-mediated excitation and mild electrical stimulation. When mild presynaptic stimulation coincided with or preceded nAChR-induced action potentials by 1-5 s, then long-term potentiation was induced. However, if the nAChR-induced action potentials fell within 1 s before the electrical stimulation, then long-term depression resulted. Outside of these time frames, the mismatch of nAChR activity and stimulation led to short-term potentiation. The results indicate that nAChRs may have various influences over excitatory events in the hippocampus. Ongoing nAChR activity likely modulates the impact of glutamate transmission and alters the probabilities for various forms of synaptic plasticity. The fine network of cholinergic fibers running through the hippocampus forms synaptic contacts onto pyramidal cells, granule cells, and interneurons, ensuring continual modulatory influence by nicotinic mechanisms throughout the hippocampal complex. Disruption of events such as those described here may contribute to the deficits associated with the decline of nicotinic cholinergic functions during degenerative diseases such as Alzheimer's dementia.

The nucleus accumbens (NAc) of the ventral striatum is involved in attention, motivation, movement, learning, reward, and addiction. GABAergic medium spiny projection neurons that make up ∼90% of the neuronal population are commonly driven by convergent bursts of afferent excitation. We monitored spiny projection neurons in mouse striatal slices while applying stimulus trains to mimic bursts of excitation. A stimulus train evoked a simple, short-lived postsynaptic response from CA1 hippocampal pyramidal neurons, but the train evoked a complex series of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) from the NAc spiny projection neurons. As is commonly seen with projection neurons, the EPSC amplitudes initially displayed facilitation followed by depression, and that pattern was sensitive to the extracellular calcium concentration. In addition, there were two other novel observations. The spiny projection neurons responded to the stimulus train with a prolonged depolarization that was accompanied by a posttrain increase of spontaneous glutamatergic synaptic activity. Blocking AMPA/kainate glutamate receptors strongly inhibited the evoked EPSP/EPSCs, the posttrain spontaneous synaptic activity, and the prolonged depolarization. A potassium channel inhibitor increased and extended the prolonged postsynaptic depolarization, causing a long-lasting depolarized plateau potential. Our results indicate that burst-like activity along ventral striatal afferents is extended in time by additional spontaneous glutamate release that is integrated by the postsynaptic spiny projection neurons into a prolonged depolarization. The results suggest that the posttrain quantal glutamate release helps to blend and maintain multiple afferent inputs. That convergent excitation is further integrated by the postsynaptic neuron into a prolonged depolarization that may contribute to the depolarized “up state” observed in vivo.

Nicotine, acting at nicotinic acetylcholine receptors (nAChRs), is the primary addictive component of tobacco. Smokers often report an anxiolytic effect of cigarettes. This relief of anxiety, attributed to nicotine, is an important contributor to relapse when smokers try to quit. Hence, the study of the anxiolytic effects of nicotine is important for understanding the mechanisms underlying nicotine addiction. Mammalian nAChRs are pentameric ion channels usually composed of alpha andbeta subunits. Taking advantage of beta4-homozygous-null mice (beta4-/-), we examined the role of the nAChR beta4 subunit in anxiety-related behaviors. The beta4-/- mice behaved as though they were less anxious than wild-type littermates on the elevated-plus and staircase mazes, tests that measure anxiety-related behaviors. To obtain an independent, physiological indication of the stress produced by several tests, we measured changes in heart rate using telemetry. Consistently with the behavioral phenotype, beta4-/- mice had a smaller heart rate increase in the elevated-plus maze than did wild-type littermates. In contrast, during social isolation, a separate test for anxiety,beta4-/- mice exhibited a greater increase in heart rate than did wild-type littermates. Finally, beta4-/- mice were indistinguishable from their wild-type littermates in the open field, the light/dark box, and the mirrored chamber. The overall results demonstrate that beta4-containing (beta4*) nAChRs influence behavioral responses during anxiety-related tests, and that this effect depends on the type of anxiety-provoking experience. Through their influence on anxiety-related behavior, beta4* nAChRs might influence both tobacco consumption and smoking relapse.

Although many psychopharmacological factors contribute to nicotine addiction, midbrain dopaminergic systems have received much attention because of their roles in reinforcement and associative learning. It is generally thought that the mesocorticolimbic dopaminergic system is important for the acquisition of behaviors that are reinforced by the salient drives of the environment or by the inappropriate stimuli of addictive drugs. Nicotine, as obtained from tobacco, can activate nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons of the mesocorticolimbic system. Using midbrain slices from rats, wild-type mice, and genetically engineered mice, we have found differences in the nAChR currents from the ventral tegmental area (VTA) and the substantia nigra compacta (SNc). Nicotinic AChRs containing the alpha7 subunit (alpha7* nAChRs) have a low expression density. Electrophysiological analysis of nAChR currents, autoradiography of [125I]-alpha-bungarotoxin binding, and in situ hybridization revealed that alpha7* nAChRs are more highly expressed in the VTA than the SNc. In contrast, beta2* nAChRs are move evenly distributed at a higher density in both the VTA and SNc. At the concentration of nicotine obtained by tobacco smokers, the slow components of current (mainly mediated by beta2* nAChRs) become essentially desensitized. However, the minority alpha7* component of the current in the VTA/SNc is not significantly desensitized by nicotine in the range < or =100 nm. These results suggest that nicotine, as obtained from tobacco, can have multiple effects on the midbrain areas by differentially influencing dopamine neurons of the VTA and SNc and differentially desensitizing alpha7* and non-alpha7 nAChRs.

Nicotine dependence has been suggested to be related to reinforcement sensitivity, which encompasses behavioral predispositions either to avoid aversive (behavioral inhibition) or to approach appetitive (behavioral activation) stimuli. Reinforcement sensitivity may shape motives for nicotine use and offer potential targets for personalized smoking cessation therapy. However, little is known regarding how reinforcement sensitivity is related to motivational processes implicated in the maintenance of smoking. Additionally, women and men differ in reinforcement sensitivity, and such difference may cause distinct relationships between reinforcement sensitivity and motivational processes for female and male smokers. In this study, the authors characterized reinforcement sensitivity in relation to affect, smoking-related reinforcement motives, and affective responses, using self-report and psychophysiological measures, in over 200 smokers before treating them. The Behavioral Inhibition/Activation Scales (BIS/BAS; Carver & White, 1994) was used to measure reinforcement sensitivity. In female and male smokers, BIS was similarly associated with negative affect and negative reinforcement of smoking. However, positive affect was positively associated with BAS Drive scores in male smokers, and this association was reversed in female smokers. BIS was positively associated with corrugator electromyographic reactivity toward negative stimuli and left frontal electroencephalogram alpha asymmetry. Female and male smokers showed similar relationships for these physiological measures. These findings suggest that reinforcement sensitivity underpins important motivational processes (e.g., affect), and gender is a moderating factor for these relationships. Future personalized smoking intervention, particularly among more dependent treatment-seeking smokers, may experiment to target individual differences in reinforcement sensitivity. (PsycINFO Database Record

Nicotinic acetylcholine receptors (nAChRs) belong to the “Cys-loop” superfamily of ligand-gated ion channels that includes GABAA, glycine, and serotonin (5-HT3) receptors. There are 16 homologous mammalian nAChR subunits encoded by a multigene family. These subunits combine to form many different nAChR subtypes with various expression patterns, diverse functional properties, and differing pharmacological characteristics. Because cholinergic innervation is pervasive and nAChR expression is extremely broad, practically every area of the brain is impinged upon by nicotinic mechanisms. This review briefly examines the structural and functional properties of the receptor/channel complex itself. The review also summarizes activation and desensitization of nAChRs by the low nicotine concentrations obtained from tobacco. Knowledge of the three-dimensional structure and the structural characteristics of channel gating has reached an advanced stage. Likewise, the basic functional properties of the channel also are reasonably well understood. It is these receptor/channel properties that underlie the participation of nAChRs in nearly every anatomical region of the mammalian brain.

The medial habenula (MHb) densely expresses nicotinic acetylcholine receptors (nAChRs) and participates in nicotine-related behaviors such as nicotine withdrawal and regulating nicotine intake. Although specific nAChR subunits are identified as being involved in withdrawal behavior, the cellular mechanisms through which nicotine acts to cause this aversive experience is unclear. Here, we demonstrate an interaction between the nicotinic and neurokinin signaling systems that may form the basis for some symptoms experienced during nicotine withdrawal. Using patch-clamp electrophysiology in mouse brain slices, we show that nicotine (1 μm) increases intrinsic excitability in MHb neurons. This nicotine-induced phenomenon requires α5-containing nAChRs and depends on intact neurokinin signaling. The effect is blocked by preincubation with neurokinin 1 (NK1; L-732138, 10 μm) and NK3 (SB222200, 2 μm) antagonists and mimicked by NK1 (substance P, 100 nm) and NK3 (neurokinin B [NKB], 100 nm) agonists. Microinjections (1 μl) of L-732138 (50 nm) and SB222200 (100 nm) into the MHb induces withdrawal behavior in chronic nicotine-treated (8.4 mg/kg/d, 2 weeks) mice. Conversely, withdrawal behavior is absent with analogous microinjections into the lateral habenula of nicotine-treated mice or in mice chronically treated with a vehicle solution. Further, chronic nicotine reduces nicotine's acute modulation of intrinsic excitability while sparing modulation by NKB. Our work elucidates the interplay between two neuromodulatory signaling systems in the brain through which nicotine acts to influence intrinsic excitability. More importantly, we document a neuroadaptation of this mechanism to chronic nicotine exposure and implicate these mechanisms collectively in the emergence of nicotine withdrawal behavior.