The insular cortex (IC) is known to underlie drug seeking and relapse for multiple drug classes, yet the precise role the IC plays in opioid use disorder (OUD) remains unclear. In preclinical models of OUD, inhibition of the IC has produced conflicting results, such that in some cases the IC seems to promote opioid seeking whereas in others the IC seems to blunt opioid seeking. These results may be related to the heterogeneity of cortical output circuits, which can have opposing functions despite their relative proximity. Thus, here we examined the role of a specific IC output circuit, from the anterior IC (aIC) to the nucleus accumbens core (NAcc), for opioid seeking. We find in mice that following 14 days of heroin self-administration and 3 days of forced abstinence, optogenetic inhibition of aIC➔NAcc terminals suppresses context-associated opioid seeking. Furthermore, the same manipulation attenuates cued opioid seeking following extinction training. Importantly, we observed no effect of aIC➔NAcc terminal inhibition on sucrose seeking. Together, our results reveal that the IC selectively controls opioid seeking through a discrete population of NAcc projecting neurons, providing the first evidence for a projection-specific role of IC circuitry in opioid seeking and relapse.

Drug self-administration has the greatest construct and predictive validity of the preclinical models for substance use disorder, providing landmark insights into the neurobiology of addiction. However, these experiments have traditionally been performed in freely moving animals, which can prohibit the incorporation of emerging neurotechnologies that require or are greatly facilitated by head restraint. Recently, we developed and validated a head-restrained approach in mice for intravenous and oral self-administration of drug and nondrug rewards. Here we present a step-by-step protocol for these experiments, including custom equipment construction, open-source software implementation and adaptation, catheter implantation, and unique considerations for conducting head-fixed self-administration experiments. To ensure that each component can be implemented by a wide range of audiences, detailed descriptions are provided so that this Protocol may serve as a standalone guide for researchers with varying levels of experience.

Avoiding danger even when competing goals are imminent supports survival, yet the mechanisms orchestrating this behavioral override remain unclear. Macdonald and colleagues1 describe a thalamostriatal microcircuit that modulates terminal dopamine release, bypassing canonical midbrain dopamine circuitry to prioritize safety.

IntroductionFear learning is critical for organisms to respond appropriately to potentially harmful stimuli. In cases of post-traumatic stress disorder (PTSD), potent or persistent trauma can facilitate future aversive learning, leading to maladaptive fear responses in generally safe contexts. Despite this knowledge, the neural circuit mechanisms that underlie stress-enhanced fear learning (SEFL) remain poorly understood.MethodsWe utilized brain clearing and light sheet imaging, mapping whole-brain expression of the immediate early gene cFos in mice to identify brain regions engaged by SEFL. Next, we used chemogenetics to test the causal contribution of a candidate target region, the paraventricular thalamus (PVT), to sensitized fear learning.ResultsSEFL was associated with reduced activity throughout the brain, most prominently within higher order thalamic nuclei. Despite the overall reduction, cFos expression in the PVT positively correlated with a principal-component-derived predictor of subsequent sensitized fear expression. Furthermore, chemogenetic inhibition of the PVT 30 days after stress exposure prevented the acquisition of SEFL and restored freezing to control levels.DiscussionOur results provide a comprehensive whole-brain SEFL-induced cFos map. Furthermore, we reveal a novel function for one identified brain region, the PVT, in sensitized fear learning.

BACKGROUND:Significant advances in neurotechnology, such as the application of two-photon (2P) imaging of biosensors in vivo, have enabled unparalleled longitudinal and high-resolution access to neural circuits that coordinate behavior in rodents. Integration of these techniques would be groundbreaking for the study of alcohol use disorder (AUD). AUD is rooted in significant neural adaptations that could be functionally monitored and manipulated at the single-cell level across the development of dependence in rodents. However, 2P imaging and related methodologies often require or are facilitated by head fixation, and a lack of head-fixed models has hindered their integration for the study of alcohol dependence. METHODS:We developed a head-fixed model in which animals learned to self-administer ethanol across ~14 days. Active lever responding resulted in a tone cue and ethanol reward, whereas responding on the inactive lever resulted in neither cue nor ethanol reward. Following acquisition, animals extinguished lever pressing across a minimum of 10 days. Finally, animals were tested separately for both cue- and ethanol-induced reinstatement of lever pressing. RESULTS:Here we show, for the first time, that in our head-fixed ethanol self-administration model, male and female mice reliably pressed an active, but not inactive, lever for an oral ethanol reward. Ethanol rewards positively correlated with blood ethanol concentrations at pharmacologically relevant levels. Furthermore, mice extinguished ethanol self-administration when the ethanol reward and cue were omitted, suggesting active lever pressing was ethanol-directed. Following extinction, presentation of the ethanol-associated cue or priming with ethanol itself invigorated reinstatement of ethanol seeking, modeling relapse in a manner that replicates decades of work in freely moving rodent studies. CONCLUSIONS:Overall, our head-fixed ethanol self-administration model will allow for incorporation of novel technologies that require or are greatly facilitated by head fixation, improving our ability to study and understand the neural adaptations and computations that underlie alcohol dependence.

Learned associations between environmental cues and reward drive motivated behavior, yet how specific cell types support this process remains unclear. Using longitudinal two-photon calcium imaging, we tracked dorsal medial prefrontal cortical astrocytes throughout the acquisition, expression, and reversal of Pavlovian sucrose conditioning. As learning progressed, astrocytes exhibited time-locked, spatially coordinated calcium signals that differentiated correct behavioral action from mistakes, evolving from broad outcome encoding to selective representation of responses associated with the reward-conditioned stimulus. Omission testing revealed that prefrontal astrocytes preferentially respond to the cue-reward association, rather than the conditioned stimulus or reward alone. When reward contingencies were reversed, astrocytic activity rapidly adapted to track the new cue-reward association and encode updated and outdated motivated behavioral actions. Finally, astrocytic ablation attenuated motivated behavior during initial associative learning and prevented persistence of conditioned reward seeking when reward contingencies were updated or unpredictable. These findings reveal prefrontal astrocytes are functionally plastic elements that regulate reward-seeking behavior across associative learning. Teaser:Prefrontal astrocytes flexibly encode the cue-reward associations that drive conditioned reward-seeking behavior.

OBJECTIVES/GOALS: Determine how a history of unpredictable foot shock in mice affects brain wide patterns of neural activation to future stressors. Additionally, we aimed to characterize how the paraventricular nucleus of the thalamus (PVT) is involved in the fear sensitization process. METHODS/STUDY POPULATION: We used a mouse model of stress enhanced fear learning, where stressed mice are first subjected to a series of unpredictable foot shocks in a novel context while control mice undergo exposure to the novel context without experiencing foot shock. Mice are then left undisturbed for 28 days, following which they are exposed to a single foot shock in a novel context. Mice are tested in the second context 24 hours after single shock, and the amount of time spent frozen in the context provides a measure of fear sensitization. Whole brain patterns of activation during the second context test will be assessed via whole brain optical clearing with antibody staining of immediate early genes. The role of the PVT in fear sensitization will be characterized using chemogenetic approaches. RESULTS/ANTICIPATED RESULTS: Our preliminary results demonstrate that mice display enhanced fear acquisition long after the initial experience of unpredictable shocks. We anticipate to identify regions previously implicated in fear learning and novel regions not previously described through our brain clearing approach. In addition, we anticipate chemogenetic inhibition of the PVT will reduce freezing to an auditory cue associated with the shock in the second context but not to the context itself. DISCUSSION/SIGNIFICANCE: Our findings will provide a comprehensive view of how a history of unpredictable stress affects whole brain processing of subsequent stressful experiences, and describe the role of the PVT in cued fear sensitization.

Cocaine use disorder (CUD) remains a serious public health crisis, with relapse vulnerability continuing to pose the largest impediment to effective clinical treatment. Relapse to cocaine seeking is often triggered by drug craving evoked by exposure to drug-associated environmental cues. Data from preclinical models of rodent self-administration (SA) and cue-induced reinstatement demonstrate that exposure to drug predictive cues following a period of withdrawal engages a large induction of glutamate release in the nucleus accumbens core (NAc), not observed during cued sucrose seeking. This profound glutamate release engages neuronal nitric oxide synthase (nNOS) expressing interneurons likely through activation of metabotropic glutamate receptor 5 (mGluR5), leading to increased production of nitric oxide (NO). Importantly, cue-induced glutamate and NO production have been linked to activation of matrix metalloproteinases (MMPs) and induction of the transient synaptic plasticity in medium spiny neurons (MSNs) required for cued cocaine seeking. Recent evidence suggests that cue-induced structural and synaptic plasticity occurs predominantly in D1 Dopamine receptor expressing MSNs, yet despite these findings, how cue-induced glutamate release is translated into D1 MSN plasticity has yet to be elucidated. We show here that knockdown of nNOS is sufficient to block cue-induced reinstatement to cocaine and prevents cue-induced functional and structural synaptic adaptions specifically in D1 receptor containing MSNs. Next, we demonstrate that knockdown of mGluR5, selectively on nitrergic interneurons in the NAc, is sufficient to block both conditioned place preference (CPP) and cue-induced reinstatement to cocaine, mechanistically linking cue-associated glutamate release to NO signaling. Finally, we demonstrate that downstream of glutamate-mediated activation of mGluR5 on nitrergic interneurons and MMP activation, expression of β3 integrin receptors on D1 MSNs is required for cued cocaine seeking. Taken together, our data provide a mechanistic link between cocaine cue-induced glutamate release, activation of nitrergic interneurons and the D1 MSN plasticity required for cued cocaine seeking,Significance Statement Relapse vulnerability to cocaine is a persistent challenge to successful treatment of CUD. Relapse precipitated by drug-associated environment cues requires synaptic plasticity in MSNs. Here, we show that knockdown of nNOS, or mGluR5 on nitrergic interneurons, or β3 integrin receptors on D1 MSNs is sufficient to block cue-induced cocaine seeking. Taken together our data support the following cocaine seeking signaling cascade.### Competing Interest StatementThe authors have declared no competing interest.

Corticostriatal projection neurons from prelimbic medial prefrontal cortex to the nucleus accumbens core critically regulate drug-seeking behaviors, yet the underlying encoding dynamics whereby these neurons contribute to drug seeking remain elusive. Here we use two-photon calcium imaging to visualize the activity of corticostriatal neurons in mice from the onset of heroin use to relapse. We find that the activity of these neurons is highly heterogeneous during heroin self-administration and seeking, with at least 8 distinct neuronal ensembles that display both excitatory and inhibitory encoding dynamics. These neuronal ensembles are particularly apparent during relapse, where excitatory responses are amplified compared to heroin self-administration. Moreover, we find that optogenetic inhibition of corticostriatal projection neurons attenuates heroin seeking regardless of the relapse trigger. Our results reveal the precise corticostriatal activity dynamics underlying drug-seeking behaviors and support a key role for this circuit in mediating relapse to drug seeking.

Significant advances in neurotechnology, such as the emergence of 2-photon imaging, have enabled unparalleled access to the complex neural circuits that coordinate behavior in rodents. Integration of these techniques would be groundbreaking for the study of animal models of alcohol use disorder (AUD), which is rooted in longitudinal brain adaptations that could be functionally monitored and manipulated at the level of neural circuits from the onset of alcohol use through dependence. However, 2-photon and related methodologies require or are often facilitated by head-fixation, and a lack of head-fixed models have hindered their integration in the study of AUD. Here we present a head-fixed alcohol self-administration model, and find that head-fixed male and female mice will reliably press an active, but not inactive, lever for an oral alcohol reward. The number of alcohol rewards obtained reliably predicted blood alcohol concentrations, at physiologically relevant levels. Furthermore, we demonstrate that mice can extinguish alcohol self-administration when the alcohol reward is omitted, suggesting active lever pressing behavior was alcohol-directed. Following extinction, presentation of alcohol-related cues or a priming reminder of alcohol itself invigorated reinstatement of alcohol seeking, modeling relapse in a manner that mimics decades of work in freely-moving rodent studies. Overall, our head-fixed alcohol self-administration model allows integration of novel technologies that require or are greatly facilitated by head-fixation, improving our ability to study and understand the neural circuits adaptations and computations that underlie AUD.### Competing Interest StatementThe authors have declared no competing interest.

Objective: An environmental context, which reliably predicts food availability, can increase the appetitive food drive within the same environment context. However, hunger is required for the development of such a context-induced feeding (CIF) response, suggesting the neural circuits sensitive to hunger link an internal energy state with a particular environment context. Since Agouti related peptide (AgRP) neurons are activated by energy deficit, we hypothesised that AgRP neurons are both necessary and sufficient to drive CIF. Methods: To examine the role of AgRP neurons in the CIF process, we used fibre photometry with GCaMP7f, chemogenetic activation of AgRP neurons, as well as optogenetic control of AgRP neurons to facilitate acute temporal control not permitted with chemogenetics. Results: A CIF response at test was only observed when mice were fasted during context training and AgRP population activity at test showed an attenuated inhibitory response to food, suggesting increased food-seeking and/or decreased satiety signalling drives the increased feeding response at test. Intriguingly, chemogenetic activation of AgRP neurons during context training did not increase CIF, suggesting precise temporal firing properties may be required. Indeed, termination of AgRP neuronal photostimulation during context training (ON-OFF in context), in the presence or absence of food, increased CIF. Moreover, photoinhibition of AgRP neurons during context training in fasted mice was sufficient to drive a subsequent CIF in the absence of food. Conclusions: Our results suggest that AgRP neurons regulate the acquisition of CIF when the acute inhibition of AgRP activity is temporally matched to context exposure. These results establish acute AgRP inhibition as a salient neural event underscoring the effect of hunger on associative learning.

A lack of behavioral suppression is the cardinal feature of all substance use disorders. Indeed, relapse results from the disinhibition of drug seeking during abstinence. However, a vast majority of research has focused on neural mechanisms that facilitate, rather than disinhibit, drug seeking. Hence, whether relapse results from drug-induced dysregulation of the neuronal mechanisms that normally guide the suppression of behavior is unknown. Here we study this by developing deep brain two-photon calcium imaging in heroin self-administering mice, wherein we longitudinally track adaptations within a paraventricular thalamo-accumbal behavioral suppression system from the onset of heroin use to relapse. We find select neuronal ensembles that become profoundly hypoactive across the development of heroin use and during relapse to heroin seeking. Electrophysiological experiments further reveal long-lasting and cell-type selective adaptations at thalamo-accumbal to parvalbumin interneuron synapses, effects that functionally disinhibit behavior. Next, we demonstrate the heroin-induced weakening of the thalamo-accumbal behavioral suppression system is mediated by thalamic µ-opioid receptors. Finally, we show that output and synapse-selective rescue of activity within this circuit prevents cue-, drug-, and stress-triggered reinstatement of heroin seeking. These data identify precise functional and physiological adaptations within a thalamic behavioral control network and reveal that restoration of this system would prevent relapse to opioid use.

By modeling neural network dynamics related to homeostatic state and BMI, we identified a novel pathway projecting from the medial prefrontal cortex (mPFC) to the lateral hypothalamus (LH) in humans. We then assessed the physiological role and dissected the function of this mPFC-LH circuit in mice. In vivo recordings of population calcium activity revealed that this glutamatergic mPFC-LH pathway is activated in response to acute stressors and inhibited during food consumption, suggesting a role in stress-related control over food intake. Consistent with this role, inhibition of this circuit increased feeding and sucrose seeking during mild stressors, but not under non-stressful conditions. Finally, chemogenetic or optogenetic activation of the mPFC-LH pathway is sufficient to suppress food intake and sucrose-seeking in mice. These studies identify a glutamatergic mPFC-LH as a novel stress-sensitive anorexigenic neural pathway involved in the cortical control of food intake.

Lack of behavioral suppression typifies substance use disorders, yet the neural circuit underpinnings of drug-induced behavioral disinhibition remain unclear. Here, we employ deep-brain two-photon calcium imaging in heroin self-administering mice, longitudinally tracking adaptations within a paraventricular thalamus to nucleus accumbens behavioral inhibition circuit from the onset of heroin use to reinstatement. We find that select thalamo-accumbal neuronal ensembles become profoundly hypoactive across the development of heroin seeking and use. Electrophysiological experiments further reveal persistent adaptations at thalamo-accumbal parvalbumin interneuronal synapses, whereas functional rescue of these synapses prevents multiple triggers from initiating reinstatement of heroin seeking. Finally, we find an enrichment of μ-opioid receptors in output- and cell-type-specific paraventricular thalamic neurons, which provide a mechanism for heroin-induced synaptic plasticity and behavioral disinhibition. These findings reveal key circuit adaptations that underlie behavioral disinhibition in opioid dependence and further suggest that recovery of this system would reduce relapse susceptibility.

An environment can have a powerful influence over appetite and feeding behaviour. For example, an environmental context, which reliably predicts food, will increase the appetitive food drive to the same environment context. Interestingly, mice are required to be hungry to develop such a context-induced feeding (CIF) response, suggesting the neural circuits sensitive to hunger play an important role to associate an internal energy state with a particular environment context. Hunger-sensing Agouti related peptide (AgRP) neurons are activated by circulating signals of energy deficit and reset to a silenced state by gut feedback mechanisms following food consumption. We hypothesised that AgRP neurons are both necessary and sufficient to drive CIF in the absence of hunger. While fasting increased CIF, chemogenetic inhibition of AgRP neurons during context acquisition prevented this effect. Intriguingly, chemogenetic activation of AgRP neurons during context acquisition did not increase CIF, suggesting precise temporal firing properties may be required. Indeed, photostimulation of AgRP neurons, only during context exposure (ON-OFF in context), increased CIF. Moreover, AgRP photostimulation prior to context exposure, coupled with the termination of photostimulation in the context in the absence of food consumption, was sufficient to drive a subsequent CIF. Our results suggest that AgRP neurons regulate the acquisition of CIF when the temporal firing properties are matched to context exposure. These results further highlight that acute AgRP inhibition is a salient neural event underscoring the effect of hunger on associative learning.

Abstract Suppression of dangerous or inappropriate reward-motivated behaviors is critical for survival, whereas therapeutic or recreational opioid use can unleash risky behavioral actions and addiction. Nevertheless, the neuronal systems that suppress maladaptive motivated behaviors remain unclear, and whether opioids disengage those systems is unknown. Using two-photon calcium imaging in vivo, we identify paraventricular thalamostriatal neuronal ensembles that are inhibited upon sucrose self-administration and seeking, yet these neurons are tonically active when behavior is suppressed by a fear-provoking predator odor, a pharmacological stressor, or inhibitory learning. Electrophysiological, optogenetic, and chemogenetic experiments reveal that thalamostriatal neurons innervate accumbal parvalbumin interneurons through synapses enriched with calcium permeable AMPA receptors, and activity within this circuit is necessary and sufficient for the suppression of sucrose seeking regardless of the behavioral suppressor administered. Furthermore, systemic or intra-accumbal opioid injections rapidly dysregulate thalamostriatal ensemble dynamics, weaken thalamostriatal synaptic innervation of downstream parvalbumin interneurons, and unleash reward-seeking behaviors in a manner that is reversed by genetic deletion of thalamostriatal µ-opioid receptors. Overall, our findings reveal a thalamostriatal to parvalbumin interneuron circuit for the suppression of reward seeking that is rapidly disengaged by opioid-driven inhibition of presynaptic thalamostriatal neurons.

The ventromedial hypothalamic (VMH) nucleus is a well-established hub for energy and glucose homeostasis. In particular, VMH neurons are thought to be important for initiating the counterregulatory response to hypoglycemia, and ex vivo electrophysiology and immunohistochemistry data indicate a clear role for VMH neurons in sensing glucose concentration. However, the temporal response of VMH neurons to physiologically relevant changes in glucose availability in vivo has been hampered by a lack of available tools for measuring neuronal activity over time. Since the majority of neurons within the VMH are glutamatergic and can be targeted using the vesicular glutamate transporter Vglut2, we expressed cre-dependent GCaMP7s in Vglut2 cre mice and examined the response profile of VMH to intraperitoneal injections of glucose, insulin, and 2-deoxyglucose (2DG). We show that reduced available glucose via insulin-induced hypoglycemia and 2DG-induced glucoprivation, but not hyperglycemia induced by glucose injection, inhibits VMH Vglut2 neuronal population activity in vivo. Surprisingly, this inhibition was maintained for at least 45 minutes despite prolonged hypoglycemia and initiation of a counterregulatory response. Thus, although VMH stimulation, via pharmacological, electrical, or optogenetic approaches, is sufficient to drive a counterregulatory response, our data suggest VMH Vglut2 neurons are not the main drivers required to do so, since VMH Vglut2 neuronal population activity remains suppressed during hypoglycemia and glucoprivation.

Agouti-related peptide (AgRP) neurons increase motivation for food, however, whether metabolic sensing of homeostatic state in AgRP neurons potentiates motivation by interacting with dopamine reward systems is unexplored. As a model of impaired metabolic-sensing, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat) in mice. We hypothesised that metabolic sensing in AgRP neurons is required to increase motivation for food reward by modulating accumbal or striatal dopamine release. Studies confirmed that Crat deletion in AgRP neurons (KO) impaired ex vivo glucose-sensing, as well as in vivo responses to peripheral glucose injection or repeated palatable food presentation and consumption. Impaired metabolic-sensing in AgPP neurons reduced acute dopamine release (seconds) to palatable food consumption and during operant responding, as assessed by GRAB-DA photometry in the nucleus accumbens, but not the dorsal striatum. Impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ~30 min in the dorsal striatum but not the nucleus accumbens. Impaired metabolic sensing in AgRP neurons suppressed motivated operant responding for sucrose rewards during fasting. Thus, metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.