Cocaine-induced projection-specific and cell type-specific adaptations in the nucleus accumbens.

Cocaine craving, seeking, and relapse are mediated, in part, by cocaine-induced adaptive changes in the brain reward circuits. The nucleus accumbens (NAc) integrates and prioritizes different emotional and motivational inputs to the reward system by processing convergent glutamatergic projections from the medial prefrontal cortex, basolateral amygdala, ventral hippocampus, and other limbic and paralimbic brain regions. Medium spiny neurons (MSNs) are the principal projection neurons in the NAc, which can be divided into two major subpopulations, namely dopamine receptor D1- versus D2-expressing MSNs, with complementing roles in reward-associated behaviors. After cocaine experience, NAc MSNs exhibit complex and differential adaptations dependent on cocaine regimen, withdrawal time, cell type, location (NAc core versus shell), and related input and output projections, or any combination of these factors. Detailed characterization of these cellular adaptations has been greatly facilitated by the recent development of optogenetic/chemogenetic techniques combined with transgenic tools. In this review, we discuss such cell type- and projection-specific adaptations induced by cocaine experience. Specifically, (1) D1 and D2 NAc MSNs frequently exhibit differential adaptations in spinogenesis, glutamatergic receptor trafficking, and intrinsic membrane excitability, (2) cocaine experience differentially changes the synaptic transmission at different afferent projections onto NAc MSNs, (3) cocaine-induced NAc adaptations exhibit output specificity, e.g., being different at NAc-ventral pallidum versus NAc-ventral tegmental area synapses, and (4) the input, output, subregion, and D1/D2 cell type may together determine cocaine-induced circuit plasticity in the NAc. In light of the projection- and cell-type specificity, we also briefly discuss ensemble and circuit mechanisms contributing to cocaine craving and relapse after drug withdrawal.

AMPA and NMDA Receptor Trafficking at Cocaine-Generated Synapses.

Cocaine experience generates AMPA receptor (AMPAR)-silent synapses in the nucleus accumbens (NAc), which are thought to be new synaptic contacts enriched in GluN2B-containing NMDA receptors (NMDARs). After drug withdrawal, some of these synapses mature by recruiting AMPARs, strengthening the newly established synaptic transmission. Silent synapse generation and maturation are two consecutive cellular steps through which NAc circuits are profoundly remodeled to promote cue-induced cocaine seeking after drug withdrawal. However, the basic cellular processes that mediate these two critical steps remains underexplored. Using a combination of electrophysiology, viral-mediated gene transfer, and confocal imaging in male rats as well as knock-in (KI) mice of both sexes, our current study characterized the dynamic roles played by AMPARs and NMDARs in generation and maturation of silent synapses on NAc medium spiny neurons after cocaine self-administration and withdrawal. We report that cocaine-induced generation of silent synapses not only required synaptic insertion of GluN2B-containing NMDARs, but also, counterintuitively, involved insertion of AMPARs, which subsequently internalized, resulting in the AMPAR-silent state on withdrawal day 1. Furthermore, GluN2B NMDARs functioned to maintain these cocaine-generated synapses in the AMPAR-silent state during drug withdrawal, until they were replaced by nonGluN2B NMDARs, a switch that allowed AMPAR recruitment and maturation of silent synapses. These results reveal dynamic interactions between AMPARs and NMDARs during the generation and maturation of silent synapses after cocaine experience and provide a mechanistic basis through which new synaptic contacts and possibly new neural network patterns created by these synapses can be manipulated for therapeutic benefit.SIGNIFICANCE STATEMENT Studies over the past decade reveal a critical role of AMPA receptor-silent, NMDA receptor-containing synapses in forming cocaine-related memories that drive cocaine relapse. However, it remains incompletely understood how AMPA and NMDA receptors traffic at these synapses during their generation and maturation. The current study characterizes a two-step AMPA receptor trafficking cascade that contributes to the generation of silent synapses in response to cocaine experience, and a two-step NMDA receptor trafficking cascade that contributes to the maturation of these synapses after cocaine withdrawal. These results depict a highly regulated cellular procedure through which nascent glutamatergic synapses are generated in the adult brain after drug experience and provide significant insight into the roles of glutamate receptors in synapse formation and maturation.

Chronic sleep fragmentation enhances habenula cholinergic neural activity.

Sleep is essential to emotional health. Sleep disturbance, particularly REM sleep disturbance, profoundly impacts emotion regulation, but the underlying neural mechanisms remain elusive. Here we show that chronic REM sleep disturbance, achieved in mice by chronic sleep fragmentation (SF), enhanced neural activity in the medial habenula (mHb), a brain region increasingly implicated in negative affect. Specifically, after a 5-day SF procedure that selectively fragmented REM sleep, cholinergic output neurons (ChNs) in the mHb exhibited increased spontaneous firing rate and enhanced firing regularity in brain slices. The SF-induced firing changes remained intact upon inhibition of glutamate, GABA, acetylcholine, and histamine receptors, suggesting cell-autonomous mechanisms independent of synaptic transmissions. Moreover, the SF-induced hyperactivity was not because of enhanced intrinsic membrane excitability, but was accompanied by depolarized resting membrane potential in mHb ChNs. Furthermore, inhibition of TASK-3 (KCNK9) channels, a subtype of two-pore domain K+ channels, mimicked the SF effects by increasing the firing rate and regularity, as well as depolarizing the resting membrane potential in mHb ChNs in control-sleep mice. These effects of TASK-3 inhibition were absent in SF mice, suggesting reduced TASK-3 activity following SF. By contrast, inhibition of small-conductance Ca2+-activated K+ (SK) channels did not produce similar effects. Thus, SF compromised TASK-3 function in mHb ChNs, which likely led to depolarized resting membrane potential and increased spontaneous firing. These results not only demonstrate that selective REM sleep disturbance leads to hyperactivity of mHb ChNs, but also identify a key molecular substrate through which REM sleep disturbance may alter affect regulation.

Cocaine-induced neural adaptations in the lateral hypothalamic melanin-concentrating hormone neurons and the role in regulating rapid eye movement sleep after withdrawal.

Sleep abnormalities are often a prominent contributor to withdrawal symptoms following chronic drug use. Notably, rapid eye movement (REM) sleep regulates emotional memory, and persistent REM sleep impairment after cocaine withdrawal negatively impacts relapse-like behaviors in rats. However, it is not understood how cocaine experience may alter REM sleep regulatory machinery, and what may serve to improve REM sleep after withdrawal. Here, we focus on the melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH), which regulate REM sleep initiation and maintenance. Using adult male Sprague-Dawley rats trained to self-administer intravenous cocaine, we did transcriptome profiling of LH MCH neurons after long-term withdrawal using RNA-sequencing, and performed functional assessment using slice electrophysiology. We found that 3 weeks after withdrawal from cocaine, LH MCH neurons exhibit a wide range of gene expression changes tapping into cell membrane signaling, intracellular signaling, and transcriptional regulations. Functionally, they show reduced membrane excitability and decreased glutamatergic receptor activity, consistent with increased expression of voltage-gated potassium channel gene Kcna1 and decreased expression of metabotropic glutamate receptor gene Grm5. Finally, chemogenetic or optogenetic stimulations of LH MCH neural activity increase REM sleep after long-term withdrawal with important differences. Whereas chemogenetic stimulation promotes both wakefulness and REM sleep, optogenetic stimulation of these neurons in sleep selectively promotes REM sleep. In summary, cocaine exposure persistently alters gene expression profiles and electrophysiological properties of LH MCH neurons. Counteracting cocaine-induced hypoactivity of these neurons selectively in sleep enhances REM sleep quality and quantity after long-term withdrawal.

Calcineurin Promotes Neuroplastic Changes in the Amygdala Associated with Weakened Cocaine-Cue Memories.

Interfering with memory reconsolidation or inducing memory extinction are two approaches for weakening maladaptive memories in disorders such as addiction and post-traumatic stress disorder. Both extinction and reconsolidation are regulated by intracellular protein kinases and phosphatases, and interfering with these signaling molecules can alter memory strength. The calcium-dependent protein phosphatase, calcineurin (CaN), has been implicated in both the consolidation and extinction of fear memories. However, the role of CaN in regulating drug-cue associative memories has not been investigated. Prior studies have demonstrated that plasticity at thalamo-lateral amygdala (T-LA) synapses is critically involved in the regulation of cocaine-cue memories. Therefore, in the present study, we tested the effects of LA administration of an activator of CaN, chlorogenic acid (CGA), on behavioral and electrophysiological indices of cocaine cue memory reconsolidation and extinction. Male, Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. The cue memory was then either briefly reactivated, extinguished, or not manipulated, followed immediately by LA infusion of CGA. Rats were tested 24 h later for cue-induced reinstatement, or LA slices were prepared for electrophysiological recordings. We found that intra-LA infusions of CGA following cue extinction or reconsolidation reduced cue-induced reinstatement, which was blocked by co-infusion of the CaN inhibitor, FK-506. Similarly, CGA infusions following cue re-exposure significantly attenuated EPSC amplitude at T-LA synapses, suggesting that CaN affects cocaine-cue memory reconsolidation and extinction by altering T-LA synaptic strength. Therefore, CaN signaling in the LA may represent a novel target for disrupting cocaine-associated memories to reduce relapse.SIGNIFICANCE STATEMENT Repetitive drug use induces synaptic plasticity that underlies the formation of long-lasting associative memories for environmental cues paired with the drug. We previously identified thalamo-amygdala synapses (T-LA) that project via the interal capsule, as an important locus for the regulation of cocaine-cue memories. These synapses are strengthened by repeated cocaine-cue pairings, but this is reversed by extinction training or by optogenetic induction of in vivo long-term depression (LTD). Here, we demonstrate that activating calcineurin, a calcium-dependent phosphatase, following the reactivation or extinction of a cocaine-cue memory, induces LTD-like changes at T-LA synapses, and a corresponding decrease in cue-induced reinstatement, suggesting that calcineurin may be a potential therapeutic target for relapse prevention.

Cortical and Thalamic Interaction with Amygdala-to-Accumbens Synapses.

The nucleus accumbens shell (NAcSh) regulates emotional and motivational responses, a function mediated, in part, by integrating and prioritizing extensive glutamatergic projections from limbic and paralimbic brain regions. Each of these inputs is thought to encode unique aspects of emotional and motivational arousal. The projections do not operate alone, but rather are often activated simultaneously during motivated behaviors, during which they can interact and coordinate in shaping behavioral output. To understand the anatomic and physiological bases underlying these interprojection interactions, the current study in mice of both sexes focused on how the basolateral amygdala projection (BLAp) to the NAcSh regulates, and is regulated by, projections from the medial prefrontal cortex (mPFCp) and paraventricular nucleus of the thalamus (PVTp). Using a dual-color SynaptoTag technique combined with a backfilling spine imaging strategy, we found that all three afferent projections primarily targeted the secondary dendrites of NAcSh medium spiny neurons, forming putative synapses. We detected a low percentage of BLAp contacts closely adjacent to mPFCp or PVTp presumed synapses, and, on some rare occasions, the BLAp formed heterosynaptic interactions with mPFCp or PVTp profiles or appeared to contact the same spines. Using dual-rhodopsin optogenetics, we detected signs of dendritic summation of BLAp with PVTp and mPFCp inputs. Furthermore, high-frequency activation of BLAp synchronous with the PVTp or mPFCp resulted in a transient enhancement of the PVTp, but not mPFCp, transmission. These results provide anatomic and functional indices that the BLAp interacts with the mPFCp and PVTp for informational processing within the NAcSh.SIGNIFICANCE STATEMENT The nucleus accumbens regulates emotional and motivational responses by integrating extensive glutamatergic projections, but the anatomic and physiological bases on which these projections integrate and interact remain underexplored. Here, we used dual-color synaptic markers combined with backfilling of nucleus accumbens medium spiny neurons to reveal some unique anatomic alignments of presumed synapses from the basolateral amygdala, medial prefrontal cortex, and paraventricular nucleus of thalamus. We also used dual-rhodopsin optogenetics in brain slices, which reveal a nonlinear interaction between some, but not all, projections. These results provide compelling anatomic and physiological mechanisms through which different glutamatergic projections to the nucleus accumbens, and possibly different aspects of emotional and motivational arousal, interact with each other for final behavioral output.

Cell-Type-Specific Regulation of Nucleus Accumbens Synaptic Plasticity and Cocaine Reward Sensitivity by the Circadian Protein, NPAS2.

The circadian transcription factor neuronal PAS domain 2 (NPAS2) is linked to psychiatric disorders associated with altered reward sensitivity. The expression of Npas2 is preferentially enriched in the mammalian forebrain, including the nucleus accumbens (NAc), a major neural substrate of motivated and reward behavior. Previously, we demonstrated that downregulation of NPAS2 in the NAc reduces the conditioned behavioral response to cocaine in mice. We also showed that Npas2 is preferentially enriched in dopamine receptor 1 containing medium spiny neurons (D1R-MSNs) of the striatum. To extend these studies, we investigated the impact of NPAS2 disruption on accumbal excitatory synaptic transmission and strength, along with the behavioral sensitivity to cocaine reward in a cell-type-specific manner. Viral-mediated knockdown of Npas2 in the NAc of male and female C57BL/6J mice increased the excitatory drive onto MSNs. Using Drd1a-tdTomato mice in combination with viral knockdown, we determined these synaptic adaptations were specific to D1R-MSNs relative to non-D1R-MSNs. Interestingly, NAc-specific knockdown of Npas2 blocked cocaine-induced enhancement of synaptic strength and glutamatergic transmission specifically onto D1R-MSNs. Last, we designed, validated, and used a novel Cre-inducible short-hairpin RNA virus for MSN-subtype-specific knockdown of Npas2 Cell-type-specific Npas2 knockdown in D1R-MSNs, but not D2R-MSNs, in the NAc reduced cocaine conditioned place preference. Together, our results demonstrate that NPAS2 regulates excitatory synapses of D1R-MSNs in the NAc and cocaine reward-related behavior.SIGNIFICANCE STATEMENT Drug addiction is a widespread public health concern often comorbid with other psychiatric disorders. Disruptions of the circadian clock can predispose or exacerbate substance abuse in vulnerable individuals. We demonstrate a role for the core circadian protein, NPAS2, in mediating glutamatergic neurotransmission at medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a region critical for reward processing. We find that NPAS2 negatively regulates functional excitatory synaptic plasticity in the NAc and is necessary for cocaine-induced plastic changes in MSNs expressing the dopamine 1 receptor (D1R). We further demonstrate disruption of NPAS2 in D1R-MSNs produces augmented cocaine preference. These findings highlight the significance of cell-type-specificity in mechanisms underlying reward regulation by NPAS2 and extend our knowledge of its function.

NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward.

The diurnal regulation of dopamine is important for normal physiology and diseases such as addiction. Here we find a novel role for the CLOCK protein to antagonize CREB-mediated transcriptional activity at the tyrosine hydroxylase (TH) promoter, which is mediated by the interaction with the metabolic sensing protein, Sirtuin 1 (SIRT1). Additionally, we demonstrate that the transcriptional activity of TH is modulated by the cellular redox state, and daily rhythms of redox balance in the ventral tegmental area (VTA), along with TH transcription, are highly disrupted following chronic cocaine administration. Furthermore, CLOCK and SIRT1 are important for regulating cocaine reward and dopaminergic (DAergic) activity, with interesting differences depending on whether DAergic activity is in a heightened state and if there is a functional CLOCK protein. Taken together, we find that rhythms in cellular metabolism and circadian proteins work together to regulate dopamine synthesis and the reward value for drugs of abuse.

Nucleus accumbens feedforward inhibition circuit promotes cocaine self-administration.

The basolateral amygdala (BLA) sends excitatory projections to the nucleus accumbens (NAc) and regulates motivated behaviors partially by activating NAc medium spiny neurons (MSNs). Here, we characterized a feedforward inhibition circuit, through which BLA-evoked activation of NAc shell (NAcSh) MSNs was fine-tuned by GABAergic monosynaptic innervation from adjacent fast-spiking interneurons (FSIs). Specifically, BLA-to-NAcSh projections predominantly innervated NAcSh FSIs compared with MSNs and triggered action potentials in FSIs preceding BLA-mediated activation of MSNs. Due to these anatomical and temporal properties, activation of the BLA-to-NAcSh projection resulted in a rapid FSI-mediated inhibition of MSNs, timing-contingently dictating BLA-evoked activation of MSNs. Cocaine self-administration selectively and persistently up-regulated the presynaptic release probability of BLA-to-FSI synapses, entailing enhanced FSI-mediated feedforward inhibition of MSNs upon BLA activation. Experimentally enhancing the BLA-to-FSI transmission in vivo expedited the acquisition of cocaine self-administration. These results reveal a previously unidentified role of an FSI-embedded circuit in regulating NAc-based drug seeking and taking.

Cascades of Homeostatic Dysregulation Promote Incubation of Cocaine Craving.

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, promoting drug relapse. This time-dependent progression of drug craving is recapitulated in rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. Although recent results suggest that functional alterations of the nucleus accumbens (NAc) contribute to incubation of drug craving, it remains poorly understood how NAc function evolves after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies on how the membrane excitability of its principal medium spiny neurons (MSNs) translates excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc MSNs. The decreased membrane excitability subsequently induces another round of SMHC, leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated cocaine seeking after drug withdrawal.SIGNIFICANCE STATEMENT Here, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk mechanism, and produces cascades of alterations in excitatory synapses and membrane properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking after drug withdrawal. These results provide a novel mechanism through which drug-induced homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and promote drug relapse.

Cholinergic system in sleep regulation of emotion and motivation.

Sleep profoundly regulates our emotional and motivational state of mind. Human brain imaging and animal model studies are providing initial insights on the underlying neural mechanisms. Here, we focus on the brain cholinergic system, including cholinergic neurons in the basal forebrain, ventral striatum, habenula, and brain stem. Although much is learned about cholinergic regulations of emotion and motivation, less is known on their interactions with sleep. Specifically, we present an anatomical framework that highlights cholinergic signaling in the integrated reward-arousal/sleep circuitry, and identify the knowledge gaps on the potential roles of cholinergic system in sleep-mediated regulation of emotion and motivation. Sleep impacts every aspect of brain functions. It not only restores cognitive control, but also retunes emotional and motivational regulation [1]. Sleep disturbance is a comorbidity and sometimes a predicting factor for various psychiatric diseases including major depressive disorder, anxiety, post-traumatic stress disorder, and drug addiction [2-9]. Although it is well recognized that sleep prominently shapes emotional and motivational regulation, the underlying neural mechanisms remain elusive. The brain cholinergic system is essential for a diverse variety of functions including cognition, learning and memory, sensory and motor processing, sleep and arousal, reward processing, and emotion regulation [10-14]. Although cholinergic functions in cognition, learning and memory, motor control, and sleep and arousal have been well established, its interaction with sleep in regulating emotion and motivation has not been extensively studied. Here we review current evidence on sleep-mediated regulation of emotion and motivation, and reveal knowledge gaps on potential contributions from the cholinergic system.

Prefrontal Cortex to Accumbens Projections in Sleep Regulation of Reward.

UNLABELLED: Sleep profoundly affects the emotional and motivational state. In humans and animals, loss of sleep often results in enhanced motivation for reward, which has direct implications for health risks as well as potential benefits. Current study aims at understanding the mechanisms underlying sleep deprivation (SDe)-induced enhancement of reward seeking. We found that after acute SDe, mice had an increase in sucrose seeking and consumption but not food intake, suggesting a selective enhancement of motivation for reward. In the nucleus accumbens (NAc), a key brain region regulating emotional and motivational responses, we observed a decrease in the ratio of the overall excitatory over inhibitory synaptic inputs onto NAc principle neurons after SDe. The shift was partly mediated by reduced glutamatergic transmission of presynaptic origin. Further analysis revealed that there was selective reduction of the glutamate release probability at the medial prefrontal cortex (mPFC)-to-NAc synapses, but not those from the hippocampus, thalamus, or the basal lateral amygdala. To reverse this SDe-induced synaptic alteration, we expressed the stabilized step function opsin (SSFO) in the mPFC; optogenetic stimulation of SSFO at mPFC-to-NAc projection terminals persistently enhanced the action potential-dependent glutamate release. Intra-NAc optogenetic stimulation of SSFO selectively at mPFC-to-NAc terminals restored normal sucrose seeking in mice after SDe without affecting food intake. These results highlight the mPFC-to-NAc projection as a key circuit-based target for sleep to regulate reward-motivated behaviors. SIGNIFICANCE STATEMENT: Sleep loss, a costly challenge of modern society, has profound physiological and psychological consequences, including altered reward processing of the brain. The current study aims at understanding the mechanisms underlying sleep deprivation-induced enhancement of reward seeking. We identify that the medial prefrontal cortex (mPFC)-to-nucleus accumbens (NAc) glutamatergic transmission is selectively weakened following acute sleep deprivation, whose restoration normalizes reward seeking in sleep-deprived mice. These results suggest a possibility of normalizing sleep deprivation-induced abnormal reward seeking by targeting specific neural projections, and they demonstrate the mPFC-to-NAc glutamatergic projection as a key circuit-based target for sleep to regulate reward-motivated behaviors.

Sleep Regulates Incubation of Cocaine Craving.

After withdrawal from cocaine, chronic cocaine users often experience persistent reduction in total sleep time, which is accompanied by increased sleep fragmentation resembling chronic insomnia. This and other sleep abnormalities have long been speculated to foster relapse and further drug addiction, but direct evidence is lacking. Here, we report that after prolonged withdrawal from cocaine self-administration, rats exhibited persistent reduction in nonrapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep, as well as increased sleep fragmentation. In an attempt to improve sleep after cocaine withdrawal, we applied chronic sleep restriction to the rats during their active (dark) phase of the day, which selectively decreased the fragmentation of REM sleep during their inactive (light) phase without changing NREM or the total amount of daily sleep. Animals with improved REM sleep exhibited decreased incubation of cocaine craving, a phenomenon depicting the progressive intensification of cocaine seeking after withdrawal. In contrast, experimentally increasing sleep fragmentation after cocaine self-administration expedited the development of incubation of cocaine craving. Incubation of cocaine craving is partially mediated by progressive accumulation of calcium-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). After withdrawal from cocaine, animals with improved REM sleep exhibited reduced accumulation of CP-AMPARs in the NAc, whereas increasing sleep fragmentation accelerated NAc CP-AMPAR accumulation. These results reveal a potential molecular substrate that can be engaged by sleep to regulate cocaine craving and relapse, and demonstrate sleep-based therapeutic opportunities for cocaine addiction. Significance statement: Sleep abnormalities are common symptoms in chronic drug users long after drug withdrawal. These withdrawal-associated sleep symptoms, particularly reduction in total sleep time and deteriorating sleep quality, have been speculated to foster relapse and further drug addiction, but direct evidence is lacking. Here we show in rats that the sleep pattern was persistently changed long after withdrawal from cocaine self-administration, and demonstrate that sleep interventions can bidirectionally regulate cocaine craving and seeking after withdrawal. We further demonstrate that glutamatergic synapses in the nucleus accumbens are potential neuronal targets for sleep intervention to influence cocaine craving after withdrawal. These results provide a strong rationale supporting sleep-based therapies for cocaine addiction.

Selective presynaptic enhancement of the prefrontal cortex to nucleus accumbens pathway by cocaine.

The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.

Cannabinoid receptor 1-expressing neurons in the nucleus accumbens.

Endocannabinoid signaling critically regulates emotional and motivational states via activation of cannabinoid receptor 1 (CB1) in the brain. The nucleus accumbens (NAc) functions to gate emotional and motivational responses. Although expression of CB1 in the NAc is low, manipulation of CB1 signaling within the NAc triggers robust emotional/motivational alterations related to drug addiction and other psychiatric disorders, and these effects cannot be exclusively attributed to CB1 located at afferents to the NAc. Rather, CB1-expressing neurons in the NAc, although sparse, appear to be critical for emotional and motivational responses. However, the cellular properties of these neurons remain largely unknown. Here, we generated a knock-in mouse line in which CB1-expressing neurons expressed the fluorescent protein td-Tomato (tdT). Using these mice, we demonstrated that tdT-positive neurons within the NAc were exclusively fast-spiking interneurons (FSIs). These FSIs were electrically coupled with each other, and thus may help synchronize populations/ensembles of NAc neurons. CB1-expressing FSIs also form GABAergic synapses on adjacent medium spiny neurons (MSNs), providing feed-forward inhibition of NAc output. Furthermore, the membrane excitability of tdT-positive FSIs in the NAc was up-regulated after withdrawal from cocaine exposure, an effect that might increase FSI-to-MSN inhibition. Taken together with our previous findings that the membrane excitability of NAc MSNs is decreased during cocaine withdrawal, the present findings suggest that the basal functional output of the NAc is inhibited during cocaine withdrawal by multiple mechanisms. As such, CB1-expressing FSIs are targeted by cocaine exposure to influence the overall functional output of the NAc.

Exposure to cocaine regulates inhibitory synaptic transmission in the nucleus accumbens.

Medium spiny neurons (MSNs) within the nucleus accumbens shell (NAc) function to gate and prioritize emotional/motivational arousals for behavioral output. The neuronal output of NAc MSNs is mainly determined by the integration of membrane excitability and excitatory/inhibitory synaptic inputs. Whereas cocaine-induced alterations at excitatory synapses and membrane excitability have been extensively examined, the overall functional output of NAc MSNs following cocaine exposure is still poorly defined because little is known about whether inhibitory synaptic input to these neurons is affected by cocaine. Here, our results demonstrate multidimensional alterations at inhibitory synapses in NAc neurons following cocaine self-administration in rats. Specifically, the amplitude of miniature IPSCs (mIPSCs) was decreased after 21 d withdrawal from 5 d cocaine self-administration. Upon re-exposure to cocaine after 21 d withdrawal, whereas the amplitude of mIPSCs remained downregulated, the frequency became significantly higher. Furthermore, the reversal potential of IPSCs, which was not significantly altered during withdrawal, became more hyperpolarized upon cocaine re-exposure. Moreover, the relative weight of excitatory and inhibitory inputs to NAc MSNs was significantly decreased after 1 d cocaine withdrawal, increased after 21 d withdrawal, and returned to the basal level upon cocaine re-exposure after 21 d withdrawal. These results, together with previous results showing cocaine-induced adaptations at excitatory synapses and intrinsic membrane excitability of NAc MSNs, may provide a relatively thorough picture of the functional state of NAc MSNs following cocaine exposure.

Dopamine triggers heterosynaptic plasticity.

As a classic neuromodulator, dopamine has long been thought to modulate, rather than trigger, synaptic plasticity. In contrast, our present results demonstrate that within the parallel projections of dopaminergic and GABAergic terminals from the ventral tegmental area to the nucleus accumbens core (NAcCo), action-potential-activated release of dopamine heterosynaptically triggers LTD at GABAergic synapses, which is likely mediated by activating presynaptically located dopamine D1 class receptors and expressed by inhibiting presynaptic release of GABA. Moreover, this dopamine-mediated heterosynaptic LTD is abolished after withdrawal from cocaine exposure. These results suggest that action-potential-dependent dopamine release triggers very different cellular consequences from those induced by volume release or pharmacological manipulation. Activation of the ventral tegmental area to NAcCo projections is essential for emotional and motivational responses. This dopamine-mediated LTD allows a flexible output of NAcCo neurons, whereas disruption of this LTD may contribute to the rigid emotional and motivational state observed in addicts during cocaine withdrawal.

Increased excitability of lateral habenula neurons in adolescent rats following cocaine self-administration.

BACKGROUND: The lateral habenula is a brain region that has been critically implicated in modulating negative emotional states and responses to aversive stimuli. Exposure to addictive drugs such as cocaine negatively impacts affective states, an effect persisting longer than acute drug effects. However, the mechanisms of this effect are poorly understood. We hypothesized that drugs of abuse, such as cocaine, may contribute to drug-induced negative affective states by altering the firing properties of lateral habenula neurons, thus changing the signaling patterns from the lateral habenula to downstream circuits. METHODS: Using whole-cell current-clamp recording of acutely prepared brain slices of rats after various periods of withdrawal from cocaine self-administration, we characterized an important heterogeneous subregion of the lateral habenula based on membrane properties. RESULTS: We found two major relevant neuronal subtypes: burst firing neurons and regular spiking neurons. We also found that lateral habenula regular spiking neurons had higher membrane excitability for at least 7 days following cocaine self-administration, likely due to a greater membrane resistance. Both the increase in lateral habenula excitability and membrane resistance returned to baseline when tested after a more prolonged period of 45 days of withdrawal. CONCLUSION: This is the first study to look at intrinsic lateral habenula neuron properties following cocaine exposure beyond acute drug effects. These results may help to explain how cocaine and other drugs negatively impact affect states.

Molecular and cellular rhythms in excitatory and inhibitory neurons in the mouse prefrontal cortex.

Previous studies have shown that there are rhythms in gene expression in the mouse prefrontal cortex (PFC); however, the contribution of different cell types and potential variation by sex has not yet been determined. Of particular interest are excitatory pyramidal cells and inhibitory parvalbumin (PV) interneurons, as interactions between these cell types are essential for regulating the excitation/inhibition balance and controlling many of the cognitive functions regulated by the PFC. In this study, we identify cell-type specific rhythms in the translatome of PV and pyramidal cells in the mouse medial PFC (mPFC) and assess diurnal rhythms in PV cell electrophysiological properties. We find that while core molecular clock genes are conserved and synchronized between cell types, pyramidal cells have nearly twice as many rhythmic transcripts as PV cells (35% vs. 18%). Rhythmic transcripts in pyramidal cells also show a high degree of overlap between sexes, both in terms of which transcripts are rhythmic and in the biological processes associated with them. Conversely, in PV cells, rhythmic transcripts from males and females are largely distinct. Moreover, we find sex-specific effects of phase on action potential properties in PV cells that are eliminated by environmental circadian disruption. Together, this study demonstrates that rhythms in gene expression and electrophysiological properties in the mouse mPFC vary both by cell type and by sex. Moreover, the biological processes associated with these rhythmic transcripts may provide insight into the unique functions of rhythms in these cells, as well as their selective vulnerabilities to circadian disruption. Significance statement: This is the first study to examine translatomic rhythms in the mouse mPFC with cell-type specificity. We find that the core molecular clock cycles in phase across cell types, indicating that previously described daily oscillations in the cortical excitation/inhibition balance are not the consequence of a phase offset between PV and pyramidal cells. Nevertheless, rhythmic transcripts and their associated biological processes differ by both sex and cell type, suggesting that molecular rhythms may play a unique role in different cell types. Therefore, our results, such as the enrichment of transcripts associated with mitochondrial function in PV cells from males, point towards possible cell and sex-specific mechanisms that could contribute to psychiatric and cognitive diseases when rhythms are disrupted.

An EEG Signature of MCH Neuron Activities Predicts Cocaine Seeking.

Background: Identifying biomarkers that predict substance use disorder (SUD) propensity may better strategize anti-addiction treatment. The melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) critically mediates interactions between sleep and substance use; however, their activities are largely obscured in surface electroencephalogram (EEG) measures, hindering the development of biomarkers. Methods: Surface EEG signals and real-time Ca2+ activities of LH MCH neurons (Ca2+MCH) were simultaneously recorded in male and female adult rats. Mathematical modeling and machine learning were then applied to predict Ca2+MCH using EEG derivatives. The robustness of the predictions was tested across sex and treatment conditions. Finally, features extracted from the EEG-predicted Ca2+MCH either before or after cocaine experience were used to predict future drug-seeking behaviors. Results: An EEG waveform derivative - a modified theta-to-delta ratio (EEG Ratio) - accurately tracks real-time Ca2+MCH in rats. The prediction was robust during rapid eye movement sleep (REMS), persisted through REMS manipulations, wakefulness, circadian phases, and was consistent across sex. Moreover, cocaine self-administration and long-term withdrawal altered EEG Ratio suggesting shortening and circadian redistribution of synchronous MCH neuron activities. In addition, features of EEG Ratio indicative of prolonged synchronous MCH neuron activities predicted lower subsequent cocaine seeking. EEG Ratio also exhibited advantages over conventional REMS measures for the predictions. Conclusions: The identified EEG Ratio may serve as a non-invasive measure for assessing MCH neuron activities in vivo and evaluating REMS; it may also serve as a potential biomarker predicting drug use propensity.