Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types.

The ventral pallidum (VP) contains GABA and glutamate neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the mechanisms by which VP cell types shape VTA activity and drive behavior. Here, we found that both VP GABA and glutamate neurons were activated during approach to reward or by delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine and glutamate neurons. Remarkably, stimulation-evoked activation was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject's own action. Conversely, VP glutamate neurons activated VTA GABA, as well as dopamine and glutamate neurons, despite driving aversion. However, VP glutamate neurons evoked dopamine in aversion-associated ventromedial nucleus accumbens (NAc), but reduced dopamine release in reward-associated dorsomedial NAc. These findings show how heterogeneous VP projections to VTA can be engaged to shape approach and avoidance behaviors.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any DA neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute DA neurodegeneration.

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta.

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of these neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter VMAT2, vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2 neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

Postnatal Phencyclidine-Induced Deficits in Decision Making Are Ameliorated by Optogenetic Inhibition of Ventromedial Orbitofrontal Cortical Glutamate Neurons.

Background: The orbitofrontal cortex (OFC) is essential for decision making, and functional disruptions within the OFC are evident in schizophrenia. Postnatal phencyclidine (PCP) administration in rats is a neurodevelopmental manipulation that induces schizophrenia-relevant cognitive impairments. We aimed to determine whether manipulating OFC glutamate cell activity could ameliorate postnatal PCP-induced deficits in decision making. Methods: Male and female Wistar rats (n = 110) were administered saline or PCP on postnatal days 7, 9, and 11. In adulthood, we expressed YFP (yellow fluorescent protein) (control), ChR2 (channelrhodopsin-2) (activation), or eNpHR 3.0 (enhanced halorhodopsin) (inhibition) in glutamate neurons within the ventromedial OFC (vmOFC). Rats were tested on the probabilistic reversal learning task once daily for 20 days while we manipulated the activity of vmOFC glutamate cells. Behavioral performance was analyzed using a Q-learning computational model of reinforcement learning. Results: Compared with saline-treated rats expressing YFP, PCP-treated rats expressing YFP completed fewer reversals, made fewer win-stay responses, and had lower learning rates. We induced similar performance impairments in saline-treated rats by activating vmOFC glutamate cells (ChR2). Strikingly, PCP-induced performance deficits were ameliorated when the activity of vmOFC glutamate cells was inhibited (halorhodopsin). Conclusions: Postnatal PCP-induced deficits in decision making are associated with hyperactivity of vmOFC glutamate cells. Thus, normalizing vmOFC activity may represent a potential therapeutic target for decision-making deficits in patients with schizophrenia.

Mesoaccumbal glutamate neurons drive reward via glutamate release but aversion via dopamine co-release.

Ventral tegmental area (VTA) projections to the nucleus accumbens (NAc) drive reward-related motivation. Although dopamine neurons are predominant, a substantial glutamatergic projection is also present, and a subset of these co-release both dopamine and glutamate. Optogenetic stimulation of VTA glutamate neurons not only supports self-stimulation but can also induce avoidance behavior, even in the same assay. Here, we parsed the selective contribution of glutamate or dopamine co-release from VTA glutamate neurons to reinforcement and avoidance. We expressed channelrhodopsin-2 (ChR2) in mouse VTA glutamate neurons in combination with CRISPR-Cas9 to disrupt either the gene encoding vesicular glutamate transporter 2 (VGLUT2) or tyrosine hydroxylase (Th). Selective disruption of VGLUT2 abolished optogenetic self-stimulation but left real-time place avoidance intact, whereas CRISPR-Cas9 deletion of Th preserved self-stimulation but abolished place avoidance. Our results demonstrate that glutamate release from VTA glutamate neurons is positively reinforcing but that dopamine release from VTA glutamate neurons can induce avoidance behavior.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types.

The ventral pallidum (VP) contains GABA and glutamate (Glut) neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the cell-type-specific mechanisms by which VP projections to VTA drive behavior. Here, we found that both VP GABA and Glut neurons were activated during approach to reward or delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine (DA) and glutamate neurons. Remarkably, this cell-type-specific recruitment was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject's own action. Conversely, VP Glut neurons activated VTA GABA, as well as DA and Glut neurons, despite driving aversion. However, VP Glut neurons evoked DA in reward-associated ventromedial nucleus accumbens (NAc), but reduced DA in aversion-associated dorsomedial NAc. These findings show how heterogeneous VP cell types can engage VTA cell types to shape approach and avoidance behaviors.

Neurotensin receptor 1-biased ligand attenuates neurotensin-mediated excitation of ventral tegmental area dopamine neurons and dopamine release in the nucleus accumbens.

Strong expression of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) in ventral tegmental area (VTA) dopamine (DA) neurons and terminals makes it an attractive target to modulate DA neuron activity and normalize DA-related pathologies. Recent studies have identified a novel class of NTR1 ligand that shows promising effects in preclinical models of addiction. A lead molecule, SBI-0654553 (SBI-553), can act as a positive allosteric modulator of NTR1 ß-arrestin recruitment while simultaneously antagonizing NTR1 Gq protein signaling. Using cell-attached recordings from mouse VTA DA neurons we discovered that, unlike neurotensin (NT), SBI-553 did not independently increase spontaneous firing. Instead, SBI-553 blocked the NT-mediated increase in firing. SBI-553 also antagonized the effects of NT on dopamine D2 auto-receptor signaling, potentially through its inhibitory effects on G-protein signaling. We also measured DA release directly, using fast-scan cyclic voltammetry in the nucleus accumbens and observed antagonist effects of SBI-553 on an NT-induced increase in DA release. Further, in vivo administration of SBI-553 did not notably change basal or cocaine-evoked DA release measured in NAc using fiber photometry. Overall, these results indicate that SBI-553 blunts NT's effects on spontaneous DA neuron firing, D2 auto-receptor function, and DA release, without independently affecting these measures. In the presence of NT, SBI-553 has an inhibitory effect on mesolimbic DA activity, which could contribute to its efficacy in animal models of psychostimulant use.

Modulation of ventromedial orbitofrontal cortical glutamatergic activity affects the explore-exploit balance and influences value-based decision-making.

The balance between exploration and exploitation is essential for decision-making. The present study investigated the role of ventromedial orbitofrontal cortex (vmOFC) glutamate neurons in mediating value-based decision-making by first using optogenetics to manipulate vmOFC glutamate activity in rats during a probabilistic reversal learning (PRL) task. Rats that received vmOFC activation during informative feedback completed fewer reversals and exhibited reduced reward sensitivity relative to rats. Analysis with a Q-learning computational model revealed that increased vmOFC activity did not affect the learning rate but instead promoted maladaptive exploration. By contrast, vmOFC inhibition increased the number of completed reversals and increased exploitative behavior. In a separate group of animals, calcium activity of vmOFC glutamate neurons was recorded using fiber photometry. Complementing our results above, we found that suppression of vmOFC activity during the latter part of rewarded trials was associated with improved PRL performance, greater win-stay responding and selecting the correct choice on the next trial. These data demonstrate that excessive vmOFC activity during reward feedback disrupted value-based decision-making by increasing the maladaptive exploration of lower-valued options. Our findings support the premise that pharmacological interventions that normalize aberrant vmOFC glutamate activity during reward feedback processing may attenuate deficits in value-based decision-making.

Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease.

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson's disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving dopamine neurons from post-mortem Parkinson's disease individuals. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson's disease.

Disruption of VGLUT1 in Cholinergic Medial Habenula Projections Increases Nicotine Self-Administration.

Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections corelease glutamate, no direct evidence has demonstrated a role for this glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 [vesicular glutamate transporter 1 (VGLUT1)] mouse was generated and used to create conditional knock-out (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Loss of Slc17a7 expression in ventral MHb cholinergic neurons was validated using fluorescent in situ hybridization, and immunohistochemistry was used to demonstrate a corresponding reduction of VGLUT1 protein in cholinergic terminals in the IPN. We also used optogenetics-assisted electrophysiology to evoke EPSCs in IPN and observed a reduction of glutamatergic currents in the cKO, supporting the functional disruption of VGLUT1 in MHb to IPN synapses. cKO mice exhibited no gross phenotypic abnormalities and displayed normal thigmotaxis and locomotor behavior in the open-field assay. When trained to lever press for food, there was no difference between control and cKO. However, when tested in a nicotine self-administration procedure, we found that the loss of VGLUT1-mediated glutamate corelease led to increased responding for nicotine. These findings indicate that glutamate corelease from ventral MHb cholinergic neurons opposes nicotine self-administration, and provide additional support for targeting this synapse to develop potential treatments for nicotine addiction.

Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB.

Methods to enhance adult neurogenesis by reprogramming glial cells into neurons enable production of new neurons in the adult nervous system. Development of therapeutically viable approaches to induce new neurons is now required to bring this concept to clinical application. Here, we successfully generate new neurons in the cortex and dentate gyrus of the aged adult mouse brain by transiently suppressing polypyrimidine tract binding protein 1 using an antisense oligonucleotide delivered by a single injection into cerebral spinal fluid. Radial glial-like cells and other GFAP-expressing cells convert into new neurons that, over a 2-month period, acquire mature neuronal character in a process mimicking normal neuronal maturation. The new neurons functionally integrate into endogenous circuits and modify mouse behavior. Thus, generation of new neurons in the dentate gyrus of the aging brain can be achieved with a therapeutically feasible approach, thereby opening prospects for production of neurons to replace those lost to neurodegenerative disease.

Ventral Pallidum GABA Neurons Mediate Motivation Underlying Risky Choice.

Pursuing rewards while avoiding danger is an essential function of any nervous system. Here, we examine a new mechanism helping rats negotiate the balance between risk and reward when making high-stakes decisions. Specifically, we focus on GABA neurons within an emerging mesolimbic circuit nexus: the ventral pallidum (VP). These neurons play a distinct role from other VP neurons in simple motivated behaviors in mice, but their role in more complex motivated behaviors is unknown. Here, we interrogate the behavioral functions of VPGABA neurons in male and female transgenic GAD1:Cre rats (and WT littermates), using a reversible chemogenetic inhibition approach. Using a behavioral assay of risky decision-making, and of the food-seeking and shock-avoidance components of this task, we show that engaging inhibitory Gi/o signaling specifically in VPGABA neurons suppresses motivation to pursue highly salient palatable foods, and possibly also motivation to avoid being shocked. In contrast, inhibiting these neurons did not affect seeking of low-value food, free consumption of palatable food, or unconditioned affective responses to shock. Accordingly, when rats considered whether to pursue food despite potential for shock in a risky decision-making task, inhibiting VPGABA neurons caused them to more readily select a small but safe reward over a large but dangerous one, an effect not seen in the absence of shock threat. Together, results indicate that VPGABA neurons are critical for high-stakes adaptive responding that is necessary for survival, but which may also malfunction in psychiatric disorders.SIGNIFICANCE STATEMENT In a dynamic world, it is essential to implement appropriate behaviors under circumstances involving rewards, threats, or both. Here, we demonstrate a crucial role for VPGABA neurons in high-stakes motivated behavior of several types. We show that this VPGABA role in motivation impacts decision-making, as inhibiting these neurons yields a conservative, risk-averse strategy not seen when the task is performed without threat of shock. These new roles for VPGABA neurons in behavior may inform future strategies for treating addiction, and other disorders of maladaptive decision-making.

Vesicular glutamate transporter modulates sex differences in dopamine neuron vulnerability to age-related neurodegeneration.

Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex- and age-related differences in DA neuron vulnerability using the genetically tractable Drosophila model. We found sex differences in age-related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. We discovered that dynamic changes in DA neuron VGLUT expression mediate these age- and sex-related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, we showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age-related DA neurodegeneration. Finally, in mice, we showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age- and PD-related neurodegeneration.

Mechanism for differential recruitment of orbitostriatal transmission during actions and outcomes following chronic alcohol exposure.

Psychiatric disease often produces symptoms that have divergent effects on neural activity. For example, in drug dependence, dysfunctional value-based decision-making and compulsive-like actions have been linked to hypo- and hyperactivity of orbital frontal cortex (OFC)-basal ganglia circuits, respectively; however, the underlying mechanisms are unknown. Here we show that alcohol-exposed mice have enhanced activity in OFC terminals in dorsal striatum (OFC-DS) associated with actions, but reduced activity of the same terminals during periods of outcome retrieval, corresponding with a loss of outcome control over decision-making. Disrupted OFC-DS terminal activity was due to a dysfunction of dopamine-type 1 receptors on spiny projection neurons (D1R SPNs) that resulted in increased retrograde endocannabinoid signaling at OFC-D1R SPN synapses reducing OFC-DS transmission. Blocking CB1 receptors restored OFC-DS activity in vivo and rescued outcome-based control over decision-making. These findings demonstrate a circuit-, synapse-, and computation-specific mechanism gating OFC activity in alcohol-exposed mice.

Genetic Probe for Visualizing Glutamatergic Synapses and Vesicles by 3D Electron Microscopy.

Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neuron's chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block-face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.

Activation of Subthalamic Nucleus Stop Circuit Disrupts Cognitive Performance.

Much evidence supports a fundamental role for the subthalamic nucleus (STN) in rapidly stopping behavior when a stop signal or surprising event occurs, but the extent to which the STN may be involved in stopping cognitive processes is less clear. Here, we used an optogenetic approach to control STN activity in a delayed-match-to-position (DMTP) task where mice had to recall a response location after a delay. We first demonstrated that a surprising event impaired performance by both slowing the latency to respond and increasing the rate of errors. We next showed that these effects could be mimicked by brief optogenetic activation of the STN. Further, inhibiting STN during surprise blocked surprise-induced slowing, although without changing surprise-induced errors. These data are consistent with the hypothesis that STN is recruited by surprise to slow responding and that this can also interrupt cognitive processes. Under normal conditions STN-mediated stopping of behavior may slow or stop ongoing cognition to facilitate cognitive reorienting and adaptive responses to unexpected sensory information, but when malfunctioning, it could produce pathologies related to over-rigidity or increased distractibility.

VTA Glutamate Neuron Activity Drives Positive Reinforcement Absent Dopamine Co-release.

Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used optogenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis, was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA mechanism by which VTA activity can support reward-seeking behaviors.

Circulating Triglycerides Gate Dopamine-Associated Behaviors through DRD2-Expressing Neurons.

Energy-dense food alters dopaminergic (DA) transmission in the mesocorticolimbic (MCL) system and can promote reward dysfunctions, compulsive feeding, and weight gain. Yet the mechanisms by which nutrients influence the MCL circuitry remain elusive. Here, we show that nutritional triglycerides (TGs), a conserved post-prandial metabolic signature among mammals, can be metabolized within the MCL system and modulate DA-associated behaviors by gating the activity of dopamine receptor subtype 2 (DRD2)-expressing neurons through a mechanism that involves the action of the lipoprotein lipase (LPL). Further, we show that in humans, post-prandial TG excursions modulate brain responses to food cues in individuals carrying a genetic risk for reduced DRD2 signaling. Collectively, these findings unveil a novel mechanism by which dietary TGs directly alter signaling in the reward circuit to regulate behavior, thereby providing a new mechanistic basis by which energy-rich diets may lead to (mal)adaptations in DA signaling that underlie reward deficit and compulsive behavior.

Ventral pallidum is essential for cocaine relapse after voluntary abstinence in rats.

Addiction is a chronic relapsing disorder, and during recovery many people experience several relapse events as they attempt to voluntarily abstain from drug. New preclinical relapse models have emerged that capture this common human experience, and mounting evidence indicates that resumption of drug seeking after voluntary abstinence recruits neural circuits distinct from those recruited during reinstatement after experimenter-imposed abstinence, or abstinence due to extinction training. Ventral pallidum (VP), a key limbic node involved in drug seeking, has well-established roles in conventional reinstatement models tested following extinction training, but it is unclear whether this region also participates in more translationally relevant models of relapse. Here we show that chemogenetic inhibition of VP neurons decreased cocaine-, context-, and cue-induced relapse tested after voluntary, punishment-induced abstinence. This effect was strongest in the most compulsive, punishment-resistant rats, and reinstatement was associated with neural activity in anatomically defined VP subregions. VP inhibition also attenuated the propensity of rats to display "abortive lever pressing," a species-typical risk assessment behavior seen here during punished drug taking, likely resulting from concurrent approach and avoidance motivations. These results indicate that VP, unlike other connected limbic brain regions, is essential for resumption of drug seeking after voluntary abstinence. Since VP inhibition effects were strongest in the most compulsively cocaine-seeking individuals, this may also indicate that VP plays a particularly important role in the most pathological, addiction-like behavior, making it an attractive target for future therapeutic interventions.