Neuropsychopharmacology

Cocaine use disorder remains a critical public health concern with limited treatment options. Astrocytes are increasingly recognized as active regulators of neurotransmission and are emerging as important contributors to addiction neurobiology. Here, we examined how chemogenetic activation of astrocytic Gq signaling within the ventral tegmental area (VTA) influences cocaine-associated behaviors in drug-naive and cocaine-experienced rats. Using a subthreshold dose of cocaine in a conditioned place preference (CPP) paradigm, we found that VTA astrocyte Gq activation facilitated the acquisition of cocaine CPP in drug-naive rats but suppressed the development of cocaine CPP in cocaine-experienced rats. Additionally, chemogenetic activation of VTA astrocyte Gq signaling suppressed voluntary cocaine intake in a self-administration paradigm. Together, these findings demonstrate that VTA astrocyte Gq signaling shapes cocaine-associated behaviors in a drug history-dependent manner, highlighting VTA astrocytes as a potential therapeutic target in cocaine use disorder.

Cocaine use disorder (CUD) remains a major public health concern with no FDA-approved pharmacotherapy, underscoring the need to define the cellular and molecular adaptations produced by voluntary cocaine taking. The nucleus accumbens (NAc) is a key substrate for cocaine reinforcement and drug-seeking behavior, but interpretation of the functional role of its cellular heterogeneity in these behaviors is limited by past bulk transcriptomic studies. Here, we used single-nucleus RNA sequencing to profile the NAc of male and female rats that self-administered intravenous cocaine for 10 consecutive days versus yoked saline controls. After quality control, we analyzed 36,766 nuclei spanning major neuronal, glial, and vascular cell populations. Pseudobulk differential-expression analyses identified 478 cocaine-associated cell type-specific transcriptional changes that were concentrated in discrete medium spiny neuron (MSN) subclasses and astrocytes. D1 Ebf1+ MSNs showed the largest transcriptomic response, accounting for [~]40% of all differential-expression events, followed by D2 Stk32a+ MSNs, astrocytes, and D1 Ppm1e+ MSNs. These responses were largely cell type-specific, indicating that cocaine self-administration engages multiple molecular programs rather than a uniform accumbens-wide transcriptional signature. Immediate-early gene module-score analyses further revealed cocaine-associated activation states in select neuronal and non-neuronal cell populations, including D1 Ebf1+ MSNs, Drd3+ neurons, Sst+ interneurons, astrocytes, and oligodendrocytes. Gene-set, pathway, and upstream-regulator analyses nominated synaptic organization, axon guidance, RAS/MAPK signaling, NMDA receptor-associated signaling, and CREB-related transcriptional regulation as candidate mechanisms of cocaine-evoked plasticity. Together, these data provide a cell type-resolved resource for understanding how voluntary cocaine taking alters the rat NAc transcriptome and identifies discrete neuronal and glial cell populations for future mechanistic studies using preclinical CUD models.

Opioid withdrawal is a powerful negative affective state that promotes continued drug use and relapse, and is often associated with hypodopaminergic states. Although corticotropin-releasing factor (CRF) signaling in the extended amygdala has been strongly implicated in stress and negative affect, how withdrawal-recruited CRF systems suppress dopamine release remains poorly understood. Here, we identify an extended amygdala-striosome-dopamine pathway engaged during fentanyl withdrawal. Naloxone-precipitated fentanyl withdrawal selectively recruited CRF neurons in the central amygdala and bed nucleus of the stria terminalis. These CRF neurons provided monosynaptic input to dorsostriatal medium spiny neurons (MSNs). Because striosomal MSNs are positioned to regulate dopamine release, we next found that CRF enhanced glutamatergic synaptic transmission onto striosomal MSNs, with CRF receptor 1 (CRFR1) signaling required for the postsynaptic strengthening of excitatory transmission. In vivo dopamine photometry further revealed that naloxone-precipitated withdrawal amplified striosome-mediated suppression of dopamine release, and this amplification was prevented by CRFR1 antagonism. Together, these findings reveal a mechanism by which withdrawal-recruited CRF signaling potentiates striosomal control of dopamine release. This extended amygdala-striosome-dopamine pathway may contribute to the hypodopaminergic negative affective state that drives opioid withdrawal and relapse vulnerability. HighlightsO_LIFentanyl withdrawal selectively recruits CeA and BNST CRF neurons C_LIO_LICeA and BNST CRF neurons provide monosynaptic input to striatal MSNs C_LIO_LICRF potentiates glutamatergic transmission onto striosomal MSNs via CRFR1 C_LIO_LICRFR1 signaling amplifies striosome-mediated dopamine suppression during withdrawal C_LI

PVT is involved in stress responses, fear, anxiety, arousal, aversion and reward. Anterior and posterior PVT (aPVT and pPVT) have different neuronal connections, molecular contents, and functional roles. PVT expressed a high level of KOR. Herein we mapped the projection targets of aPVT KOR(+) neurons and explored the behavioral significance of aPVT KOR. Using KOR-iCre mice and Cre-dependent anterograde tracer, we found that KOR(+) glutamatergic neurons in aPVT primarily projected to NAc, CeA, BNST, PFC, and reticular nucleus of the thalamus (RT). 3-D images showed the pathway emanating from aPVT to the ventral RT, through NAc, and out to the other regions, indicating widespread axonal collateralization. We conditionally knock-downed KOR (KOR cKD) in aPVT by injection of AAV-eGFP-Cre or AAV-eGFP (control) into aPVT of Oprk1lox/lox mice. [3H]U69,593 receptor autoradiography revealed substantial KOR cKD in aPVT. In both male and female mice, the KOR cKD in aPVT significantly reduced anxiety-like behaviors in the elevated plus-maze test, cue-induced freezing after fear conditioning and naloxone-precipitated morphine withdrawal-associated jumps. KOR cKD attenuated U50,488H-induced conditioned place aversion in males only, while having no effect on forced swim immobility or the U50,488H-induced visceral analgesic and antipruritic effects in either sex. Thus, our results reveal for the first time that KOR-mediated inhibition of aPVT neurons may mediate morphine withdrawal, anxiety, and cue-induced fear in both sexes but contribute to KOR agonist-induced aversion only in males. Notably, our findings reveal a previously unrecognized role for aPVT in regulating morphine withdrawal, acting in a manner distinct from pPVT.

Cannabis is the most common illicit drug abused worldwide, and its consumption has substantially increased among pregnant women. We previously demonstrated that male preadolescent offspring prenatally exposed to {Delta}9-tetrahydrocannabinol (THC), a model of prenatal cannabinoid exposure (PCE), exhibit a mesolimbic dopamine (DA) neuron dysfunction contributing to at-risk psychotic-like (endo)phenotypes that are unmasked by acute THC exposure at preadolescence. Dysregulation of mesocortical DA signaling along with prefrontal cortex (PFC) function is also a central feature of psychotic disorders. Furthermore, studies investigating the impact of PCE on PFC in the offspring at preadolescence, a window of heightened plasticity and vulnerability, are limited. To fill this gap, we applied a multiscale analysis of mesocortical DA transmission and PFC function in PCE preadolescent offspring by integrating behavioral, neurochemical, electrophysiological, and molecular approaches. PCE enhanced spontaneous repetitive behaviors in a male-specific manner. PCE also abolished sex differences in the intrinsic excitability of PFC pyramidal neurons and Netrin-1 expression. In addition, PCE altered the expression of genes associated with endocannabinoid signaling without changing basal and THC-induced extracellular levels of DA in the PFC. Collectively, these findings demonstrate that prenatal THC exposure disrupts both proper maturation and sexual differentiation of PFC circuitry, thus extending the impact of PCE from previously described mesolimbic abnormalities to mesocortical pathway. Finally, our data identify early cortical molecular and cellular alterations that may contribute to neuropsychiatric vulnerability later in life. Highlights* Preadolescent male rats exposed in utero to THC display repetitive behavior * Prenatal cannabinoid exposure (PCE) does not alter dopamine transmission in the PFC * PCE potentiates AMPA-mediated transmission in male pyramidal cells * PCE abolishes sex differences in Netrin-1 expression levels in the PFC

Relapse following prolonged abstinence is a primary challenge in the treatment of opioid and cocaine use disorders, driven in part by enduring dysfunction of medial prefrontal cortex (mPFC) circuits that impair inhibitory control over drug-seeking. The molecular substrates underlying this dysfunction, and whether they differ across drug classes, remain unknown. Here, we performed label-free quantitative proteomics of mPFC synaptosomes isolated from rats after 30-day abstinence following intravenous heroin or cocaine self-administration to profile synaptic adaptations that may contribute to relapse vulnerability. Heroin abstinence induced extensive synaptic proteomic remodeling characterized by coordinated downregulation of mitochondrial proteins involved in oxidative phosphorylation, including pyruvate dehydrogenase complex subunits that regulate carbon entry into mitochondrial metabolism. Targeted metabolomic profiling of whole mPFC revealed accumulation of upstream glycolytic and pentose phosphate pathway intermediates, consistent with altered pyruvate utilization and mitochondrial oxidation. Several bioenergetic metabolites also correlated positively with the severity of escalation of heroin intake. Consistent with the bioenergetic remodeling observed during protracted heroin abstinence, whole-cell patch-clamp recordings from layer V mPFC pyramidal neurons revealed lasting suppression of intrinsic excitability and a decreased spontaneous excitatory synaptic activity. Cocaine abstinence, by contrast, produced limited changes in synaptic bioenergetics while inducing a distinct cytoskeletal remodeling signature. Overall, these findings identify synaptic bioenergetic remodeling as a previously underappreciated feature of prolonged heroin abstinence and reveal a marked divergence in the molecular adaptations induced by heroin versus cocaine within the mPFC. These results implicate mitochondrial bioenergetic pathways as therapeutic targets for reducing relapse vulnerability specifically associated with opioid use disorder.

N, N-dimethyltryptamine (DMT) and 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT) are structurally related tryptamine psychedelics with emerging therapeutic potential, yet their comparative acute pharmacology and longer-term neuroplastic effects remain incompletely defined. Here we show that DMT produces a bell-shaped dose-response curve in the mouse head-twitch response (HTR) assay, whereas 5-MeO-DMT elicits a monotonic increase. Selective antagonism at 5-HT2A or 5-HT1D receptors, or agonism at 5-HT1A, robustly attenuates HTR for both compounds without abolishing their ability to reduce marble burying, a screening assay for OCD-like behavior. Acutely, both agents elevate TrkB phosphorylation in a region-specific manner, with broader engagement by DMT across default-mode-network and hippocampal territories. Twelve days after a single dose, both compounds increase synaptic proteins (PSD-95, synaptophysin; SV2A for DMT), while DMT uniquely lowers hippocampal BDNF and reprograms frontal-cortex glutathione and energy metabolism. These findings demonstrate that acute hallucinogenic-like activity and selected therapeutic-like behavioral and plasticity outcomes can be pharmacologically dissociated, informing the rational design of more tolerable, scalable psychedelic-based treatments.

Impulsive-compulsive disorders (ICDs), including pathological gambling, hypersexuality, and compulsive buying, are frequently precipitated by dopamine D2/3 receptor agonists such as pramipexole (PPX), yet the molecular mechanisms that confer individual vulnerability remain poorly understood. Impulsive choice, a core dimension of ICDs, is modulated by dopaminergic signaling within corticostriatal circuits, but the microRNAs (miRs) that potentially translate this signaling into persistent behavioral change have not been identified. Here, we combined a delay discounting task (DDT) with high-throughput miR sequencing in the dorsal striatum and nucleus accumbens (NAcc) of rats stratified by baseline impulsivity and subchronic PPX treatment. PPX increased impulsive choice selectively in low- and mid-impulsive rats, whereas high-impulsive rats remained unaffected, consistent with a ceiling effect. Among the differentially expressed miRs, miR-10a-5p emerged as the strongest candidate: it was constitutively elevated in high-impulsive rats and upregulated by PPX in low- and mid-impulsive animals in both regions, thereby paralleling the trait-dependent behavioral effect of the drug. In vivo viral-mediated overexpression of miR-10a-5p confirmed its predicted downregulation of the PI3K-AKT-mTOR and BDNF pathways in the striatum, and, critically, overexpression restricted to the NAcc, but not the dorsal striatum, was sufficient to increase impulsive choice, recapitulating the pro-impulsive effect of PPX. These findings identify miR-10a-5p as a critical molecular regulator of impulsivity through its activity in the NAcc, providing a mechanistic link between dopaminergic perturbation, trait vulnerability, and ICDs, and opening new avenues for the development of miR-directed therapeutic strategies for these disorders.

Post-traumatic stress disorder (PTSD) is a highly prevalent, debilitating psychiatric condition. Existing treatments are ineffective for many patients. 3,4- methylenedioxymethamphetamine (MDMA)-assisted psychotherapy has demonstrated substantial clinical efficacy but relies on prolonged, resource-intensive therapeutic protocols that limit scalability and accessibility. Here, we investigated whether combining MDMA with exposure-based intervention could enhance therapeutic efficiency in a preclinical model of PTSD-like behavior. Using a learned helplessness paradigm in mice, we identified trauma-susceptible individuals based on persistent escape failures following inescapable stress. Traumatised mice subsequently received brief treatment regimens consisting of MDMA or saline vehicle administered with or without exposure to the traumatic cue. Behavioral outcomes were tracked longitudinally using active avoidance performance as the primary endpoint, complemented by assays of anxiety- like, depressive-like, cognitive, and social behaviors. MDMA treatment markedly reduced trauma-associated behavioral deficits. MDMA combined with exposure produced rapid and sustained recovery compared to control conditions. Statistical analyses revealed significant treatment- and time-dependent effects on avoidance behavior, indicating accelerated resilience acquisition in MDMA-treated groups. Additional behavioral assays demonstrated dose-dependent effects of MDMA on anxiety- and depression-related measures. Together, these findings provide proof-of-principle that pharmacological modulation with MDMA can enhance exposure-driven behavioral recovery, supporting a strategy to integrate MDMA into more efficient and accessible PTSD treatment frameworks. This work establishes a preclinical foundation for clinical studies aimed at optimizing MDMA-assisted interventions to improve scalability and patient access.

A single dose of psilocybin can induce long-lasting antidepressant effects. The neurobiological mechanisms underlying such sustained antidepressant effect remain insufficiently understood, particularly at the level of synaptic function and drug-target specificity. Here, we aimed to delineate single-dose psilocybin-induced excitatory synaptic plasticity. Synaptic plasticity was assessed by whole-cell patch-clamp recording of excitatory synaptic transmission 24 h after treating mice with single-dose psilocybin. We correlated the recordings with transcriptomics data and used a conditional single-vector CRISPR/SaCas9-dependent knock-out strategy to validate the role of the 5-HT2A receptor. Psilocybin selectively increased the frequency of miniature excitatory postsynaptic currents in specific cortical sub-regions and in the amygdala. Frequency correlated with the expression levels of psilocin-targeted serotonin receptors, when expression heterogeneity between cortical subregions and along the anterior-posterior axis was accounted for. Post-synaptic Htr2a knock-out in the insular/orbitofrontal cortex precluded psilocybin-induced 24-h plasticity. These findings demonstrate that lasting psilocybin-induced effects on excitatory synaptic transmission manifest with brain-region specificity, likely reflecting a functional consequence of synapse formation. This work establishes a foundation for a circuit-specific, mechanistic understanding of functional aspects of psilocybin-induced neuroplasticity.

BackgroundAlthough nicotine reinforcement is often attributed to mesolimbic dopamine neurons in the ventral tegmental area, accumulating evidence indicates that additional brain circuits contribute to its reinforcing effects. AimsThe hypothalamic supramammillary region (SuM) has been implicated as one such substrate, yet the cellular targets and circuit mechanisms through which nicotine engages this region remain poorly understood. MethodsWe combined RNAscope in situ hybridization to identify nicotinic acetylcholine receptor (nAChR) subunits, intravenous nicotine self-administration in mice to determine doses that reliably support reinforcement, and fiber photometry to monitor calcium activity in SuM VGluT2 neurons in vivo. ResultsMice exhibited reliable nicotine self-administration across a range of doses under fixed-ratio and progressive-ratio schedules. RNAscope analysis revealed prominent expression of the {beta}2 nAChR subunit in VGluT2-expressing neurons projecting from the SuM to the medial septum. Fiber photometry recordings showed that reinforcing doses of nicotine produced rapid, infusion-locked increases in GCaMP signals in SuM VGluT2 neurons. ConclusionsThese findings identify nAChR-expressing SuM neurons as a candidate circuit substrate engaged by reinforcing doses of nicotine and extend current models of nicotine reinforcement beyond canonical mesolimbic dopamine pathways.

Background and aimsSubstance-use addiction models have demonstrated that a progressive transition from reward-guided to habitual and ultimately compulsive behaviour is mediated by a ventral-to-dorsal striatal shift in behavioural control. While symptomatic and neural similarities between substance and digital technology-based disorders have been hypothesised, the causal role of the striatum in the latter remains unknown. We here employ a validated mouse light self-administration paradigm with a loss-of-function approach to determine the role of the dorsal striatum in behavioural persistence towards non-food, non-drug reinforcers. MethodsMice received a control virus or a virus expressing the inhibitory designer receptor (hM4Di) into the dorsal striatum (caudate-putamen). The chemogenetic actuator clozapine N-oxide (CNO) was injected systemically shortly before selected sessions, silencing striatal neuronal firing in vivo in hM4Di-expressing mice. During operant training, lever presses were reinforced by light under fixed-ratio schedules of reinforcement (FR1, FR3, and FR5). ResultsOn initial training days, CNO reduced light self-administration in striatal hM4Di-expressing mice. CNO had a negligible effect in control mice under FR3 and FR5 but attenuated responding in hM4Di mice, which showed response recovery on days without CNO. Linear mixed-effects models confirmed an improvement in light self-administration across days, with a stronger detrimental effect of CNO in hM4Di mice. DiscussionDorsal striatum silencing attenuates light self-administration without impairing response acquisition. Thus, like drug self-administration, light self-administration relies partly on dorsal striatal neurons. Our work bridges the gap between animal models and human neuroimaging studies reporting shared brain mechanisms underlying non-drug and drug habits.

Nearly five decades ago, it was first observed that long-term memory consolidation requires waves of transcriptional activity and protein synthesis, with the first two waves occurring within hours after learning. While numerous studies have examined the effects of protein synthesis inhibitors, the contribution of epigenetic mechanisms in these waves remains poorly understood. Here, we aimed to determine the role of HDAC3, a key modulator of memory, in these two phases of gene expression, as well as its functional link with the transcription factor NF-{kappa}B, one of its deacetylation targets, and a critical player in memory formation. Pharmacological inhibition of HDAC3 with RGFP966, either immediately or 6 hours after training, enhanced memory persistence in the NOR task in mice. Conversely, inhibition of NF-kB with BAY 11-7082 impaired memory at the same time points. Moreover, RGFP966 injection increased the nuclear proportion of NF-{kappa}B in the CA1 region of the hippocampus, suggesting a functional link between HDAC3 activity and NF-{kappa}B nuclear translocation. To our knowledge, this study provides the first in vivo evidence of this relationship during memory consolidation, extending previous findings from cell culture and electrophysiological studies in brain slices.

Background and Hypothesis. Motor alterations represent an important component of psychotic disorders. Chronic cannabis use, a key risk factor for psychosis, is also associated with sensorimotor dysfunctions. Yet, the hypothesis of a common sensorimotor disturbance remains underinvestigated. Study Design. In this study, we examined submovements, elementary units of motor output, to search for common subclinical impairments in these populations. Patients with psychosis (n = 17), heavy cannabis users (n = 21), and healthy controls (n = 17) performed a continuous visuomotor synchronization task, consisting in tracking a dot moving on a screen with a finger. Study Results. Individuals with psychosis and cannabis users exhibited less frequent and more variable submovements compared with healthy controls. Furthermore, when interacting with a pre-recorded human kinematic profile, both groups exhibited attenuated responses to the observed submovements. This alteration was found to be more pronounced in patients with psychosis. Conclusions. These findings suggest that submovement analysis may reveal subtle, shared alterations in sensorimotor integration in psychosis and chronic cannabis use, providing an objective window onto motor dysfunction not readily captured by current clinical tools.

GLP-1 agonist drug repurposing efforts may establish new clinical niches in managing psychiatric and neurological disorders. However, a comprehensive understanding of GLP-1 neurobiology and an appreciation of specific neural mechanisms by which GLP-1 agonists might provide therapeutic effects is limited and stands as a barrier to these efforts. When considering current preclinical research evaluating GLP-1 agonist central mechanisms, a considerable knowledge gap remains regarding which specific cellular populations and systems define potential therapeutic effects in the brain. In this research, we used cFos immunohistochemistry to identify specific neuronal populations that exhibited altered cellular activity following acute administration of semaglutide to rats. We found that the orexin/hypocretin and basal forebrain cholinergic systems are activated following acute semaglutide administration in male and female young adult rats (3-5 months). Informed by the results of our histological analysis, we next employed in vivo microdialysis to test our hypothesis that semaglutide would acutely increase acetylcholine release in the rodent hippocampus. Here, we report that semaglutide acutely increases acetylcholine efflux in the ventral hippocampus of conscious and freely moving rats regardless of biological sex in both young adult and aged rats (23-26 months). Given the relevance of hippocampal cholinergic neurotransmission in learning and memory, our research mechanistically connects GLP-1 agonists with established targets in cognitive decline and dementia.

Dysregulation of the delta-type glutamate receptor GluD1 and N-methyl-D-aspartate receptors (NMDARs) is implicated in neuropsychiatric disorders including schizophrenia and intellectual disability, and GluD1 modulates NMDAR response in hippocampal neurons. However, the precise mechanisms by which GluD1 influences specific NMDAR subtypes remain undefined, representing a critical gap given the reliance of synaptic plasticity and cognition on NMDAR composition. GluN2A- and GluN2B-containing NMDARs are essential for synaptic long-term potentiation (LTP) and contextual learning and memory. Here, we used CRISPR/Cas9 to generate GluD1 knockout (KO) in cultured hippocampal neurons and observed a selective decrease in GluN2B-containing NMDAR responses. In acute hippocampal slices, GluD1 KO similarly reduced GluN2B-containing NMDAR currents at ventral CA1[->]subiculum synapses and impaired LTP at these synapses. In vivo, region-specific GluD1 deficiency in the ventral subiculum disrupted long-term contextual memory, indicating a critical role for GluD1 in cognitive processes. These findings demonstrate that GluD1 is indispensable for preserving GluN2B-containing NMDAR function, synaptic plasticity, and memory, providing molecular insight into how GluD1 regulates NMDAR subtypes implicated in synaptic dysfunction in neuropsychiatric disorders. Understanding this mechanism will guide the development of therapeutic strategies that selectively target GluD1-dependent modulation of NMDAR subtypes in brain disease.

Background Prenatal cannabidiol (CBD) consumption is increasing, driven by a perception of safety relative to delta-9-tetrahydrocannabinol (THC). However, the neurodevelopmental risks of gestational CBD remain largely uncharacterized. Methods Using a sex-disaggregated framework, we investigated adult (P100-140) mouse offspring following in utero exposure (GD5-18; 3 mg/kg) to THC or CBD. Behavioral strategies were evaluated through risk-assessment and repetitive behavior tasks, coupled with targeted electrophysiological mapping of medial prefrontal cortex Layer 5 neurons, the primary hub for approach-avoidance arbitration. Results We found that increased repetitive behavior was a universal feature of prenatal cannabinoids exposure. Alterations in risk appraisal emerged uniquely in CBD-exposed females and appeared dissociated from classical anxiety metrics. At the circuit level, THC and CBD were linked to an absence of endocannabinoid long-term depression (eCB-LTD). In males, CBD exposure coincided with a bidirectional plasticity collapse characterized by functional saturation, elevated AMPA/NMDA ratios, and slowed NMDAR activation kinetics. This ceiling effect may represent a top-down constraint on the prefrontal output circuit, potentially limiting the synaptic flexibility typically associated with adaptive behavioral transitions. In contrast, females exhibited compound-specific reorganizations of E/I balance. CBD-exposed females displayed a scaled-up architecture that preserved net E/I balance, whereas THC was associated with a pro-excitatory phenotype through the collapse of inhibitory control. Conclusions Despite a shared loss of eCB-LTD, distinct synaptic remodeling might underlie divergent alterations in risk assessment and behavioral flexibility. This sex-specific circuit rewiring provides a neurobiological framework for the long-term behavioral risks associated with gestational cannabinoid exposure.

Chronic cocaine triggers changes in brain function that persist long after drug taking has ceased. Relapse to substance use during abstinence can be triggered by drug-associated cues, implicating the drug-free period after chronic cocaine as a time when the probability of relapse is modulated by plasticity mechanisms. Here, we studied the regulation and function of transcriptional plasticity activated in a time-dependent manner in dopaminergic and glutamatergic neurons of the mouse ventral tegmental area (VTA) over a drug-free period after chronic cocaine. We used in vivo dCas9/CRISPR inhibition to demonstrate a causal role for Brain-Derived Neurotrophic Factor transcription in the incubation of cocaine seeking during abstinence, and we used single-nucleus sequencing to identify a program of gene regulation induced in VTA neurons selectively by the prolonged absence of cocaine. These data advance the understanding of plasticity mechanisms that modulate reward functions of VTA neurons, which may be a main factor propagating relapse in substance use disorders.

Major depressive disorder and bipolar depression are disabling illnesses associated with impaired neuroplasticity, mitochondrial dysfunction, and altered cellular bioenergetics. Available pharmacotherapies often have delayed onset, incomplete efficacy, and limited effects on underlying plasticity and energetic systems. Non-selective thyroid hormone therapies provide evidence that enhancing central thyroid hormone signaling may improve depressive symptoms, but systemic cardiovascular, skeletal, and metabolic effects limit their broader use. Rapid-acting treatments such as esketamine and psychedelics further support neuroplasticity as a therapeutic strategy, although psychotomimetic, hallucinogenic, and implementation burdens may constrain widespread use. Elunetirom is a brain-targeted, fatty acid amide hydrolase-sensitive prodrug of LL-340001, a potent thyroid hormone receptor activator as demonstrated in transfected cell-line and brain-slice target-engagement assays. We evaluated elunetirom and LL-340001 in primary cortical and mature hippocampal neuronal cultures. LL-340001 increased MAP-2-positive neuron number, neurite length, branching, and neurite extremities in immature cortical neurons. In mature hippocampal neurons, elunetirom rapidly increased neurite network and synapse number within 24 hours, with effects persisting through 72 hours, while LL-340001 preserved synapse number following amyloid-{beta}1-42 challenge. LL-340001 also increased nuclear PGC-1 and NRF2, the number of functional mitochondria measured by MitoTracker, and ATP content. Pharmacologic inhibition indicated that the LL-340001-induced increase in cortical MAP-2-positive neuron number was sensitive to TrkB inhibition but not 5-HT2A antagonism. Together, these findings indicate that elunetirom and LL- 340001 engage complementary neuroplasticity and mitochondrial biogenesis-related programs in neuronal systems and support further investigation of brain-targeted thyromimetic signaling as a mechanistically differentiated approach for major depressive disorder and bipolar depression.

Psychedelic drugs offer a potentially promising avenue for novel therapeutics for the treatment of psychiatric conditions, including for mood and anxiety disorders. Emerging clinical studies have prompted increased preclinical research into mechanisms of action. However, post-acute behavioral outcomes in rodents remain inconsistent and difficult to reproduce across laboratories. To address this, we collected a large dataset of behavioral responses to psilocybin in mice (N=693) and used data-driven analyses to identify biological and experimental factors that modulated behavioral readouts. We examined the effects of sex, age, strain, stress protocol, and 5-HT1B signaling on the response to a single high dose of psilocybin measured in acute (head twitch responding and locomotion) or post-acute (sucrose preference, elevated plus maze, novelty-suppressed feeding) behavioral outcomes. We found that psilocybin displays a robust acute behavioral response and a more variable post-acute behavioral profile with a most robust response in sucrose preference. Sex, stress model, and 5-HT1BR activation emerged as key modulators of post-acute antidepressant-like and anxiolytic effects. Random forest classification was narrowly able to predict drug treatment based on post-acute behavioral outcomes and biological and experimental factors. These findings suggest that the heterogeneity seen in the response to psilocybin in mice reflects meaningful biological and experimental variables. Accounting for these factors may enhance translational relevance by identifying the conditions under which therapeutic-like effects are most robust and can support more reproducible models for understanding the neural mechanisms underlying the persisting behavioral effects of psilocybin.