Elucidating an Affective Pain Circuit that Creates a Threat Memory.

Animals learn to avoid harmful situations by associating a neutral stimulus with a painful one, resulting in a stable threat memory. In mammals, this form of learning requires the amygdala. Although pain is the main driver of aversive learning, the mechanism that transmits pain signals to the amygdala is not well resolved. Here, we show that neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus are critical for relaying pain signals to the central nucleus of amygdala and that this pathway may transduce the affective motivational aspects of pain. Genetic silencing of CGRP neurons blocks pain responses and memory formation, whereas their optogenetic stimulation produces defensive responses and a threat memory. The pain-recipient neurons in the central amygdala expressing CGRP receptors are also critical for establishing a threat memory. The identification of the neural circuit conveying affective pain signals may be pertinent for treating pain conditions with psychiatric comorbidities.

Circuit coordination of opposing neuropeptide and neurotransmitter signals.

Fast-acting neurotransmitters and slow, modulatory neuropeptides are co-released from neurons in the central nervous system, albeit from distinct synaptic vesicles1. The mechanisms of how co-released neurotransmitters and neuropeptides that have opposing actions-for example, stimulatory versus inhibitory-work together to exert control of neural circuit output remain unclear. This has been difficult to resolve owing to the inability to selectively isolate these signalling pathways in a cell- and circuit-specific manner. Here we developed a genetic-based anatomical disconnect procedure that utilizes distinct DNA recombinases to independently facilitate CRISPR-Cas9 mutagenesis2 of neurotransmitter- and neuropeptide-related genes in distinct cell types in two different brain regions simultaneously. We demonstrate that neurons within the lateral hypothalamus that produce the stimulatory neuropeptide neurotensin and the inhibitory neurotransmitter GABA (γ-aminobutyric acid) utilize these signals to coordinately activate dopamine-producing neurons of the ventral tegmental area. We show that GABA release from lateral hypothalamus neurotensin neurons inhibits GABA neurons within the ventral tegmental area, disinhibiting dopamine neurons and causing a rapid rise in calcium, whereas neurotensin directly generates a slow inactivating calcium signal in dopamine neurons that is dependent on the expression of neurotensin receptor 1 (Ntsr1). We further show that these two signals work together to regulate dopamine neuron responses to maximize behavioural responding. Thus, a neurotransmitter and a neuropeptide with opposing signals can act on distinct timescales through different cell types to enhance circuit output and optimize behaviour.

An endogenous opioid circuit determines state-dependent reward consumption.

µ-Opioid peptide receptor (MOPR) stimulation alters respiration, analgesia and reward behaviour, and can induce substance abuse and overdose1-3. Despite its evident importance, the endogenous mechanisms for MOPR regulation of consummatory behaviour have remained unknown4. Here we report that endogenous MOPR regulation of reward consumption in mice acts through a specific dorsal raphe to nucleus accumbens projection. MOPR-mediated inhibition of raphe terminals is necessary and sufficient to determine consummatory response, while select enkephalin-containing nucleus accumbens ensembles are engaged prior to reward consumption, suggesting that local enkephalin release is the source of the endogenous MOPR ligand. Selective modulation of nucleus accumbens enkephalin neurons and CRISPR-Cas9-mediated disruption of enkephalin substantiate this finding. These results isolate a fundamental endogenous opioid circuit for state-dependent consumptive behaviour and suggest alternative mechanisms for opiate modulation of reward.

Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.

Pathogenic gating pore current conducted by autism-related mutations in the NaV1.2 brain sodium channel.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social and communication deficits and repetitive behaviors. The genetic heterogeneity of ASD presents a challenge to the development of an effective treatment targeting the underlying molecular defects. ASD gating charge mutations in the KCNQ/KV7 potassium channel cause gating pore currents (Igp) and impair action potential (AP) firing of dopaminergic neurons in brain slices. Here, we investigated ASD gating charge mutations of the voltage-gated SCN2A/NaV1.2 brain sodium channel, which ranked high among the ion channel genes with mutations in individuals with ASD. Our results show that ASD mutations in the gating charges R2 in Domain-II (R853Q), and R1 (R1626Q) and R2 (R1629H) in Domain-IV of NaV1.2 caused Igp in the resting state of ~0.1% of the amplitude of central pore current. The R1626Q mutant also caused significant changes in the voltage dependence of fast inactivation, and the R1629H mutant conducted proton-selective Igp. These potentially pathogenic Igp were exacerbated by the absence of the extracellular Mg2+ and Ca2+. In silico simulation of the effects of these mutations in a conductance-based single-compartment cortical neuron model suggests that the inward Igp reduces the time to peak for the first AP in a train, increases AP rates during a train of stimuli, and reduces the interstimulus interval between consecutive APs, consistent with increased neural excitability and altered input/output relationships. Understanding this common pathophysiological mechanism among different voltage-gated ion channels at the circuit level will give insights into the underlying mechanisms of ASD.

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

Functional circuit architecture underlying parental behaviour.

Parenting is essential for the survival and wellbeing of mammalian offspring. However, we lack a circuit-level understanding of how distinct components of this behaviour are coordinated. Here we investigate how galanin-expressing neurons in the medial preoptic area (MPOAGal) of the hypothalamus coordinate motor, motivational, hormonal and social aspects of parenting in mice. These neurons integrate inputs from a large number of brain areas and the activation of these inputs depends on the animal's sex and reproductive state. Subsets of MPOAGal neurons form discrete pools that are defined by their projection sites. While the MPOAGal population is active during all episodes of parental behaviour, individual pools are tuned to characteristic aspects of parenting. Optogenetic manipulation of MPOAGal projections mirrors this specificity, affecting discrete parenting components. This functional organization, reminiscent of the control of motor sequences by pools of spinal cord neurons, provides a new model for how discrete elements of a social behaviour are generated at the circuit level.

Autism-associated mutations in KV7 channels induce gating pore current.

Autism spectrum disorder (ASD) adversely impacts >1% of children in the United States, causing social interaction deficits, repetitive behaviors, and communication disorders. Genetic analysis of ASD has advanced dramatically through genome sequencing, which has identified >500 genes with mutations in ASD. Mutations that alter arginine gating charges in the voltage sensor of the voltage-gated potassium (KV) channel KV7 (KCNQ) are among those frequently associated with ASD. We hypothesized that these gating charge mutations would induce gating pore current (also termed ω-current) by causing an ionic leak through the mutant voltage sensor. Unexpectedly, we found that wild-type KV7 conducts outward gating pore current through its native voltage sensor at positive membrane potentials, owing to a glutamine in the third gating charge position. In bacterial and human KV7 channels, gating charge mutations at the R1 and R2 positions cause inward gating pore current through the resting voltage sensor at negative membrane potentials, whereas mutation at R4 causes outward gating pore current through the activated voltage sensor at positive potentials. Remarkably, expression of the KV7.3/R2C ASD-associated mutation in vivo in midbrain dopamine neurons of mice disrupts action potential generation and repetitive firing. Overall, our results reveal native and mutant gating pore current in KV7 channels and implicate altered control of action potential generation by gating pore current through mutant KV7 channels as a potential pathogenic mechanism in autism.

Catecholaminergic Innervation of the Lateral Nucleus of the Cerebellum Modulates Cognitive Behaviors.

The cerebellum processes neural signals related to rewarding and aversive stimuli, suggesting that the cerebellum supports nonmotor functions in cognitive and emotional domains. Catecholamines are a class of neuromodulatory neurotransmitters well known for encoding such salient stimuli. Catecholaminergic modulation of classical cerebellar functions have been demonstrated. However, a role for cerebellar catecholamines in modulating cerebellar nonmotor functions is unknown. Using biochemical methods in male mice, we comprehensively mapped TH+ fibers throughout the entire cerebellum and known precerebellar nuclei. Using electrochemical (fast scan cyclic voltammetry), and viral/genetic methods to selectively delete Th in fibers innervating the lateral cerebellar nucleus (LCN), we interrogated sources and functional roles of catecholamines innervating the LCN, which is known for its role in supporting cognition. The LCN has the most TH+ fibers in cerebellum, as well as the most change in rostrocaudal expression among the cerebellar nuclei. Norepinephrine is the major catecholamine measured in LCN. Distinct catecholaminergic projections to LCN arise only from locus coeruleus, and a subset of Purkinje cells that are positive for staining of TH. LC stimulation was sufficient to produce catecholamine release in LCN. Deletion of Th in fibers innervating LCN (LCN-Th-cKO) resulted in impaired sensorimotor integration, associative fear learning, response inhibition, and working memory in LCN-Th-cKO mice. Strikingly, selective inhibition of excitatory LCN output neurons with inhibitory designer receptor exclusively activated by designer drugs led to facilitation of learning on the same working memory task impaired in LCN-Th-cKO mice. Collectively, these data demonstrate a role for LCN catecholamines in cognitive behaviors.SIGNIFICANCE STATEMENT Here, we report on interrogating sources and functional roles of catecholamines innervating the lateral nucleus of the cerebellum (LCN). We map and quantify expression of TH, the rate-limiting enzyme in catecholamine synthesis, in the entire cerebellar system, including several precerebellar nuclei. We used cyclic voltammetry and pharmacology to demonstrate sufficiency of LC stimulation to produce catecholamine release in LCN. We used advanced viral techniques to map and selectively KO catecholaminergic neurotransmission to the LCN, and characterized significant cognitive deficits related to this manipulation. Finally, we show that inhibition of excitatory LCN neurons with designer receptor exclusively activated by designer drugs, designed to mimic Gi-coupled catecholamine GPCR signaling, results in facilitation of a working memory task impaired in LCN-specific TH KO mice.

Repetitive blast mild traumatic brain injury increases ethanol sensitivity in male mice and risky drinking behavior in male combat veterans.

BACKGROUND: Mild traumatic brain injury (mTBI) is common in civilians and highly prevalent among military service members. mTBI can increase health risk behaviors (e.g., sensation seeking, impulsivity) and addiction risk (e.g., for alcohol use disorder (AUD)), but how mTBI and substance use might interact to promote addiction risk remains poorly understood. Likewise, potential differences in single vs. repetitive mTBI in relation to alcohol use/abuse have not been previously examined. METHODS: Here, we examined how a history of single (1×) or repetitive (3×) blast exposure (blast-mTBI) affects ethanol (EtOH)-induced behavioral and physiological outcomes using an established mouse model of blast-mTBI. To investigate potential translational relevance, we also examined self-report responses to the Alcohol Use Disorders Identification Test-Consumption questions (AUDIT-C), a widely used measure to identify potential hazardous drinking and AUD, and used a novel unsupervised machine learning approach to investigate whether a history of blast-mTBI affected drinking behaviors in Iraq/Afghanistan Veterans. RESULTS: Both single and repetitive blast-mTBI in mice increased the sedative properties of EtOH (with no change in tolerance or metabolism), but only repetitive blast potentiated EtOH-induced locomotor stimulation and shifted EtOH intake patterns. Specifically, mice exposed to repetitive blasts showed increased consumption "front-loading" (e.g., a higher rate of consumption during an initial 2-h acute phase of a 24-h alcohol access period and decreased total daily intake) during an intermittent 2-bottle choice condition. Examination of AUDIT-C scores in Iraq/Afghanistan Veterans revealed an optimal 3-cluster solution: "low" (low intake and low frequency), "frequent" (low intake and high frequency), and "risky" (high intake and high frequency), where Veterans with a history of blast-mTBI displayed a shift in cluster assignment from "frequent" to "risky," as compared to Veterans who were deployed to Iraq/Afghanistan but had no lifetime history of TBI. CONCLUSIONS: Together, these results offer new insight into how blast-mTBI may give increase AUD risk and highlight the increased potential for adverse health risk behaviors following repetitive blast-mTBI.

Genetic identification of a neural circuit that suppresses appetite.

Appetite suppression occurs after a meal and in conditions when it is unfavourable to eat, such as during illness or exposure to toxins. A brain region proposed to play a role in appetite suppression is the parabrachial nucleus, a heterogeneous population of neurons surrounding the superior cerebellar peduncle in the brainstem. The parabrachial nucleus is thought to mediate the suppression of appetite induced by the anorectic hormones amylin and cholecystokinin, as well as by lithium chloride and lipopolysaccharide, compounds that mimic the effects of toxic foods and bacterial infections, respectively. Hyperactivity of the parabrachial nucleus is also thought to cause starvation after ablation of orexigenic agouti-related peptide neurons in adult mice. However, the identities of neurons in the parabrachial nucleus that regulate feeding are unknown, as are the functionally relevant downstream projections. Here we identify calcitonin gene-related peptide-expressing neurons in the outer external lateral subdivision of the parabrachial nucleus that project to the laterocapsular division of the central nucleus of the amygdala as forming a functionally important circuit for suppressing appetite. Using genetically encoded anatomical, optogenetic and pharmacogenetic tools, we demonstrate that activation of these neurons projecting to the central nucleus of the amygdala suppresses appetite. In contrast, inhibition of these neurons increases food intake in circumstances when mice do not normally eat and prevents starvation in adult mice whose agouti-related peptide neurons are ablated. Taken together, our data demonstrate that this neural circuit from the parabrachial nucleus to the central nucleus of the amygdala mediates appetite suppression in conditions when it is unfavourable to eat. This neural circuit may provide targets for therapeutic intervention to overcome or promote appetite.

Reversal of Alcohol-Induced Dysregulation in Dopamine Network Dynamics May Rescue Maladaptive Decision-making.

Alcohol is the most commonly abused substance among adolescents, promoting the development of substance use disorders and compromised decision-making in adulthood. We have previously demonstrated, with a preclinical model in rodents, that adolescent alcohol use results in adult risk-taking behavior that positively correlates with phasic dopamine transmission in response to risky options, but the underlying mechanisms remain unknown. Here, we show that adolescent alcohol use may produce maladaptive decision-making through a disruption in dopamine network dynamics via increased GABAergic transmission within the ventral tegmental area (VTA). Indeed, we find that increased phasic dopamine signaling after adolescent alcohol use is attributable to a midbrain circuit, including the input from the pedunculopontine tegmentum to the VTA. Moreover, we demonstrate that VTA dopamine neurons from adult rats exhibit enhanced IPSCs after adolescent alcohol exposure corresponding to decreased basal dopamine levels in adulthood that negatively correlate with risk-taking. Building on these findings, we develop a model where increased inhibitory tone on dopamine neurons leads to a persistent decrease in tonic dopamine levels and results in a potentiation of stimulus-evoked phasic dopamine release that may drive risky choice behavior. Based on this model, we take a pharmacological approach to the reversal of risk-taking behavior through normalization of this pattern in dopamine transmission. These results isolate the underlying circuitry involved in alcohol-induced maladaptive decision-making and identify a novel therapeutic target. SIGNIFICANCE STATEMENT: One of the primary problems resulting from chronic alcohol use is persistent, maladaptive decision-making that is associated with ongoing addiction vulnerability and relapse. Indeed, studies with the Iowa Gambling Task, a standard measure of risk-based decision-making, have reliably shown that alcohol-dependent individuals make riskier, more maladaptive choices than nondependent individuals, even after periods of prolonged abstinence. Using a preclinical model, in the current work, we identify a selective disruption in dopamine network dynamics that may promote maladaptive decision-making after chronic adolescent alcohol use and demonstrate its pharmacological reversal in adulthood. Together, these results highlight a novel neural mechanism underlying heightened risk-taking behavior in alcohol-dependent individuals and provide a potential therapeutic target for further investigation.

Overexpression of the type 1 adenylyl cyclase in the forebrain leads to deficits of behavioral inhibition.

The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition.

Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons.

The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(-/-) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects.

A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward.

Visualization of plasticity in fear-evoked calcium signals in midbrain dopamine neurons.

Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium indicator GCaMP3. We observed calcium transients in a subset of dopamine neurons to an unconditioned fear stimulus on the first day of Pavlovian fear conditioning. On the second day, calcium transients occurred in response to conditioned and unconditioned stimuli. These results demonstrate plasticity in dopamine neuron calcium signals and the occurrence of activity-dependent processes in these neurons during fear conditioning.

Genetic reconstruction of dopamine D1 receptor signaling in the nucleus accumbens facilitates natural and drug reward responses.

The dopamine D1 receptor (D1R) facilitates reward acquisition and its alteration leads to profound learning deficits. However, its minimal functional circuit requirement is unknown. Using conditional reconstruction of functional D1R signaling in D1R knock-out mice, we define distinct requirements of D1R in subregions of the nucleus accumbens (NAc) for specific dimensions of reward. We demonstrate that D1R expression in the core region of the NAc (NAc(Core)), but not the shell (NAc(Shell)), enhances selectively a unique form of pavlovian conditioned approach and mediates D1R-dependent cocaine sensitization. However, D1R expression in either the NAc(Core) or the NAc(Shell) improves instrumental responding for reward. In contrast, neither NAc(Core) nor NAc(Shell) D1R is sufficient to promote motivation to work for reward in a progressive ratio task or for motor learning. These results highlight dissociated circuit requirements of D1R for dopamine-dependent behaviors.

Balanced NMDA receptor activity in dopamine D1 receptor (D1R)- and D2R-expressing medium spiny neurons is required for amphetamine sensitization.

Signaling through N-methyl-D-aspartate-type glutamate receptors (NMDARs) is essential for the development of behavioral sensitization to psychostimulants such as amphetamine (AMPH). However, the cell type and brain region in which NMDAR signaling is required for AMPH sensitization remain unresolved. Here we use selective inactivation of Grin1, the gene encoding the essential NR1 subunit of NMDARs, in dopamine neurons or their medium spiny neuron (MSN) targets, to address this issue. We show that NMDAR signaling in dopamine neurons is not required for behavioral sensitization to AMPH. Conversely, removing NMDARs from MSNs that express the dopamine D1 receptor (D1R) significantly attenuated AMPH sensitization, and conditional, virus-mediated restoration of NR1 in D1R neurons in the nucleus accumbens (NAc) of these animals rescued sensitization. Interestingly, sensitization could also be restored by virus-mediated inactivation of NR1 in all remaining neurons in the NAc of animals lacking NMDARs on D1R neurons, or by removing NMDARs from all MSNs. Taken together, these data indicate that unbalanced loss of NMDAR signaling in D1R MSNs alone prevents AMPH sensitization, whereas a balanced loss of NMDARs from both D1R and dopamine D2 receptor-expressing (D2R) MSNs is permissive for sensitization.

Valence and Salience Encoding in the Central Amygdala.

The central amygdala (CeA) has emerged as an important brain region for regulating both negative (fear and anxiety) and positive (reward) affective behaviors. The CeA has been proposed to encode affective information in the form of valence (whether the stimulus is good or bad) or salience (how significant is the stimulus), but the extent to which these two types of stimulus representation occur in the CeA is not known. Here, we used single cell calcium imaging in mice during appetitive and aversive conditioning and found that majority of CeA neurons (~65%) encode the valence of the unconditioned stimulus (US) with a smaller subset of cells (~15%) encoding the salience of the US. Valence and salience encoding of the conditioned stimulus (CS) was also observed, albeit to a lesser extent. These findings show that the CeA is a site of convergence for encoding oppositely valenced US information.

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.