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.

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.

Opioid addiction treatment is often hampered by the severe dysphoria of opioid withdrawal, but withdrawal treatments are limited by incomplete understanding of brain mechanisms involved. One area frequently implicated in withdrawal symptoms is the central amygdala, whose capsular portion (CeC) is particularly strongly activated during withdrawal. Additionally, a ventral posterior striatal region that resides near CeC, the interstitial nucleus of the posterior limb of the anterior commissure (IPACc), is also activated as strikingly as CeC. However, it is still unknown how these regions are activated, nor whether their activation explains the high intensity of withdrawal dysphoria. Using RNAscope, we found that c-fos expression is induced in the parabrachial nucleus (PB), a key glutamatergic afferent of CeC, after precipitated morphine withdrawal. Chemogenetic inhibition of PB glutamatergic neurons (VG2PB) nearly eliminated withdrawal-induced CeC c-Fos, without affecting IPACc c-Fos, indicating these two nuclei are activated by distinct sources. Furthermore, VG2PB inhibition markedly reduced somatic (jumping) and modestly reduced affective (place avoidance) withdrawal behavior. On the other hand, inhibition of CeC-projecting PB neuronal subtypes expressing calcitonin gene-related peptide (CGRP) or mu opioid receptor (MOR) reduced place avoidance without affecting jumping, indicating their specific role in withdrawal aversion. Strikingly, simultaneous inhibition of VG2PB and posterior striatal region containing IPACc robustly reduced withdrawal-induced place avoidance much more than the modest effects of either inhibition alone, suggesting their cooperative action in driving aversion. Our data suggests that PB-CeC circuit and posterior striatal area constitute a cooperative system driving opioid withdrawal aversion.

Mu opioid agonists, the most widely used opioids, produce analgesia, respiratory depression, constipation, and reward leading to substance abuse. Like all opioid receptor family members, mu receptors are Gi/o-coupled and inhibitory, yet mu-mediated disinhibition of GABAergic neurons propagates downstream activation that differs between naive and dependent animals and across receptor subtypes. Here we used TRAP2/Ai9 reporter mice, which deposit tdTomato in active neurons via the c-Fos locus, with tissue clearing and light sheet microscopy to map whole-brain neuronal activation after acute morphine (10 mg/kg), an escalating twice-daily morphine regimen producing dependence, or the NOP receptor agonist Ro 64-6198. Despite Gi/o coupling, all treatments raised global activation relative to vehicle, though this whole-brain increase reached significance only in males, and acute morphine engaged the largest number of regions, all increased with none decreased. Sex differences were prominent: males were more sensitive than females across treatments, with morphine engaging mainly pain- and reward-related midbrain circuitry in males and hypothalamic circuitry in females. In dependent mice, activation was lower across several regions, including a cluster of cerebellar regions that trended toward being turned off relative to acute morphine. NOP receptor activation produced a far weaker profile. Although chronic morphine produced less activation than acute morphine, it produced more widespread co-activation, and while hub composition differed across conditions, global network organization was preserved. These results reveal whole-brain changes in neuronal activation and establish a TRAP2/Ai9 model that renders activated regions genetically accessible for future site-directed investigation.

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.

Opioid Use Disorder (OUD) continues to be a pressing public health crisis, marked by high relapse rates and limited treatment options. Ketamine, a non-competitive N-Methyl-D-Aspartate receptor (NMDAR) antagonist, has emerged as a promising therapeutic for numerous psychiatric illnesses due to its robust antidepressant properties and glutamate-mediated synaptic plasticity. However, ketamine's therapeutic mechanisms remain elusive, with emerging evidence implicating the endogenous opioid system. Furthermore, few studies have investigated ketamine's potential as an intervention for OUD. Accordingly, this study assessed ketamine's ability to rewire endogenous opioid and glutamatergic circuitry and reduce heroin-seeking activity in male Long-Evans rats. Using a multi-cohort design, we investigated ketamine's effects on opioid-related transcriptional, protein, and signaling properties in key brain regions associated with reward and addiction, and subsequently on heroin-seeking using a self-administration paradigm. To investigate mechanisms of plasticity, we also quantified transcriptional changes to NMDAR subunits. Molecular analyses demonstrated increased expression of endogenous opioid system-related genes and endogenous opioid peptides at the protein level, elevated mu-opioid receptor protein levels, and enhanced G protein activity, suggesting increased receptor function. Notably, transcriptional changes were particularly prominent in the shell subregion of the nucleus accumbens (NAc). We also revealed that ketamine altered NMDAR subunit expression in the medial prefrontal cortex and NAc, indicating circuit-level synaptic remodeling. Behavioral analyses revealed that ketamine significantly reduced heroin-primed, but not cue-induced, seeking activity. Together, these findings suggest ketamine reduces acute relapse vulnerability to opioids through coordinated modulation of opioid and glutamatergic systems, highlighting its potential as a novel intervention for OUD.

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.

Dynamic regulation of midbrain dopamine neuron activity is necessary for diverse processes including motivation, novelty detection, reinforcement learning, and cognitive flexibility. By setting the strength of synaptic inputs to dopaminergic neurons, endocannabinoid signaling is essential for regulating dopaminergic activity. Prenatal exposure to {Delta}9-tetrahydrocannabinol (THC), the main psychoactive substance in cannabis, is known to induce abnormal dopaminergic activity and increased susceptibility to psychopathology. However, how prenatal cannabinoid exposure (PCE) affects endocannabinoid-mediated synaptic plasticity of dopamine neurons remains largely unknown. Here, we use a rat model of PCE to directly determine this. We found that endocannabinoid-mediated synaptic plasticity at excitatory synapses on dopamine neurons of the ventral tegmental area (VTA) was absent in PCE male rat offspring, where the presynaptic nanoscale architecture of excitatory afferents onto VTA dopamine neurons was reorganized to impair the control of type-1 cannabinoid receptors on glutamate release. We demonstrate that, in response to postsynaptic depolarization, PCE dopamine cells switch to nitric oxide (NO) rather than endocannabinoid signaling to induce opposing forms of synaptic plasticity at inhibitory and excitatory inputs. We disclosed a NO-dependent long-term potentiation of GABAA-receptor-mediated synaptic transmission, and a long-term depression of glutamatergic synapses requiring presynaptic activation of GABAB-receptors. These PCE-induced reciprocal forms of synaptic plasticity reshape the balance of excitatory and inhibitory control over dopamine neurons, potentially contributing to increased vulnerability to psychiatric disorders.

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.

Adolescence is characterized by increased novelty seeking, impulsivity, locomotion and risk-taking, behaviours that facilitate the transition to adulthood but may also increase vulnerability to psychiatric illness. Maturation of dopamine (DA) transmission within the striatum is thought to contribute to these behavioural changes. However, the developmental trajectory of striatal DA release remains unclear, with conflicting evidence regarding age- and sex-dependent changes, particularly across dorsal and ventral striatal regions. Complicating interpretation, many studies classify mice younger than 2 months as 'adults,' potentially obscuring important developmental differences. Here, we examined how age, sex and striatal subregion influence DA release during adolescence and early adulthood. DA release was measured in dorsal and ventral striatal brain slices from male and female C57BL/6J mice at 2 and 4 months of age, corresponding to adolescence and young adulthood. Ultrafast imaging of the genetically encoded DA sensor, dLight, was used to quantify extracellular DA transients evoked by low- and high-frequency electrical stimulation. Glutamatergic and cholinergic transmission was pharmacologically blocked to isolate DA release intrinsic to dopaminergic axons. DA release measures were related to exploratory and locomotor behaviour. We identified a male-specific increase in dorsal striatal DA release between 2 and 4 months of age that coincided with increased locomotion and risk-taking. In contrast, DA release in the ventral striatum did not show comparable age- or sex-dependent changes. These differences were not attributable to altered glutamatergic or cholinergic contributions to DA release. Together, these findings demonstrate that maturation of striatal DA transmission is region- and sex-dependent, with prominent developmental changes occurring in the dorsal striatum during the transition from adolescence to adulthood. These findings help reconcile conflicting reports of developmental DA release and emphasize the importance of considering age, sex and striatal subregion when defining mature dopaminergic function in rodents.

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.

The ventral subiculum (vSub), a major output node of the hippocampal formation, has been implicated in diverse neurological disorders. Disruption of excitatory-inhibitory balance is a common feature of neurological disorders, with altered GABAergic signaling representing a critical component. More recently, the vSub has gained increasing attention as a component of neural circuits underlying emotional and motivational regulation, through its extensive connections with regions implicated in mental health disorders such as addiction and anxiety. Our previous work in models of alcohol use disorder and comorbid anxiety identified an important role for GABAergic inhibition in regulating vSub excitability, prompting us to investigate the functional consequences of recruiting the broader local GABAergic population. Although previous studies have examined individual interneuron subtypes, the network consequences of activating the global population of local GABAergic neurons remain poorly understood. To address this question, we expressed channelrhodopsin using an mDlx-dependent strategy to target GABAergic neurons in the ventral hippocampus and performed in vivo electrophysiological recordings from vSub neurons in anesthetized rats. Repeated activation of this GABAergic population at 20 Hz, a stimulation frequency commonly used in behavioral optogenetic studies, produced robust suppression of pyramidal neuron firing during stimulation. This direct inhibition was accompanied by network-dependent increases in post-stimulus activity that varied as a function of baseline activity, sex, and trial number. Complementary ex vivo electrophysiological experiments further supported network-dependent effects and revealed short-term synaptic plasticity that evolved across repeated stimulation trials, reflecting contributions from both pre- and postsynaptic mechanisms. Together, these findings demonstrate that recruitment of the broader GABAergic population produces effects extending beyond direct inhibition and can engage dynamic network responses that depend on stimulation history and circuit state. These findings highlight the importance of stimulation frequency, timing, and network context when interpreting the functional consequences of GABAergic circuit manipulation.

GABAergic inhibitory neurons play a central role in shaping network dynamics, and accordingly, their dysfunction is implicated in the pathophysiology of various psychiatric, neurodevelopmental, and neurological disorders. In the past decade, studies on vasoactive intestinal polypeptide-expressing inhibitory neurons (VIP+ neurons) have positioned them as critical components of this circuitry. In particular, VIP+ neurons have been proposed to function within a canonical disinhibitory circuit motif, preferentially inhibiting other interneuron subtypes, to ultimately modulate the gain and activity of local pyramidal neurons. However, their contribution to psychiatrically relevant behavioral endophenotypes is still not fully understood, limiting our ability to target these neurons for therapeutic purposes. In the present study, we systematically assessed the functional contribution of VIP+ neurons to affective and social behaviors. To this end, we used a chemogenetic approach to manipulate the activity of VIP+ neurons in core corticolimbic regions, including the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC). The consequences of either activating or inhibiting VIP+ neurons in these regions were observed while mice underwent a battery of behavioral tasks assessing anxiety-related avoidance behaviors, social interest and recognition, and associative fear learning. While sustained modulation of VIP+ neurons had no significant effect on anxiety-related or social behaviors in any of the brain regions assessed, it produced a robust reduction in associative fear learning specifically in the mPFC. These findings demonstrate that the consequences of sustained VIP+ neuron modulation depend on both anatomical location and behavioural context. Moreover, they identify VIP+ neurons in the mPFC as potentially clinically relevant targets for interventions aimed at modulating fear-related circuits.

Adverse childhood experiences like social isolation (SI) contribute to long term negative health outcomes and the development of severe psychiatric disorder. Adolescence is a unique period of coordinated social and motivational system development driven by dopamine (DA) signaling. Microglia, immune cells of the brain, may play a unique role in regulating SI-induced DA system changes and associated behavioral dysfunction. Male microglia refine dopamine D1 receptors (D1rs) in the nucleus accumbens (NAc) of adolescent male rats shaping play behavior. Here we characterized the molecular developmental impacts of postweaning SI from P21-P38, with a resocialization period to P54. We tested the role of microglial engulfment of D1rs in mediating behavioral consequences of SI using neutrophil inhibitory factor (NIF) to block complement-mediated engulfment required for D1r pruning. We found sex specific impacts of SI on brain development and the DA system. In SI males, microglia engulf more NAc D1rs than group housed (GH) controls. Within GH animals, NAc D1r density is positively correlated to play and allogrooming. In SI animals, NAc D1r density is negatively correlated to biting behavior, and is not correlated to play or allogrooming. Pharmacologically inhibiting microglial engulfment of D1r via bilateral NAc NIF injection prevents observed D1r:behavior correlations in GH and SI animals. NIF treatment interacts with SI, exaggerating aggressive-like behavior, showing a role for microglia in regulating aggressive-like behavior via complement pruning in adolescence. Microglia may play a unique role in regulating behavior that drive negative health outcomes in the face of developmental stress, including SI.

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.

The nucleus accumbens (NAc) is a key structure translating dopaminergic inputs into social avoidance following repeated social defeat. Within the NAc, dopamine (DA) release is modulated in part by cholinergic interneurons (CINs). CINs express the vesicular glutamate transporter type 3 (VGLUT3), which supports glutamate co-release and regulates vesicular acetylcholine (ACh) loading, thereby fine-tuning cholinergic signaling. A rare human VGLUT3-p.T8I missense variant is associated with compulsive behaviors toward food and drugs of abuse. We hypothesized that p.T8I disrupts NAc ACh dynamics and affects social avoidance following social defeat. Male and female mice expressing the VGLUT3-p.T8I variant were exposed to a 10-day chronic social defeat stress (CSDS) paradigm. Social avoidance toward the aggressor strain and a C57BL/6 (B6) conspecific was measured using the social interaction and 3-chamber tests, and anxiety-like behavior was evaluated in the elevated zero maze. Fiber photometry was used to monitor ACh and DA dynamics in the NAc medial shell (NAcMsh) during CSDS and social interaction. After CSDS, VGLUT3T8I/T8I males showed aggressor strain-specific social avoidance and increased anxiety-like behavior compared to WT mice. Mutants showed blunted CSDS-induced ACh increase during attacks but enhanced ACh release upon re-exposure to the CD1 aggressor strain correlating with avoidance severity. DA dynamics were minimally affected. Females, regardless of genotype, displayed a male-mutant-like phenotype. In males, the VGLUT3-p.T8I variant alters NAcMsh ACh signaling and promotes selective social avoidance toward aggressors and elevated anxiety after CSDS, highlighting a potential cholinergic mechanism underlying stress-induced social avoidance with broader relevance to psychiatric disorders.