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.

Activation of CGRP neurons in the parabrachial nucleus suppresses addictive behavior.

Punishment such as electric shock or physical discipline employs a mixture of physical pain and emotional distress to induce behavior modification. However, a neural circuit that produces behavior modification by selectively focusing the emotional component, while bypassing the pain typically induced by peripheral nociceptor activation, is not well studied. Here, we show that genetically silencing the activity of neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus blocks the suppression of addictive-like behavior induced by footshock. Furthermore, activating CGRP neurons suppresses not only addictive behavior induced by self-stimulating dopamine neurons but also behavior resulting from self-administering cocaine, without eliciting nocifensive reactions. Moreover, among multiple downstream targets of CGRP neurons, terminal activation of CGRP in the central amygdala is effective, mimicking the results of cell body stimulation. Our results indicate that unlike conventional electric footshock, stimulation of CGRP neurons does not activate peripheral nociceptors but effectively curb addictive behavior.

Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization.

ΔfosB is an alternatively spliced product of the FosB gene that is essential for dopamine-induced reward pathways and that acts as a master switch for addiction. However, the molecular mechanisms of its generation and regulation by dopamine signaling are unknown. Here, we report that dopamine D1 receptor signaling synergizes with the activin/ALK4/Smad3 pathway to potentiate the generation of ΔFosB mRNA in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) via activation of the RNA-binding protein PCBP1, a regulator of mRNA splicing. Concurrent activation of PCBP1 and Smad3 by D1 and ALK4 signaling induced their interaction, nuclear translocation, and binding to sequences in exon-4 and intron-4 of FosB mRNA. Ablation of either ALK4 or PCBP1 in MSNs impaired ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in response to repeated co-stimulation of D1 and ALK4 receptors. Finally, ALK4 is required in NAc MSNs of adult mice for behavioral sensitization to cocaine. These findings uncover an unexpected mechanism for ΔFosB generation and drug-induced sensitization through convergent dopamine and ALK4 signaling.

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.

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants.

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.

Persistent binding at dopamine transporters determines sustained psychostimulant effects.

Psychostimulants interacting with the dopamine transporter (DAT) can be used illicitly or for the treatment of specific neuropsychiatric disorders. However, they can also produce severe and persistent adverse events. Often, their pharmacological properties in vitro do not fully correlate to their pharmacological profile in vivo. Here, we investigated the pharmacological effects of enantiomers of pyrovalerone, α-pyrrolidinovalerophenone, and 3,4-methylenedioxypyrovalerone as compared to the traditional psychostimulants cocaine and methylphenidate, using a variety of in vitro, computational, and in vivo approaches. We found that in vitro drug-binding kinetics at DAT correlate with the time-course of in vivo psychostimulant action in mice. In particular, a slow dissociation (i.e., slow koff) of S-enantiomers of pyrovalerone analogs from DAT predicts their more persistent in vivo effects when compared to cocaine and methylphenidate. Overall, our findings highlight the critical importance of drug-binding kinetics at DAT for determining the in vivo profile of effects produced by psychostimulant drugs.

Fine-Regional Role of the Claustrum in Anxiety and Higher Sensitivity to Cocaine in Adolescent Cocaine-Exposed Male Mice during Adulthood.

Adolescent cocaine exposure (ACE) induces anxiety and higher sensitivity to substances abuse during adulthood. Here, we show that the claustrum is crucial for controlling these psychiatric problems in male mice. In anxiety-like behavioral tests, the CaMKII-positive neurons in the median portion of the claustrum (MClaustrum) were triggered, and local suppression of these neurons reduced the anxiety-like behavior in ACE mice during adulthood. In contrast, the CaMKII-positive neurons in the anterior portion of the claustrum (AClaustrum) were more activated in response to subthreshold dose of cocaine induced conditioned place preference (CPP), and local suppression of these neurons blocked the acquisition of cocaine CPP in ACE mice during adulthood. Our findings for the first time identified the fine-regional role of the claustrum in regulating the anxiety and susceptibility to cocaine in ACE mice during adulthood, extending our understanding of the claustrum in substance use disorder.

Inactivation of ERK1/2 Signaling in Dopaminergic Neurons by Map Kinase Phosphatase MKP3 Regulates Dopamine Signaling and Motivation for Cocaine.

The mesolimbic dopamine system is a crucial component of reward and reinforcement processing, including the psychotropic effects of drugs of abuse such as cocaine. Drugs of abuse can activate intracellular signaling cascades that engender long-term molecular changes to brain reward circuitry, which can promote further drug use. However, gaps remain about how the activity of these signaling pathways, such as ERK1/2 signaling, can affect cocaine-induced neurochemical plasticity and cocaine-associated behaviors specifically within dopaminergic cells. To enable specific modulation of ERK1/2 signaling in dopaminergic neurons of the ventral tegmental area, we utilize a viral construct that Cre dependently expresses Map kinase phosphatase 3 (MKP3) to reduce the activity of ERK1/2, in combination with transgenic rats that express Cre in tyrosine hydroxylase (TH)-positive cells. Following viral transfection, we found an increase in the surface expression of the dopamine transporter (DAT), a protein associated with the regulation of dopamine signaling, dopamine transmission, and cocaine-associated behavior. We found that inactivation of ERK1/2 reduced post-translational phosphorylation of the DAT, attenuated the ability of cocaine to inhibit the DAT, and decreased motivation for cocaine without affecting associative learning as tested by conditioned place preference. Together, these results indicate that ERK1/2 signaling plays a critical role in shaping the dopamine response to cocaine and may provide additional insights into the function of dopaminergic neurons. Further, these findings lay important groundwork toward the assessment of how signaling pathways and their downstream effectors influence dopamine transmission and could ultimately provide therapeutic targets for treating cocaine use disorders.

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

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

Circuit-Wide Gene Network Analysis Reveals Sex-Specific Roles for Phosphodiesterase 1b in Cocaine Addiction.

Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b (Pde1b), a Ca2+/calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine.

Tmod2 Is a Regulator of Cocaine Responses through Control of Striatal and Cortical Excitability and Drug-Induced Plasticity.

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance abuse and intellectual disability in humans. Here, we mine the KOMP2 data and find that Tmod2 knock-out mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping shows that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knock-outs, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole-brain MR imaging shows differences in brain volume across multiple regions, although transcriptomic experiments did not reveal perturbations in gene coexpression networks. Detailed electrophysiological characterization of Tmod2 KO neurons showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data, collected from both males and females, provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.

Vagus Nerve Stimulation (VNS) Modulates Synaptic Plasticity in the Infralimbic Cortex via Trk-B Receptor Activation to Reduce Drug-Seeking in Male Rats.

Drugs of abuse cause changes in the prefrontal cortex (PFC) and associated regions that impair inhibitory control over drug-seeking. Breaking the contingencies between drug-associated cues and the delivery of the reward during extinction learning reduces relapse. Vagus nerve stimulation (VNS) has previously been shown to enhance extinction learning and reduce drug-seeking. Here we determined the effects of VNS-mediated release of brain-derived neurotrophic factor (BDNF) on extinction and cue-induced reinstatement in male rats trained to self-administer cocaine. Pairing 10 d of extinction training with VNS facilitated extinction and reduced drug-seeking behavior during reinstatement. Rats that received a single extinction session with VNS showed elevated BDNF levels in the medial PFC as determined via an enzyme-linked immunosorbent assay. Systemic blockade of tropomyosin receptor kinase B (TrkB) receptors during extinction, via the TrkB antagonist ANA-12, decreased the effects of VNS on extinction and reinstatement. Whole-cell recordings in brain slices showed that cocaine self-administration induced alterations in the ratio of AMPA and NMDA receptor-mediated currents in Layer 5 pyramidal neurons of the infralimbic cortex (IL). Pairing extinction with VNS reversed cocaine-induced changes in glutamatergic transmission by enhancing AMPAR currents, and this effect was blocked by ANA-12. Our study suggests that VNS consolidates the extinction of drug-seeking behavior by reversing drug-induced changes in synaptic AMPA receptors in the IL, and this effect is abolished by blocking TrkB receptors during extinction, highlighting a potential mechanism for the therapeutic effects of VNS in addiction.

Serotonin Signaling in Hippocampus during Initial Cocaine Abstinence Drives Persistent Drug Seeking.

The initiation of abstinence after chronic drug self-administration is stressful. Cocaine-seeking behavior on the first day of the absence of the expected drug (Extinction Day 1, ED1) is reduced by blocking 5-HT signaling in dorsal hippocampal cornu ammonis 1 (CA1) in both male and female rats. We hypothesized that the experience of ED1 can substantially influence later relapse behavior and that dorsal raphe (DR) serotonin (5-HT) input to CA1 may be involved. We inhibited 5-HT1A/1B receptors (WAY-100635 plus GR-127935), or DR input (chemogenetics), in CA1 on ED1 to test the role of this pathway on cocaine-seeking persistence 2 weeks later. We also inhibited 5-HT1A or 5-HT1B receptors in CA1 during conditioned place preference (CPP) for cocaine, to examine mechanisms involved in the persistent effects of ED1 manipulations. Inhibition of DR inputs, or 5-HT1A/1B signaling, in CA1 decreased drug seeking on ED1 and decreased cocaine seeking 2 weeks later revealing that 5-HT signaling in CA1 during ED1 contributes to persistent drug seeking during abstinence. In addition, 5-HT1B antagonism alone transiently decreased drug-associated memory performance when given prior to a CPP test, whereas similar antagonism of 5-HT1A alone had no such effect but blocked CPP retrieval on a test 24 h later. These CPP findings are consistent with prior work showing that DR inputs to CA1 augment recall of the drug-associated context and drug seeking via 5-HT1B receptors and prevent consolidation of the updated nondrug context via 5-HT1A receptors. Thus, treatments that modulate 5-HT-dependent memory mechanisms in CA1 during initial abstinence may facilitate later maintenance of abstinence.

Astrocytes modulate cerebral blood flow and neuronal response to cocaine in prefrontal cortex.

Cocaine affects both cerebral blood vessels and neuronal activity in brain. Cocaine can also disrupt astrocytes, which modulate neurovascular coupling-a process that regulates cerebral hemodynamics in response to neuronal activation. However, separating neuronal and astrocytic effects from cocaine's direct vasoactive effects has been challenging, partially due to limitations of neuroimaging techniques able to differentiate vascular from neuronal and glial effects at high temporal and spatial resolutions. Here, we used a newly-developed multi-channel fluorescence and optical coherence Doppler microscope (fl-ODM) that allows for simultaneous measurements of neuronal and astrocytic activities (reflected by the intracellular calcium changes in neurons Ca2+N and astrocytes Ca2+A, respectively) alongside their vascular interactions in vivo to address this challenge. Using green and red genetically-encoded Ca2+ indicators differentially expressed in astrocytes and neurons, fl-ODM enabled concomitant imaging of large-scale astrocytic and neuronal Ca2+ fluorescence and 3D cerebral blood flow velocity (CBFv) in vascular networks in the mouse cortex. We assessed cocaine's effects in the prefrontal cortex (PFC) and found that the CBFv changes triggered by cocaine were temporally correlated with astrocytic Ca2+A activity. Chemogenetic inhibition of astrocytes during the baseline state resulted in blood vessel dilation and CBFv increases but did not affect neuronal activity, suggesting modulation of spontaneous blood vessel's vascular tone by astrocytes. Chemogenetic inhibition of astrocytes during a cocaine challenge prevented its vasoconstricting effects alongside the CBFv decreases, but it also attenuated the neuronal Ca2+N increases triggered by cocaine. These results document a role of astrocytes both in regulating vascular tone and consequently blood flow, at baseline and for modulating the vasoconstricting and neuronal activation responses to cocaine in the PFC. Strategies to inhibit astrocytic activity could offer promise for ameliorating vascular and neuronal toxicity from cocaine misuse.

Memory consolidation drives the enhancement of remote cocaine memory via prefrontal circuit.

Remote memory usually decreases over time, whereas remote drug-cue associated memory exhibits enhancement, increasing the risk of relapse during abstinence. Memory system consolidation is a prerequisite for remote memory formation, but neurobiological underpinnings of the role of consolidation in the enhancement of remote drug memory are unclear. Here, we found that remote cocaine-cue associated memory was enhanced in rats that underwent self-administration training, together with a progressive increase in the response of prelimbic cortex (PrL) CaMKII neurons to cues. System consolidation was required for the enhancement of remote cocaine memory through PrL CaMKII neurons during the early period post-training. Furthermore, dendritic spine maturation in the PrL relied on the basolateral amygdala (BLA) input during the early period of consolidation, contributing to remote memory enhancement. These findings indicate that memory consolidation drives the enhancement of remote cocaine memory through a time-dependent increase in activity and maturation of PrL CaMKII neurons receiving a sustained BLA input.

mPFC DUSP1 mediates adolescent cocaine exposure-induced higher sensitivity to drug in adulthood.

Adolescent cocaine abuse increases the risk for developing addiction in later life, but the underlying molecular mechanism remains poorly understood. Here, we establish adolescent cocaine-exposed (ACE) male mouse models. A subthreshold dose of cocaine (sdC) treatment, insufficient to produce conditioned place preference (CPP) in adolescent mice, induces CPP in ACE mice during adulthood, along with more activated CaMKII-positive neurons, higher dual specificity protein kinase phosphatase-1 (Dusp1) mRNA, lower DUSP1 activity, and lower DUSP1 expression in CaMKII-positive neurons in the medial prefrontal cortex (mPFC). Overexpressing DUSP1 in CaMKII-positive neurons suppresses neuron activity and blocks sdC-induced CPP in ACE mice during adulthood. On the contrary, depleting DUSP1 in CaMKII-positive neurons activates more neurons and further enhances sdC-induced behavior in ACE mice during adulthood. Also, ERK1/2 might be a downstream signal of DUSP1 in the process. Our findings reveal a role of mPFC DUSP1 in ACE-induced higher sensitivity to the drug in adult mice. DUSP1 might be a potential pharmacological target to predict or treat the susceptibility to addictive drugs caused by adolescent substance use.

Dopamine D2 receptor signaling in the brain modulates circadian liver metabolomic profiles.

SignificanceWe analyzed the liver metabolome of mice deficient in the expression of the dopamine D2 receptor (D2R) in striatal medium spiny neurons (iMSN-D2RKO) and found profound changes in the liver circadian metabolome compared to control mice. Additionally, we show activation of dopaminergic circuits by acute cocaine administration in iMSN-D2RKO mice reprograms the circadian liver metabolome in response to cocaine. D2R signaling in MSNs is key for striatal output and essential for regulating the first response to the cellular and rewarding effects of cocaine. Our results suggest changes in dopamine signaling in specific striatal neurons evoke major changes in liver physiology. Dysregulation of liver metabolism could contribute to an altered allostatic state and therefore be involved in continued use of drugs.

A high-affinity cocaine binding site associated with the brain acid soluble protein 1.

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine's behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.

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.

Prelimbic Cortex Activity during a Distress Tolerance Task Predicts Cocaine-Seeking Behavior in Male, But Not Female Rats.

Distress tolerance (DT) is defined as the ability to persist in challenging goal-directed behavior in the face of stress, and individuals with low DT exhibit heightened drug-seeking behavior. However, no preclinical studies have examined the neurobiology underlying this phenomenon. To assess this, in vivo electrophysiology was used in Long Evans male and female rats during a DT task to record neural activity in the prelimbic cortex (PrL), a brain region implicated in drug-seeking. Rats were first assessed for DT, defined as the amount of time elapsed before rats quit seeking reward in an increasingly difficult operant task. Subsequently, rats underwent 2 weeks of self-administration for either water/saline or cocaine for 6 h/day. Animals then began a 1 month period of experimenter-imposed abstinence to induce heightened drug-seeking behavior. On day 28 of abstinence, DT and neural activity were reassessed; and on day 30, cocaine-seeking behavior was examined under extinction. Males had significantly higher DT than females and exhibited significantly more phasic PrL activity during the DT task. Furthermore, in male rats with a history of cocaine, PrL activity shifted to track DT; and this change in activity significantly correlated with the change in DT. Additionally, male (but not female) rats with low DT after 28 d of abstinence had significantly heightened drug-seeking behavior. Finally, PrL activity during the DT task predicted cocaine-seeking behavior. Collectively, these data demonstrate an important role for the PrL in DT in males, and link this neural activity and behavior to drug-seeking, particularly in males.SIGNIFICANCE STATEMENT Distress tolerance (DT) is defined as the ability to persist in challenging goal-directed behavior in the face of stress, and individuals with low DT exhibit heightened drug-seeking. Here, we investigated the role of the prelimbic cortex (PrL) in DT and its relationship to cocaine-seeking in male and female rats. We found that males had significantly higher DT than females and exhibited significantly more PrL activity during the DT task. Furthermore, male (but not female) rats with low DT after 28 d of abstinence had significantly heightened drug-seeking behavior. Finally, PrL activity during the DT task predicted cocaine-seeking. These data demonstrate an important role for the PrL in DT and link this neural activity and behavior to drug-seeking in males.