Coordinating brain-distributed network activities in memory resistant to extinction.

Certain memories resist extinction to continue invigorating maladaptive actions. The robustness of these memories could depend on their widely distributed implementation across populations of neurons in multiple brain regions. However, how dispersed neuronal activities are collectively organized to underpin a persistent memory-guided behavior remains unknown. To investigate this, we simultaneously monitored the prefrontal cortex, nucleus accumbens, amygdala, hippocampus, and ventral tegmental area (VTA) of the mouse brain from initial recall to post-extinction renewal of a memory involving cocaine experience. We uncover a higher-order pattern of short-lived beta-frequency (15-25 Hz) activities that are transiently coordinated across these networks during memory retrieval. The output of a divergent pathway from upstream VTA glutamatergic neurons, paced by a slower (4-Hz) oscillation, actuates this multi-network beta-band coactivation; its closed-loop phase-informed suppression prevents renewal of cocaine-biased behavior. Binding brain-distributed neural activities in this temporally structured manner may constitute an organizational principle of robust memory expression.

A Hippocampus-Accumbens Tripartite Neuronal Motif Guides Appetitive Memory in Space.

Retrieving and acting on memories of food-predicting environments are fundamental processes for animal survival. Hippocampal pyramidal cells (PYRs) of the mammalian brain provide mnemonic representations of space. Yet the substrates by which these hippocampal representations support memory-guided behavior remain unknown. Here, we uncover a direct connection from dorsal CA1 (dCA1) hippocampus to nucleus accumbens (NAc) that enables the behavioral manifestation of place-reward memories. By monitoring neuronal ensembles in mouse dCA1→NAc pathway, combined with cell-type selective optogenetic manipulations of input-defined postsynaptic neurons, we show that dCA1 PYRs drive NAc medium spiny neurons and orchestrate their spiking activity using feedforward inhibition mediated by dCA1-connected parvalbumin-expressing fast-spiking interneurons. This tripartite cross-circuit motif supports spatial appetitive memory and associated NAc assemblies, being independent of dorsal subiculum and dispensable for both spatial novelty detection and reward seeking. Our findings demonstrate that the dCA1→NAc pathway instantiates a limbic-motor interface for neuronal representations of space to promote effective appetitive behavior.

Combined Social and Spatial Coding in a Descending Projection from the Prefrontal Cortex.

Social behaviors are crucial to all mammals. Although the prelimbic cortex (PL, part of medial prefrontal cortex) has been implicated in social behavior, it is not clear which neurons are relevant or how they contribute. We found that PL contains anatomically and molecularly distinct subpopulations that target three downstream regions that have been implicated in social behavior: the nucleus accumbens (NAc), amygdala, and ventral tegmental area. Activation of NAc-projecting PL neurons (PL-NAc), but not the other subpopulations, decreased the preference for a social target. To determine what information PL-NAc neurons convey, we selectively recorded from them and found that individual neurons were active during social investigation, but only in specific spatial locations. Spatially specific manipulation of these neurons bidirectionally regulated the formation of a social-spatial association. Thus, the unexpected combination of social and spatial information within the PL-NAc may contribute to social behavior by supporting social-spatial learning.

Molecular and Circuit-Dynamical Identification of Top-Down Neural Mechanisms for Restraint of Reward Seeking.

Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well, even for rewards of high value. In humans and rodents, the medial prefrontal cortex (mPFC) has been implicated in suppressing reward seeking; however, despite vital significance in health and disease, the neural circuitry through which mPFC regulates reward seeking remains incompletely understood. Here, we show that a specific subset of superficial mPFC projections to a subfield of nucleus accumbens (NAc) neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. A highly resolved subpopulation of these top-down projecting neurons, identified by 2-photon Ca2+ imaging and activity-dependent labeling to recruit the relevant neurons, was found capable of suppressing reward seeking. This natural activity-resolved mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.

Brain activity of professional investors signals future stock performance.

A major aspiration of investors is to better forecast stock performance. Interestingly, emerging "neuroforecasting" research suggests that brain activity associated with anticipatory reward relates to market behavior and population-wide preferences, including stock price dynamics. In this study, we extend these findings to professional investors processing comprehensive real-world information on stock investment options while making predictions of long-term stock performance. Using functional MRI, we sampled investors' neural responses to investment cases and assessed whether these responses relate to future performance on the stock market. We found that our sample of investors could not successfully predict future market performance of the investment cases, confirming that stated preferences do not predict the market. Stock metrics of the investment cases were not predictive of future stock performance either. However, as investors processed case information, nucleus accumbens (NAcc) activity was higher for investment cases that ended up overperforming in the market. These findings remained robust, even when controlling for stock metrics and investors' predictions made in the scanner. Cross-validated prediction analysis indicated that NAcc activity could significantly predict future stock performance out-of-sample above chance. Our findings resonate with recent neuroforecasting studies and suggest that brain activity of professional investors may help in forecasting future stock performance.

The Neuroscience of Drug Reward and Addiction.

Drug consumption is driven by a drug's pharmacological effects, which are experienced as rewarding, and is influenced by genetic, developmental, and psychosocial factors that mediate drug accessibility, norms, and social support systems or lack thereof. The reinforcing effects of drugs mostly depend on dopamine signaling in the nucleus accumbens, and chronic drug exposure triggers glutamatergic-mediated neuroadaptations in dopamine striato-thalamo-cortical (predominantly in prefrontal cortical regions including orbitofrontal cortex and anterior cingulate cortex) and limbic pathways (amygdala and hippocampus) that, in vulnerable individuals, can result in addiction. In parallel, changes in the extended amygdala result in negative emotional states that perpetuate drug taking as an attempt to temporarily alleviate them. Counterintuitively, in the addicted person, the actual drug consumption is associated with an attenuated dopamine increase in brain reward regions, which might contribute to drug-taking behavior to compensate for the difference between the magnitude of the expected reward triggered by the conditioning to drug cues and the actual experience of it. Combined, these effects result in an enhanced motivation to "seek the drug" (energized by dopamine increases triggered by drug cues) and an impaired prefrontal top-down self-regulation that favors compulsive drug-taking against the backdrop of negative emotionality and an enhanced interoceptive awareness of "drug hunger." Treatment interventions intended to reverse these neuroadaptations show promise as therapeutic approaches for addiction.

Epigenetic function during heroin self-administration controls future relapse-associated behavior in a cell type-specific manner.

Opioid use produces enduring associations between drug reinforcement/euphoria and discreet or diffuse cues in the drug-taking environment. These powerful associations can trigger relapse in individuals recovering from opioid use disorder (OUD). Here, we sought to determine whether the epigenetic enzyme, histone deacetylase 5 (HDAC5), regulates relapse-associated behavior in an animal model of OUD. We examined the effects of nucleus accumbens (NAc) HDAC5 on both heroin- and sucrose-seeking behaviors using operant self-administration paradigms. We utilized cre-dependent viral-mediated approaches to investigate the cell-type-specific effects of HDAC5 on heroin-seeking behavior, gene expression, and medium spiny neuron (MSN) cell and synaptic physiology. We found that NAc HDAC5 functions during the acquisition phase of heroin self-administration to limit future relapse-associated behavior. Moreover, overexpressing HDAC5 in the NAc suppressed context-associated and reinstated heroin-seeking behaviors, but it did not alter sucrose seeking. We also found that HDAC5 functions within dopamine D1 receptor-expressing MSNs to suppress cue-induced heroin seeking, and within dopamine D2 receptor-expressing MSNs to suppress drug-primed heroin seeking. Assessing cell-type-specific transcriptomics, we found that HDAC5 reduced expression of multiple ion transport genes in both D1- and D2-MSNs. Consistent with this observation, HDAC5 also produced firing rate depression in both MSN classes. These findings revealed roles for HDAC5 during active heroin use in both D1- and D2-MSNs to limit distinct triggers of drug-seeking behavior. Together, our results suggest that HDAC5 might limit relapse vulnerability through regulation of ion channel gene expression and suppression of MSN firing rates during active heroin use.

Father's care uniquely influences male neurodevelopment.

Mammalian infants depend on parental care for survival, with numerous consequences for their behavioral development. We investigated the epigenetic and neurodevelopmental mechanisms mediating the impact of early biparental care on development of alloparenting behavior, or caring for offspring that are not one's own. We find that receiving high parental care early in life leads to slower epigenetic aging of both sexes and widespread male-specific differential expression of genes related to synaptic transmission and autism in the nucleus accumbens. Examination of parental care composition indicates that high-care fathers promote a male-specific increase in excitatory synapses and increases in pup retrieval behavior as juveniles. Interestingly, females raised by high-care fathers have the opposite behavioral response and display fewer pup retrievals. These results support the concept that neurodevelopmental trajectories are programmed by different features of early-life parental care and reveal that male neurodevelopmental processes are uniquely sensitive to care by fathers.

Transcriptional signatures of early-life stress and antidepressant treatment efficacy.

Individuals with a history of early-life stress (ELS) tend to have an altered course of depression and lower treatment response rates. Research suggests that ELS alters brain development, but the molecular changes in the brain following ELS that may mediate altered antidepressant response have not been systematically studied. Sex and gender also impact the risk of depression and treatment response. Here, we leveraged existing RNA sequencing datasets from 1) blood samples from depressed female- and male-identifying patients treated with escitalopram or desvenlafaxine and assessed for treatment response or failure; 2) the nucleus accumbens (NAc) of female and male mice exposed to ELS and/or adult stress; and 3) the NAc of mice after adult stress, antidepressant treatment with imipramine or ketamine, and assessed for treatment response or failure. We find that transcriptomic signatures of adult stress after a history of ELS correspond with transcriptomic signatures of treatment nonresponse, across species and multiple classes of antidepressants. Transcriptomic correspondence with treatment outcome was stronger among females and weaker among males. We next pharmacologically tested these predictions in our mouse model of early-life and adult social defeat stress and treatment with either chronic escitalopram or acute ketamine. Among female mice, the strongest predictor of behavior was an interaction between ELS and ketamine treatment. Among males, however, early experience and treatment were poor predictors of behavior, mirroring our bioinformatic predictions. These studies provide neurobiological evidence for molecular adaptations in the brain related to sex and ELS that contribute to antidepressant treatment response.

An Ascending Excitatory Circuit from the Dorsal Raphe for Sensory Modulation of Pain.

The dorsal raphe nucleus (DRN) is an important nucleus in pain regulation. However, the underlying neural pathway and the function of specific cell types remain unclear. Here, we report a previously unrecognized ascending facilitation pathway, the DRN to the mesoaccumbal dopamine (DA) circuit, for regulating pain. Chronic pain increased the activity of DRN glutamatergic, but not serotonergic, neurons projecting to the ventral tegmental area (VTA) (DRNGlu-VTA) in male mice. The optogenetic activation of DRNGlu-VTA circuit induced a pain-like response in naive male mice, and its inhibition produced an analgesic effect in male mice with neuropathic pain. Furthermore, we discovered that DRN ascending pathway regulated pain through strengthened excitatory transmission onto the VTA DA neurons projecting to the ventral part of nucleus accumbens medial shell (vNAcMed), thereby activated the mesoaccumbal DA neurons. Correspondingly, optogenetic manipulation of this three-node pathway bilaterally regulated pain behaviors. These findings identified a DRN ascending excitatory pathway that is crucial for pain sensory processing, which can potentially be exploited toward targeting pain disorders.

Sex-Specific Mechanisms Underlie Long-Term Potentiation at Hippocampus→Medium Spiny Neuron Synapses in the Medial Shell of the Nucleus Accumbens.

Sex differences have complicated our understanding of the neurobiological basis of many behaviors that are key for survival. As such, continued elucidation of the similarities and differences between sexes is necessary to gain insight into brain function and vulnerability. The connection between the hippocampus (Hipp) and nucleus accumbens (NAc) is a crucial site where modulation of neuronal activity mediates reward-related behavior. Our previous work demonstrated that long-term potentiation (LTP) of Hipp→NAc synapses is rewarding, and mice can establish learned associations between LTP of these synapses and the contextual environment in which LTP occurred. Here, we investigated sex differences in the mechanisms underlying Hipp→NAc LTP using whole-cell electrophysiology and pharmacology. We observed similarities in basal synaptic strength between males and females and found that LTP occurs postsynaptically with similar magnitudes in both sexes. However, key sex differences emerged as LTP in males required NMDA receptors (NMDAR), whereas LTP in females utilized an NMDAR-independent mechanism involving L-type voltage-gated Ca2+ channels (VGCCs) and estrogen receptor α (ERα). We also uncovered sex-similar features as LTP in both sexes depended on CaMKII activity and occurred independently of dopamine-1 receptor (D1R) activation. Our results have elucidated sex-specific molecular mechanisms for LTP in an integral pathway that mediates reward-related behaviors, emphasizing the importance of considering sex as a variable in mechanistic studies. Continued characterization of sex-specific mechanisms underlying plasticity will offer novel insight into the neurophysiological basis of behavior, with significant implications for understanding how diverse processes mediate behavior and contribute to vulnerability to developing psychiatric disorders.

State- and Circuit-Dependent Opponent Processing of Fear.

The presence of valence coding neurons in the basolateral amygdala (BLA) that form distinct projections to other brain regions implies functional opposition between aversion and reward during learning. However, evidence for opponent interactions in fear learning is sparse and may only be apparent under certain conditions. Here we test this possibility by studying the roles of the BLA→central amygdala (CeA) and BLA→nucleus accumbens (Acb) pathways in fear learning in male rats. First, we assessed the organization of these pathways in the rat brain. BLA→CeA and BLA→Acb pathways were largely segregated in the BLA but shared overlapping molecular profiles. Then we assessed activity of the BLA→CeA and BLA→Acb pathways during two different forms of fear learning-fear learning in a neutral context and fear learning in a reward context. BLA→CeA neurons were robustly recruited by footshock regardless of where fear learning occurred, whereas recruitment of BLA→Acb neurons was state-dependent because footshock only recruited this pathway in a reward context. Finally, we assessed the causal roles of activity in these pathways in fear learning. Photoinhibition of the BLA→CeA pathway during the footshock US impaired fear learning, regardless of where fear learning occurred. In contrast, photoinhibition of the BLA→Acb pathway augmented fear learning, but only in the reward context. Taken together, our findings show circuit- and state-dependent opponent processing of fear. Footshock activity in the BLA→Acb pathway limits how much fear is learned.

Distinct Action Signals by Subregions in the Nucleus Accumbens during STOP-Change Performance.

The nucleus accumbens (NAc) is thought to contribute to motivated behavior by signaling the value of reward-predicting cues and the delivery of anticipated reward. The NAc is subdivided into core and shell, with each region containing different populations of neurons that increase or decrease firing to rewarding events. While there are numerous theories of functions pertaining to these subregions and cell types, most are in the context of reward processing, with fewer considering that the NAc might serve functions related to action selection more generally. We recorded from single neurons in the NAc as rats of both sexes performed a STOP-change task that is commonly used to study motor control and impulsivity. In this task, rats respond quickly to a spatial cue on 80% of trials (GO) and must stop and redirect planned movement on 20% of trials (STOP). We found that the activity of reward-excited neurons signaled accurate response direction on GO, but not STOP, trials and that these neurons exhibited higher precue firing after correct trials. In contrast, reward-inhibited neurons significantly represented response direction on STOP trials at the time of the instrumental response. Finally, the proportion of reward-excited to reward-inhibited neurons and the strength of precue firing decreased as the electrode traversed the NAc. We conclude that reward-excited cells (more common in core) promote proactive action selection, while reward-inhibited cells (more common in shell) contribute to accurate responding on STOP trials that require reactive suppression and redirection of behavior.

Cell- and Pathway-Specific Disruptions in the Accumbens of Fragile X Mouse.

Fragile X syndrome (FXS) is a genetic cause of intellectual disability and autism spectrum disorder. The mesocorticolimbic system, which includes the prefrontal cortex (PFC), basolateral amygdala (BLA), and nucleus accumbens core (NAcC), is essential for regulating socioemotional behaviors. We employed optogenetics to compare the functional properties of the BLA→NAcC, PFC→NAcC, and reciprocal PFC↔BLA pathways in Fmr1-/y::Drd1a-tdTomato male mice. In FXS mice, the PFC↔BLA reciprocal pathway was unaffected, while significant synaptic modifications occurred in the BLA/PFC→NAcC pathways. We observed distinct changes in D1 striatal projection neurons (SPNs) and separate modifications in D2 SPNs. In FXS mice, the BLA/PFC→NAcC-D2 SPN pathways demonstrated heightened synaptic strength. Focusing on the BLA→NAcC pathway, linked to autistic symptoms, we found increased AMPAR and NMDAR currents and elevated spine density in D2 SPNs. Conversely, the amplified firing probability of BLA→NAcC-D1 SPNs was not accompanied by increased synaptic strength, AMPAR and NMDAR currents, or spine density. These pathway-specific alterations resulted in an overall enhancement of excitatory-to-spike coupling, a physiologically relevant index of how efficiently excitatory inputs drive neuronal firing, in both BLA→NAcC-D1 and BLA→NAcC-D2 pathways. Finally, the absence of fragile X messenger ribonucleoprotein 1 (FMRP) led to impaired long-term depression specifically in BLA→D1 SPNs. These distinct alterations in synaptic transmission and plasticity within circuits targeting the NAcC highlight the potential role of postsynaptic mechanisms in selected SPNs in the observed circuit-level changes. This research underscores the heightened vulnerability of the NAcC in the context of FMRP deficiency, emphasizing its pivotal role in the pathophysiology of FXS.

Sex Differences in Dopamine Release in Nucleus Accumbens and Dorsal Striatum Determined by Chronic Fast-Scan Cyclic Voltammetry: Effects of Social Housing and Repeated Stimulation.

We investigated sex differences in dopamine (DA) release in the nucleus accumbens (NAc) and dorsolateral striatum (DLS) using a chronic 16-channel carbon fiber electrode and fast-scan cyclic voltammetry (FSCV). Electrical stimulation-induced (ES; 60 Hz) DA release was recorded in the NAc of single- or pair-housed male and female rats. When core (NAcC) and shell (NAcS) were recorded simultaneously, there was greater ES DA release in NAcC of pair-housed females compared with single females and males. Housing did not affect ES NAc DA release in males. In contrast, there was significantly more ES DA release from the DLS of female rats than male rats. This was true prior to and after treatment with methamphetamine. Furthermore, in castrated (CAST) males and ovariectomized (OVX) females, there were no sex differences in ES DA release from the DLS, demonstrating the hormone dependence of this sex difference. However, in the DLS of both intact and gonadectomized rats, DA reuptake was slower in females than that in males. Finally, DA release following ES of the medial forebrain bundle at 60 Hz was studied over 4 weeks. ES DA release increased over time for both CAST males and OVX females, demonstrating sensitization. Using this novel 16-channel chronic FSCV electrode, we found sex differences in the effects of social housing in the NAcS, sex differences in DA release from intact rats in DLS, and sex differences in DA reuptake in DLS of intake and gonadectomized rats, and we report sensitization of ES-induced DA release in DLS in vivo.

Functional states of prelimbic and related circuits during the acquisition of a GO/noGO task in rats.

GO/noGO tasks enable assessing decision-making processes and the ability to suppress a specific action according to the context. Here, rats had to discriminate between 2 visual stimuli (GO or noGO) shown on an iPad screen. The execution (for GO) or nonexecution (for noGO) of the selected action (to touch or not the visual display) were reinforced with food. The main goal was to record and to analyze local field potentials collected from cortical and subcortical structures when the visual stimuli were shown on the touch screen and during the subsequent activities. Rats were implanted with recording electrodes in the prelimbic cortex, primary motor cortex, nucleus accumbens septi, basolateral amygdala, dorsolateral and dorsomedial striatum, hippocampal CA1, and mediodorsal thalamic nucleus. Spectral analyses of the collected data demonstrate that the prelimbic cortex was selectively involved in the cognitive and motivational processing of the learning task but not in the execution of reward-directed behaviors. In addition, the other recorded structures presented specific tendencies to be involved in these 2 types of brain activity in response to the presentation of GO or noGO stimuli. Spectral analyses, spectrograms, and coherence between the recorded brain areas indicate their specific involvement in GO vs. noGO tasks.

The Neurobiology of Activational Aspects of Motivation: Exertion of Effort, Effort-Based Decision Making, and the Role of Dopamine.

Motivational processes are complex and multifaceted, with both directional and activational aspects. Behavioral activation and exertion of effort are functions that enable organisms to overcome obstacles separating them from significant outcomes. In a complex environment, organisms make cost/benefit decisions, assessing work-related response costs and reinforcer preference. Animal studies have challenged the general idea that dopamine (DA) is best viewed as the reward transmitter and instead have illustrated the involvement of DA in activational and effort-related processes. Mesocorticolimbic DA is a key component of the effort-related motivational circuitry that includes multiple neurotransmitters and brain areas. Human studies have identified brain areas and transmitter systems involved in effort-based decision making and characterized the reduced selection of high-effort activities associated with motivational symptoms of depression and schizophrenia. Animal and human research on the neurochemistry of behavioral activation and effort-related processes makes an important conceptual contribution by illustrating the dissociable nature of distinct aspects of motivation.

Brain tract structure predicts relapse to stimulant drug use.

Diffusion tractography allows identification and measurement of structural tracts in the human brain previously associated with motivated behavior in animal models. Recent findings indicate that the structural properties of a tract connecting the midbrain to nucleus accumbens (NAcc) are associated with a diagnosis of stimulant use disorder (SUD), but not relapse. In this preregistered study, we used diffusion tractography in a sample of patients treated for SUD (n = 60) to determine whether qualities of tracts projecting from medial prefrontal, anterior insular, and amygdalar cortices to NAcc might instead foreshadow relapse. As predicted, reduced diffusion metrics of a tract projecting from the right anterior insula to the NAcc were associated with subsequent relapse to stimulant use, but not with previous diagnosis. These findings highlight a structural target for predicting relapse to stimulant use and further suggest that distinct connections to the NAcc may confer risk for relapse versus diagnosis.

Local accumbens in vivo imaging during deep brain stimulation reveals a strategy-dependent amelioration of hedonic feeding.

Impulsive overeating is a common, disabling feature of eating disorders. Both continuous deep brain stimulation (DBS) and responsive DBS, which limits current delivery to pathological brain states, have emerged as potential therapies. We used in vivo fiber photometry in wild-type, Drd1-cre, and A2a-cre mice to 1) assay subtype-specific medium spiny neuron (MSN) activity of the nucleus accumbens (NAc) during hedonic feeding of high-fat food, and 2) examine DBS strategy-specific effects on NAc activity. D1, but not D2, NAc GCaMP activity increased immediately prior to high-fat food approach. Responsive DBS triggered a GCaMP surge throughout the stimulation period and durably reduced high-fat intake. However, with continuous DBS, this surge decayed, and high-fat intake reemerged. Our results argue for a stimulation strategy-dependent modulation of D1 MSNs with a more sustained decrease in consumption with responsive DBS. This study illustrates the important role in vivo imaging can play in understanding effects of such novel therapies.

Infralimbic Projections to the Nucleus Accumbens Shell and Amygdala Regulate the Encoding of Cocaine Extinction Learning.

Prior evidence indicates that the infralimbic cortex (IL) mediates the ongoing inhibition of cocaine seeking following self-administration and extinction training in rats, specifically through projections to the nucleus accumbens shell (NAshell). Our own data indicate that IL activity immediately following an unreinforced lever press is critical for encoding the extinction contingencies in such procedures. Whether extinction encoding requires activity in the IL exclusively or also activity in its outputs, such as those to the NAshell and amygdala, is unknown. To address this issue, we used a closed-loop optogenetic approach in female and male Sprague Dawley rats to silence IL-NAshell or IL-amygdala activity following an unreinforced lever press during extinction training. Optical illumination (20 s) was given either immediately after a lever press or following a 20 s delay. IL-NAshell inhibition immediately following an unreinforced lever press increased lever pressing during extinction training and impaired retention of extinction learning, as assessed during subsequent extinction sessions without optical inhibition. Likewise, IL-amygdala inhibition given in the same manner impaired extinction retention during sessions without inhibition. Control experiments indicate that critical encoding of extinction learning does not require activity in these pathways beyond the initial 20 s post-lever press period, as delayed IL-NAshell and IL-amygdala inhibition had no effect on extinction learning. These results suggest that a larger network extending from the IL to the NAshell and amygdala is involved in encoding extinction contingencies following cocaine self-administration.SIGNIFICANCE STATEMENT Infralimbic cortex (IL) activity following an unreinforced lever press during extinction learning encodes the extinction of cocaine-seeking behavior. However, the larger circuitry controlling such encoding has not been investigated. Using closed-loop optogenetic pathway targeting, we found that inhibition of IL projections to the nucleus accumbens shell and to the amygdala impaired the extinction of cocaine seeking. Importantly, these effects were only observed when activity was disrupted during the first 20 s post-lever press and not when given following a 20 s delay. These findings suggest that successful cocaine extinction encoding requires activity across a larger circuit beyond simply inputs to the IL.