Engaging high-school students in scientific conferences.

Scientific meetings rarely involve the local community and have minimal educational and scientific impacts on it. Here, we report the successful engagement of high-school students in scientific conferences. To promote science education and trust in science, we call upon conference attendees and organizers to involve high-school students in their meetings.

Multimodal Interrogation of Ventral Pallidum Projections Reveals Projection-Specific Signatures and Effects on Cocaine Reward.

The ventral pallidum (VP) is a central hub in the reward circuitry with diverse projections that have different behavioral roles attributed mostly to the connectivity with the downstream target. However, different VP projections may represent, as in the striatum, separate neuronal populations that differ in more than just connectivity. In this study, we performed in mice of both sexes a multimodal dissection of four major projections of the VP-to the lateral hypothalamus (VP→LH), ventral tegmental area (VP→VTA), lateral habenula (VP→LHb), and mediodorsal thalamus (VP→MDT)-with physiological, anatomical, genetic, and behavioral tools. We also tested for physiological differences between VP neurons receiving input from nucleus accumbens medium spiny neurons (MSNs) that express either the D1 (D1-MSNs) or the D2 (D2-MSNs) dopamine receptor. We show that each VP projection (1) when inhibited during a cocaine conditioned place preference (CPP) test affects performance differently, (2) receives a different pattern of inputs using rabies retrograde labeling, (3) shows differentially expressed genes using RNA sequencing, and (4) has projection-specific characteristics in excitability and synaptic input characteristics using whole-cell patch clamp. VP→LH and VP→VTA projections have different effects on CPP and show low overlap in circuit tracing experiments, as VP→VTA neurons receive more striatal input, while VP→LH neurons receive more olfactory input. Additionally, VP→VTA neurons are less excitable, while VP→LH neurons are more excitable than the average VP neuron, a difference driven mainly by D2-MSN-responding neurons. Thus, VP→VTA and VP→LH neurons may represent largely distinct populations of VP neurons.

Cocaine induces input and cell-type-specific synaptic plasticity in ventral pallidum-projecting nucleus accumbens medium spiny neurons.

Cocaine use and abstinence induce long-term synaptic alterations in the excitatory input to nucleus accumbens (NAc) medium spiny neurons (MSNs). The NAc regulates reward-related behaviors through two parallel projections to the ventral pallidum (VP)-originating in D1 or D2-expressing MSNs (D1-MSNs→VP; D2-MSNs→VP). The activity of these projections depends on their excitatory synaptic inputs, but it is not known whether and how abstinence from cocaine affects the excitatory transmission to D1-MSNs→VP and D2-MSNs→VP. Here we examined different forms of cocaine-induced synaptic plasticity in the inputs from the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) to NAc D1-MSNs→VP and putative D2-MSNs→VP (pD2-MSNs→VP) in the core and shell subcompartments of the NAc. We used the whole-cell patch-clamp technique to record excitatory postsynaptic currents from D1-tdTomato mice injected with ChR2 in either the BLA or the mPFC and retrograde tracer (RetroBeads) in the VP. We found that cocaine conditioned place preference (CPP) followed by abstinence potentiated the excitatory input from the BLA and mPFC to both D1-MSNs→VP and pD2-MSNs→VP. Interestingly, while the strengthening of the inputs to D1-MSNs→VP was of postsynaptic origin and manifested as increased AMPA to NMDA ratio, in pD2-MSNs→VP plasticity was predominantly presynaptic and was detected as changes in the paired-pulse ratio and coefficient of variation. Lastly, some of the changes were sex-specific. Overall our data show that abstinence from cocaine changes the excitatory inputs to both D1-MSNs→VP and pD2-MSNs→VP but with different mechanisms. This may help understand how circuits converging into the VP change after cocaine exposure.

Ventral pallidum cellular and pathway specificity in drug seeking.

The ventral pallidum (VP) is central to the reinforcing effects across a variety of drugs and relapse to drug seeking. Emerging studies from animal models of reinstatement reveal a complex neurobiology of the VP that contributes to different aspects of relapse to drug seeking. This review builds on classical understanding of the VP as part of the final common pathway of relapse but also discusses the properties of the VP as an independent structure. These include VP neural anatomical subregions, cellular heterogeneity, circuitry, neurotransmitters and peptides. Collectively, this review provides a current understanding of the VP from molecular to circuit level architecture that contributes to both the appetitive and aversive symptoms of drug addiction. We show the complex neurobiology of the VP in drug seeking, emphasizing its critical role in addiction, and review strategic approaches that target the VP to reduce relapse rates.

The role of the nucleus accumbens and ventral pallidum in feeding and obesity.

Obesity is a growing global epidemic that stems from the increasing availability of highly-palatable foods and the consequent enhanced calorie consumption. Extensive research has shown that brain regions that are central to reward seeking modulate feeding and evidence linking obesity to pathology in such regions have recently started to accumulate. In this review we focus on the contribution of two major interconnected structures central to reward processing, the nucleus accumbens and the ventral pallidum, to obesity. We first review the known literature linking these structures to feeding behavior, then discuss recent advances connecting pathology in the nucleus accumbens and ventral pallidum to obesity, and finally examine the similarities and differences between drug addiction and obesity in the context of these two structures. The understanding of how pathology in brain regions involved in reward seeking and consumption may drive obesity and how mechanistically similar obesity and addiction are, is only now starting to be revealed. We hope that future research will advance knowledge in the field and open new avenues to studying and treating obesity.

Metaplasticity in the Ventral Pallidum as a Potential Marker for the Propensity to Gain Weight in Chronic High-Calorie Diet.

A major driver of obesity is the increasing palatability of processed foods. Although reward circuits promote the consumption of palatable food, their involvement in obesity remains unclear. The ventral pallidum (VP) is a key hub in the reward system that encodes the hedonic aspects of palatable food consumption and participates in various proposed feeding circuits. However, there is still no evidence for its involvement in developing diet-induced obesity. Here we examine, using male C57BL6/J mice and patch-clamp electrophysiology, how chronic high-fat high-sugar (HFHS) diet changes the physiology of the VP and whether mice that gain the most weight differ in their VP physiology from others. We found that 10-12 weeks of HFHS diet hyperpolarized and decreased the firing rate of VP neurons without a major change in synaptic inhibitory input. Within the HFHS group, the top 33% weight gainers (WGs) had a more hyperpolarized VP with longer latency to fire action potentials on depolarization compared with bottom 33% of weight gainers (i.e., non-weight gainers). WGs also showed synaptic potentiation of inhibitory inputs both at the millisecond and minute ranges. Moreover, we found that the tendency to potentiate the inhibitory inputs to the VP might exist in overeating mice even before exposure to HFHS, thus making it a potential property of being an overeater. These data point to the VP as a critical player in obesity and suggest that hyperpolarized membrane potential of, and potentiated inhibitory inputs to, VP neurons may play a significant role in promoting the overeating of palatable food.SIGNIFICANCE STATEMENT In modern world, where highly palatable food is readily available, overeating is often driven by motivational, rather than metabolic, needs. It is thus conceivable that reward circuits differ between obese and normal-weight individuals. But is such difference, if it exists, innate or does it develop with overeating? Here we reveal synaptic properties in the ventral pallidum, a central hub of reward circuits, that differ between mice that gain the most and the least weight when given unlimited access to highly palatable food. We show that these synaptic differences also exist without exposure to palatable food, potentially making them innate properties that render some more susceptible than others to overeat. Thus, the propensity to overeat may have a strong innate component embedded in reward circuits.

Accumbens neuroimmune signaling and dysregulation of astrocytic glutamate transport underlie conditioned nicotine-seeking behavior.

Nicotine self-administration is associated with decreased expression of the glial glutamate transporter (GLT-1) and the cystine-glutamate exchange protein xCT within the nucleus accumbens core (NAcore). N-acetylcysteine (NAC) has been shown to restore these proteins in a rodent model of drug addiction and relapse. However, the specific molecular mechanisms driving its inhibitory effects on cue-induced nicotine reinstatement are unknown. Here, we confirm that extinction of nicotine-seeking behavior is associated with impaired NAcore GLT-1 function and expression and demonstrates that reinstatement of nicotine seeking rapidly enhances membrane fraction GLT-1 expression. Extinction and cue-induced reinstatement of nicotine seeking was also associated with increased tumor necrosis factor alpha (TNFα) and decreased glial fibrillary acidic protein (GFAP) expression in the NAcore. NAC treatment (100 mg/kg/day, i.p., for 5 d) inhibited cue-induced nicotine seeking and suppressed AMPA to NMDA current ratios, suggesting that NAC reduces NAcore postsynaptic excitability. In separate experiments, rats received NAC and an antisense vivo-morpholino to selectively suppress GLT-1 expression in the NAcore during extinction and were subsequently tested for cue-induced reinstatement of nicotine seeking. NAC treatment rescued NAcore GLT-1 expression and attenuated cue-induced nicotine seeking, which was blocked by GLT-1 antisense. NAC also reduced TNFα expression in the NAcore. Viral manipulation of the NF-κB pathway, which is downstream of TNFα, revealed that cue-induced nicotine seeking is regulated by NF-κB pathway signaling in the NAcore independent of GLT-1 expression. Ultimately, these results are the first to show that immunomodulatory mechanisms may regulate known nicotine-induced alterations in glutamatergic plasticity that mediate cue-induced nicotine-seeking behavior.

Chronic calorie-dense diet drives differences in motivated food seeking between obesity-prone and resistant mice.

Obesity results from overconsumption of energy, partly because of the inability to refrain from highly palatable rewarding foods. Even though palatable food is available to everyone, only a fraction of the population develops obesity. We previously showed that following chronic exposure to highly palatable food animals that gained the most weight also showed addictive-like motivation to seek for palatable food. An important question remains-is this extreme, addictive-like, motivation to consume palatable food the cause or the consequence of diet-induced obesity? Here, we show that obesity-prone (OP) mice exhibit higher motivation for palatable food consumption compared with obesity-resistant mice even before developing obesity, but that the full manifestation of this high motivation to eat is expressed only after chronic exposure to high-fat-high-sugar (HFHS) diet. HFHS diet also impairs performance in the operant food-seeking task selectively in OP mice, an impairment that persists even after 2 weeks of abstinence from HFHS food. Overall, our data suggest that while some aspects of food motivation are high in OP mice already before developing obesity, the chronic exposure to HFHS food accentuates it and drives the development of obesity.

Cocaine Dysregulates Dynorphin Modulation of Inhibitory Neurotransmission in the Ventral Pallidum in a Cell-Type-Specific Manner.

Cocaine-driven changes in the modulation of neurotransmission by neuromodulators are poorly understood. The ventral pallidum (VP) is a key structure in the reward system, in which GABA neurotransmission is regulated by opioid neuropeptides, including dynorphin. However, it is not known whether dynorphin acts differently on different cell types in the VP and whether its effects are altered by withdrawal from cocaine. Here, we trained wild-type, D1-Cre, A2A-Cre, or vGluT2-Cre:Ai9 male and female mice in a cocaine conditioned place preference protocol followed by 2 weeks of abstinence, and then recorded GABAergic synaptic input evoked either electrically or optogenetically onto identified VP neurons before and after applying dynorphin. We found that after cocaine CPP and abstinence dynorphin attenuated inhibitory input to VPGABA neurons through a postsynaptic mechanism. This effect was absent in saline mice. Furthermore, this effect was seen specifically on the inputs from nucleus accumbens medium spiny neurons expressing either the D1 or the D2 dopamine receptor. Unlike its effect on VPGABA neurons, dynorphin surprisingly potentiated the inhibitory input on VPvGluT2 neurons, but this effect was abolished after cocaine CPP and abstinence. Thus, dynorphin has contrasting influences on GABA input to VPGABA and VPvGluT2 neurons and these influences are affected differentially by cocaine CPP and abstinence. Collectively, our data suggest a role for dynorphin in withdrawal through its actions in the VP. As VPGABA and VPvGluT2 neurons have contrasting effects on drug-seeking behavior, our data may indicate a complex role for dynorphin in withdrawal from cocaine.SIGNIFICANCE STATEMENT The ventral pallidum consists mainly of GABAergic reward-promoting neurons, but it also encloses a subgroup of aversion-promoting glutamatergic neurons. Dynorphin, an opioid neuropeptide abundant in the ventral pallidum, shows differential modulation of GABA input to GABAergic and glutamatergic pallidal neurons and may therefore affect both the rewarding and aversive aspects of withdrawal. Indeed, abstinence after repeated exposure to cocaine alters dynorphin actions in a cell-type-specific manner; after abstinence dynorphin suppresses the inhibitory drive on the "rewarding" GABAergic neurons but ceases to modulate the inhibitory drive on the "aversive" glutamatergic neurons. This reflects a complex role for dynorphin in cocaine reward and abstinence.

Projection-Specific Potentiation of Ventral Pallidal Glutamatergic Outputs after Abstinence from Cocaine.

The ventral pallidum (VP) is a central node in the reward system that is strongly implicated in reward and addiction. Although the majority of VP neurons are GABAergic and encode reward, recent studies revealed a novel glutamatergic neuronal population in the VP [VP neurons expressing the vesicular glutamate transporter 2 (VPVGluT2)], whose activation generates aversion. Withdrawal from drugs has been shown to induce drastic synaptic changes in neuronal populations associated with reward, such as the ventral tegmental area (VTA) or nucleus accumbens neurons, but less is known about cocaine-induced synaptic changes in neurons classically linked with aversion. Here, we demonstrate that VPVGluT2 neurons contact different targets with different intensities, and that cocaine conditioned place preference (CPP) training followed by abstinence selectively potentiates their synapses on targets that encode aversion. Using whole-cell patch-clamp recordings combined with optogenetics in male and female transgenic mice, we show that VPVGluT2 neurons preferentially contact aversion-related neurons, such as lateral habenula neurons and VTA GABAergic neurons, with minor input to reward-related neurons, such as VTA dopamine and VP GABA neurons. Moreover, after cocaine CPP and abstinence, the VPVGluT2 input to the aversion-related structures is potentiated, whereas the input to the reward-related structures is depressed. Thus, cocaine CPP followed by abstinence may allow VPVGluT2 neurons to recruit aversion-related targets more readily and therefore be part of the mechanism underlying the aversive symptoms seen after withdrawal.SIGNIFICANCE STATEMENT The biggest problem in drug addiction is the high propensity to relapse. One central driver for relapse events is the negative aversive symptoms experienced by addicts during withdrawal. In this work, we propose a possible mechanism for the intensification of aversive feelings after withdrawal that involves the glutamatergic neurons of the ventral pallidum. We show not only that these neurons are most strongly connected to aversive targets, such as the lateral habenula, but also that, after abstinence, their synapses on aversive targets are strengthened, whereas the synapses on other rewarding targets are weakened. These data illustrate how after abstinence from cocaine, aversive pathways change in a manner that may contribute to relapse.

Loss of Plasticity in the D2-Accumbens Pallidal Pathway Promotes Cocaine Seeking.

Distinct populations of D1- and D2-dopamine receptor-expressing medium spiny neurons (D1-/D2-MSNs) comprise the nucleus accumbens, and activity in D1-MSNs promotes, whereas activity in D2-MSNs inhibits, motivated behaviors. We used chemogenetics to extend D1-/D2-MSN cell specific regulation to cue-reinstated cocaine seeking in a mouse model of self-administration and relapse, and found that either increasing activity in D1-MSNs or decreasing activity in D2-MSNs augmented cue-induced reinstatement. Both D1- and D2-MSNs provide substantial GABAergic innervation to the ventral pallidum, and chemogenetic inhibition of ventral pallidal neurons blocked the augmented reinstatement elicited by chemogenetic regulation of either D1- or D2-MSNs. Because D1- and D2-MSNs innervate overlapping populations of ventral pallidal neurons, we next used optogenetics to examine whether changes in synaptic plasticity in D1- versus D2-MSN GABAergic synapses in the ventral pallidum could explain the differential regulation of VP activity. In mice trained to self-administer cocaine, GABAergic LTD was abolished in D2-, but not in D1-MSN synapses. A µ opioid receptor antagonist restored GABA currents in D2-, but not D1-MSN synapses of cocaine-trained mice, indicating that increased enkephalin tone on presynaptic µ opioid receptors was responsible for occluding the LTD. These results identify a behavioral function for D1-MSN innervation of the ventral pallidum, and suggest that losing LTDGABA in D2-MSN, but not D1-MSN input to ventral pallidum may promote cue-induced reinstatement of cocaine-seeking. SIGNIFICANCE STATEMENT: More than 90% of ventral striatum is composed of two cell types, those expressing dopamine D1 or D2 receptors, which exert opposing roles on motivated behavior. Both cell types send GABAergic projections to the ventral pallidum and were found to differentially promote cue-induced reinstatement of cocaine seeking via the ventral pallidum. Furthermore, after cocaine self-administration, synaptic plasticity was selectively lost in D2, but not D1 inputs to the ventral pallidum. The selective impairment in D2 afferents may promote the influence of D1 inputs to drive relapse to cocaine seeking.

Optogenetic inhibition of cortical afferents in the nucleus accumbens simultaneously prevents cue-induced transient synaptic potentiation and cocaine-seeking behavior.

Animal models of relapse reveal that the motivation to seek drug is regulated by enduring morphological and physiological changes in the nucleus accumbens, as well as transient synaptic potentiation in the accumbens core (NAcore) that parallels drug-seeking behavior. The current study sought to examine the link between the behavioral and synaptic consequences of cue-induced cocaine seeking by optically silencing glutamatergic afferents to the NAcore from the prelimbic cortex (PL). Adeno-associated virus coding for the inhibitory opsin archaerhodopsin was microinjected into PL, and optical fibers were targeted to NAcore. Animals were trained to self-administer cocaine followed by extinction training, and then underwent cue-induced reinstatement in the presence or absence of 15 min of optically induced inhibition of PL fibers in NAcore. Inhibiting the PL-to-NAcore projection blocked reinstated behavior and was paralleled by decreased dendritic spine head diameter and AMPA/NMDA ratio relative to sham-laser control rats. Interestingly, while spine density was elevated after extinction training, no further effects were observed by cued reinstatement or optical inhibition. These findings validate the critical role for PL afferents to the NAcore in simultaneously regulating both reinstated behavior and the associated transient synaptic potentiation.

Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections.

It is widely accepted that D1 dopamine receptor-expressing striatal neurons convey their information directly to the output nuclei of the basal ganglia, whereas D2-expressing neurons do so indirectly via pallidal neurons. Combining optogenetics and electrophysiology, we found that this architecture does not apply to mouse nucleus accumbens projections to the ventral pallidum. Thus, current thinking attributing D1 and D2 selectivity to accumbens projections akin to dorsal striatal pathways needs to be reconsidered.

Synaptic plasticity mediating cocaine relapse requires matrix metalloproteinases.

Relapse to cocaine use necessitates remodeling excitatory synapses in the nucleus accumbens and synaptic reorganization requires matrix metalloproteinase (MMP) degradation of the extracellular matrix proteins. We found enduring increases in MMP-2 activity in rats after withdrawal from self-administered cocaine and transient increases in MMP-9 during cue-induced cocaine relapse. Cue-induced heroin and nicotine relapse increased MMP activity, and increased MMP activity was required for both cocaine relapse and relapse-associated synaptic plasticity.

α2δ-1 signaling in nucleus accumbens is necessary for cocaine-induced relapse.

Relapse to cocaine seeking is associated with potentiated excitatory synapses in nucleus accumbens. α2δ-1 is an auxiliary subunit of voltage-gated calcium channels that affects calcium-channel trafficking and kinetics, initiates extracellular signaling cascades, and promotes excitatory synaptogenesis. Previous data demonstrate that repeated exposure to alcohol, nicotine, methamphetamine, and morphine upregulates α2δ-1 in reward-related brain regions, but it was unclear whether this alteration generalized to cocaine. Here, we show that α2δ-1 protein was increased in nucleus accumbens after cocaine self-administration and extinction compared with saline controls. Furthermore, the endogenous ligand thrombospondin-1, responsible for the synaptogenic properties of the α2δ-1 receptor, was likewise elevated. Using whole-cell patch-clamp recordings of EPSCs in nucleus accumbens, we demonstrated that gabapentin, a specific α2δ-1 antagonist, preferentially reduced the amplitude and increased the paired-pulse ratio of EPSCs evoked by electrical stimulation in slices from cocaine-experienced rats compared with controls. In vivo, gabapentin microinjected in the nucleus accumbens core attenuated cocaine-primed but not cue-induced reinstatement. Importantly, gabapentin's effects on drug seeking were not due to a general depression of spontaneous or cocaine-induced locomotor activity. Moreover, gabapentin had no effect on reinstatement of sucrose seeking. These data indicate that α2δ-1 contributes specifically to cocaine-reinstated drug seeking, and identifies this protein as a target for the development of cocaine relapse medications. These results also inform ongoing discussion in the literature regarding efficacy of gabapentin as a candidate addiction therapy.

Cocaine dysregulates opioid gating of GABA neurotransmission in the ventral pallidum.

The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and µ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.

Rapid, transient synaptic plasticity in addiction.

Chronic use of addictive drugs produces enduring neuroadaptations in the corticostriatal glutamatergic brain circuitry. The nucleus accumbens (NAc), which integrates cortical information and regulates goal-directed behavior, undergoes long-term morphological and electrophysiological changes that may underlie the increased susceptibility for relapse in drug-experienced individuals even after long periods of withdrawal. Additionally, it has recently been shown that exposure to cues associated with drug use elicits rapid and transient morphological and electrophysiological changes in glutamatergic synapses in the NAc. This review highlights these dynamic drug-induced changes in this pathway that are specific to a drug seeking neuropathology, as well as how these changes impair normal information processing and thereby contribute to the uncontrollable motivation to relapse. Future directions for relapse prevention and pharmacotherapeutic targeting of the rapid, transient synaptic plasticity in relapse are discussed. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Optogenetic evidence that pallidal projections, not nigral projections, from the nucleus accumbens core are necessary for reinstating cocaine seeking.

The core subcompartment of the nucleus accumbens (NAcore) contributes significantly to behavioral responses following motivationally relevant stimuli, including drug-induced, stress-induced, and cue-induced reinstatement of cocaine seeking. Projections from NAcore that could carry information necessary to initiate reinstated cocaine seeking include outputs via the indirect pathway to the dorsolateral subcompartment of the ventral pallidum (dlVP) and through the direct pathway to the medial substantia nigra (SN). Here we used an optogenetic strategy to determine whether the dlVP or nigral projections from the NAcore are necessary for cocaine seeking initiated by a cocaine and conditioned cue combination in rats extinguished from cocaine self-administration. Rats were pretreated in the NAcore with an adeno-associated virus expressing the inhibitory opsin archaerhodopsin, and fiber-optic cannulae were implanted above the indirect pathway axon terminal field in the dlVP, or the direct pathway terminal field in the SN. Inhibiting the indirect pathway to the dlVP, but not the direct pathway to the SN, prevented cocaine-plus-cue-induced reinstatement. We also examined projections back to the NAcore from the ventral tegmental area (VTA) and dlVP. Inhibiting the dlVP to NAcore projection did not alter, while inhibiting VTA afferents abolished reinstated cocaine seeking. Localization of green fluorescent protein reporter expression and whole-cell patch electrophysiology were used to verify opsin expression. These data reveal a circuit involving activation of VTA inputs to the NAcore and NAcore projections through the indirect pathway to the dlVP as critical for cocaine-plus-cue-induced reinstatement of cocaine seeking.