Addiction Biology

Cannabis is one of the most widely used psychoactive substances worldwide, and cannabis use disorder (CUD) is increasingly recognized as a major public health concern. Clinical evidence indicates that women may be particularly vulnerable to developing addiction, exhibiting a faster transition from initial drug use to loss of control and increased relapse vulnerability. However, females remain underrepresented in both preclinical and clinical research, limiting our understanding of the neurobiological mechanisms underlying this susceptibility. Here, we investigated sex differences in behavioral and epigenetic susceptibility to cannabinoid addiction using a mouse operant self-administration model with the synthetic cannabinoid agonist WIN 55,212-2. Female mice displayed increased addiction-like behavior, characterized by greater persistence of responding during drug-free periods and enhanced compulsive-like drug seeking compared to males. miRNA profiling in the medial prefrontal cortex (mPFC) identified a female-specific epigenetic signature associated with the addiction-like phenotype, including downregulation of mmu-miR-669j, mmu-miR-7036b-5p, mmu-miR-878-3p, and mmu-miR-7017-3p, together with upregulation of mmu-miR-3092-5p in addicted females. Functional enrichment analyses of predicted target genes revealed pathways related to synaptic organization, axon guidance, neurotransmission, and structural plasticity. Together, these findings demonstrate sex-dependent differences in vulnerability to cannabinoid addiction-like behavior and identify a specific miRNA signature in the mPFC associated with this phenotype, highlighting potential targets for the development of sex-specific therapeutic strategies.

IntroductionNeural cue reactivity is increasingly being investigated as a biomarker of treatment response and relapse prediction in addiction disorders. Whilst aberrant brain responses to salient cues (e.g. drugs) have been widely reported in addiction, it is unclear whether these brain responses persist during longer-term abstinence, how they compare between substance use disorder and obesity, and relate to potential differences in eating behaviours. As part of the Gut Hormones in ADDiction (GHADD) neuroimaging study, we investigated how salient cue reactivity to drugs or food, craving and eating behaviours compare in three clinical populations where alterations have been previously observed: abstinent nicotine use disorder (NUD) and alcohol use disorder (AUD), and obesity. MethodsThis study compared group differences in salient cue reactivity and eating behaviours between ex-smokers (n=25, ExS), adults with alcohol dependence who are abstinent (n=26, AAD), adults with obesity who were actively dieting (n=26, OB). Participants completed a high-energy food, preferred alcohol and cigarette functional magnetic resonance imaging (fMRI) cue reactivity task, along with eating behaviour questionnaires, appetite visual analogues scales and an ad libitum test meal. ResultsExS exhibited greater blood oxygen level dependent (BOLD) signal to high-energy food pictures in several reward processing regions in both whole brain and region of interest (ROI) analyses, compared with the OB and AAD groups, with no difference in their appeal rating. Compared with the OB group, ExS exhibited greater BOLD signal to cigarette pictures in the frontal gyrus, orbitofrontal cortex, frontal pole and insula, with no difference in their appeal rating. There were no group differences in preferred alcohol cue reactivity. The AAD group rated sweet taste as more pleasant, and consumed more calories from sweet dishes in the ad libitum meal than the OB and ExS groups. ConclusionsThe presence of heightened cue reactivity to high-energy foods in ex-smokers could contribute to post-quitting weight gain after smoking cessation. Neuroimaging findings were consistent with persistence of some salient drug cue reactivity, despite absence of craving, after medium term abstinence in ExS, but not in AAD. This study also adds to the body of evidence supporting a sweet taste preference endophenotype predisposing individuals to AUD. These changes in eating behaviour in NUD and AUD may provide targets for treatments to reduce substance misuse and facilitate abstinence.

Regular cannabis use has been associated with alterations in reward-related neural processes, yet findings remain inconsistent and the relationship between neural activity and behavioural performance is not fully understood. The present study aimed to characterise neural and behavioural correlates of reward processing in regular cannabis users (CU) compared with matched non-users (NU) using the Monetary Incentive Delay Task (MIDT). Firstly, we assessed behavioural performance through reaction times, accuracy and monetary earnings to determine whether potential neural alterations were reflected in task performance. Secondly, focusing on reward-related brain regions, we examined group differences in BOLD functional MRI activity during anticipation and outcome phases separately for monetary win and loss conditions. Finally, we explored the association between behavioural performance and neural activation. Our findings indicate that regular cannabis use is associated with altered engagement of key nodes within the mesocorticolimbic circuit during both anticipatory and outcome phases of reward processing, accompanied by impaired behavioural performance. Particularly, compared with NU, CU showed (I) lower striatal activity during anticipation of monetary win and higher ventral striatum and frontal pole activity during anticipation of monetary loss; (II) greater VTA activation during outcome of successful monetary win and loss avoidance and lower frontal pole activity during outcome of unsuccessful loss avoidance; (III) impaired behavioural performance, reflected in lower monetary rewards and a trend towards slower reaction times and reduced accuracy; (IV) disrupted brain-behaviour coupling. Results from this study may help inform future research on the neurobiological mechanisms underlying changes in reward function and the resultant behavioural consequences of cannabis use.

BackgroundSubstance use disorders (SUDs), including alcohol and drug dependence, and smoking, pose a public health threat with their high prevalence and comorbidity with other diseases, and contribution to mortality. SUDs are highly correlated, and their genetic background is shared to some degree. ObjectivesWe aimed to investigate the genetic associations of previously reported loci for a wide range of SUDs in an unstudied Ukrainian population. MethodsWe collected data from 595 individuals (339 women, 253 men), including 321 participants from two rehab centres. Based on clinical review and questionnaire data we defined drug dependence, alcohol dependence, alcohol abuse, binge drinking, smoking, opiate, amphetamine, cannabis, and hallucinogen use, along with several intermediary alcohol use and smoking variables considering the amount of use and the level of dependence. We genotyped COMT-rs4680, ADH1B-ADH1C-rs1789891, and HTR2A-rs6313, and applied logistic and ordered logistic regression assuming an additive inheritance model, controlling for the recruitment group, other substance uses, age, and sex, in the association analyses. ResultsWe replicate (P<0.05) the associations at COMT-rs4680 with smoking status (OR[95% CI]=1.56[1.01-2.41], P=0.047) and heaviness (1.37[1.04-1.80], P=0.026), and at ADH1B-ADH1C-rs1789891 and HTR2A-rs6313 with alcohol dependence (1.69[1.03-2.76], P=0.038 and 0.66[0.47-0.92, P=0.016], respectively). Furthermore, we provide evidence for an association at HTR2A-rs6313 with hallucinogen use (0.58[0.35-0.98], P=0.040). ConclusionIn this study on multiple SUDs we shed light on the genetic background of SUDs in Ukrainians and provide further evidence that variation at COMT is mainly associated with smoking, at ADH1B-ADH1C with alcohol-related variables, whereas HTR2A is a more general SUD-associated locus. HighlightsO_LIWe present the first genetic study of substance use disorders in Ukrainians C_LIO_LIWe replicate the associations at COMT-rs4680 with smoking status and heaviness, and ADH1B-ADH1C-rs1789891 and HTR2A-rs6313 with alcohol dependence C_LIO_LIWe provide evidence for an association at HTR2A-rs6313 with hallucinogen use C_LI

The endogenous peptide dynorphin (Dyn) and its target the kappa opioid receptor (KOR) play a crucial role in regulating factors related to stress and reward. The KOR is expressed in multiple cell types in the nucleus accumbens (NAc), including presynaptic dopamine (DA) terminals, where it inhibits DA release modulates the function of the DA transporter (DAT). The Dyn/KOR system is upregulated by exposure to drugs of abuse including the DAT inhibitor, cocaine, and their activity is integrally involved in negative affective states associated with withdrawal from substance abuse. We aimed to better understand the impact of the Dyn/KOR system on presynaptic DA terminals and potential effects on DAT interactions with cocaine by measuring the impact of the KOR agonist U50,488 on electrically-evoked DA release and subsequent reuptake in NAc slices from C57BL6/J mice. We showed that superfusion of U50,488 inhibited DA release and markedly reduced cocaine-induced inhibition of DA reuptake, indicating tolerance to cocaine effects. We replicated this finding in the NAc of rhesus macaques using the DAT/NET inhibitor nomifensine, demonstrating that these mechanisms are conserved across DAT inhibitors and in non-human primates. KOR activation results in phosphorylation of the Threonine-53 site on the DAT, a process thought to mediate its impact on DAT function. We tested whether this phosphorylation site is required for the KOR-mediated reduction cocaine effects. To tackle this question, we employed a knock-in mouse line with an Alanine-53 on the DAT (DAT-T53A), rendering that residue insensitive to phosphorylation. We show that DAT-T53A mice have enhanced DA release and uptake, and U50,488 has a reduced inhibitory effect on peak DA release. Remarkably, U50,488 no longer modified the effect of cocaine on uptake in these mice, demonstrating the dependence of this effect on phosphorylated Threonine-53 and highlighting a potential mechanism underlying cocaine tolerance.

Opioid addiction is characterized by a strong motivational drive to obtain the drug, traditionally attributed to neuroadaptations within mesocorticolimbic reward circuits. Increasing evidence suggests that opioid-induced plasticity extends beyond these pathways, implicating the cerebellum in addiction, although its contribution to drug seeking remains poorly defined. Here, we hypothesized that morphine self-administration would induce cerebellum-associated behavioral alterations alongside structural and functional remodeling. Male Wistar rats (N = 27) were trained to self-administer morphine (0.1 mg/kg/infusion) or saline under fixed-ratio (FR1, 3 h/day, 20 days) and progressive-ratio (PR 9-4, 6 h/day, 25 days) schedules. Behavioral assessments included open field, elevated plus maze, novel object recognition, and Morris water maze tasks. Structural and functional magnetic resonance imaging was acquired longitudinally using a 7T scanner. Morphine self-administering rats showed a progressive increase in infusions and motivation. Exploratory activity increased without affecting anxiety-like behavior or recognition memory. In the Morris water maze, spatial learning was preserved; however, rats exhibited increased Whishaws error (path complexity) and a shift from serial to disorganized navigation strategies, indicating impaired movement sequencing. We also found cerebellar volume changes in Crus1, 7Cb, and 8Cb that were accompanied by altered cerebello-cerebral functional connectivity with insular, striatal, hippocampal, motor, and reticular networks. Multivariate analysis further suggested a brain-behavior covariance pattern in which motivational measures showed the strongest contribution, involving a distributed structural network including the cerebellum and insular cortex. These findings indicate that morphine self-administration preserves drug-seeking motivation while disrupting the organization of goal-directed actions, alongside cerebellar remodeling and altered cerebello-cerebral coupling.

Background: Alcohol use disorder (AUD) is associated with altered gene expression across diverse cell types in reward-related brain regions, including the ventral tegmental area (VTA), which is rich in dopaminergic neurons. The VTA plays a central role in reward processing, learning, and memory; however, cell type-specific gene expression changes within the VTA remain uncharacterized. Methods: We applied single-nucleus RNA sequencing (snRNA-seq) to profile transcriptomic alterations associated with AUD in the VTA. Postmortem VTA samples from four individuals of European ancestry [two with AUD (one male, one female) and two matched controls (one male, one female)] were analyzed using the 10X Genomics Chromium Fixed RNA Profiling protocol. Differentially expressed genes (DEGs) were identified using Seurat, and enriched KEGG pathways was assessed by gene set enrichment analysis. Results: Nuclei were classified into six major cell types: astrocytes, endothelial cells, mature neurons, microglia, oligodendrocytes, and oligodendrocyte precursor cells (OPCs). At thresholds of P < 0.05 and |fold change| > 2.0, we identified 547 DEGs in astrocytes, 727 DEGs in endothelial cells, 715 DEGs in mature neurons, 421 DEGs in microglia, 263 DEGs in oligodendrocytes, and 432 DEGs in OPCs. DEGs across VTA cell types were enriched for pathways related to mitochondrial function, neurodegeneration, and synaptic signaling. Notably, DEGs in mature neurons were enriched for addiction-related pathways. Further subdivision of mature neurons into dopaminergic, GABAergic, glutamatergic, and unclassified subtypes revealed 526, 930, 896, and 569 DEGs, respectively. Neuronal DEGs indicate a convergence on mitochondrial/oxidative phosphorylation and neurodegeneration-related pathways across subtypes, whereas addiction- and synapse-related pathways show dopaminergic neuron-specific enrichment. Conclusions: This study provides the first cell type-resolved transcriptomic profiling of the human VTA, revealing AUD-associated gene expression alterations across neuronal, glial, and endothelial cells. The observed cell type-specific changes in synaptic plasticity and addiction-related genes offer new insights into molecular mechanisms underlying AUD pathophysiology.

Background: Alcohol use disorder is associated with dysregulated glutamatergic signaling within mesocorticolimbic circuits that govern reinforcement and excessive ethanol intake. Group II metabotropic glutamate receptors (mGlu2/3) act primarily as presynaptic autoreceptors that regulate glutamate release. However, how voluntary alcohol intake alters mGlu2/3 expression within reward circuitry remains unclear. Methods and Results: We examined the effects of operant alcohol self-administration on mGlu2/3 protein expression and assessed the functional impact of group II receptor modulation on binge-like ethanol intake. Male C57BL/6J mice self-administered sweetened ethanol or sucrose under behaviorally matched conditions for 35 days. Immediately after the final session, tissue punches from the nucleus accumbens (NAc), amygdala, and prefrontal cortex were collected for Western blot analysis. Operant ethanol self-administration selectively reduced mGlu2/3 protein expression in the NAc, with no changes detected in the amygdala or prefrontal cortex. Both monomeric and dimeric mGlu2/3 protein levels were reduced, and a composite index revealed coordinated downregulation of receptor expression. In separate cohorts, systemic administration of the mGlu2/3 agonist LY379268 dose-dependently reduced binge-like ethanol intake in a limited-access home-cage drinking model, whereas positive allosteric modulation of mGlu2 receptors with LY487379 was ineffective. Conclusions: These results show that low-dose operant ethanol self-administration produces an ethanol- and region-specific reduction of mGlu2/3 protein expression in the NAc and that pharmacological activation of group II receptors, potentially involving mGlu3-specific receptors, is sufficient to suppress binge-like ethanol consumption. These data identify presynaptic mGlu2/3 dysregulation as a mechanism contributing to ethanol-related behaviors and support group II metabotropic glutamate receptors as therapeutic targets for alcohol use disorder.

BackgroundIndividual vulnerability to addiction is driven by neuroadaptations within dopaminergic circuits. G protein-coupled receptor kinases (GRKs), specifically GRK2 and GRK3 regulate D2 receptor (D2R) signaling and trafficking, but their role in amphetamine (AMPH)-induced locomotor sensitization remains unclear. This study aimed to determine whether GRK2/3 inhibition alters locomotor sensitization, and its associated molecular correlates across striatal regions. MethodsAdult rats (n = 39) were assigned to saline, acute AMPH, or repeated AMPH groups and received intraperitoneal administration of vehicle or the GRK2/3 inhibitor Cmpd101 (1.0 mg/kg intraperitoneally, i.p.). Locomotor activity was assessed under basal and injection conditions to evaluate sensitization. Protein levels of D2R, GRK2, and GRK5 were quantified across striatal regions using Western blot and analyzed with linear mixed models. ResultsCmpd101 did not significantly affect the acute hyperlocomotor effects of AMPH or the expression of AMPH sensitization. At the molecular level, Cmpd101 had no effect on D2R levels and produced selective, region-dependent changes in GRK2 and GRK5. Notably, GRK2/3 inhibition altered the relationship between protein expression and the degree of locomotor sensitization in a region-specific manner, rather than inducing consistent changes in absolute protein levels. ConclusionsGRK2/3 inhibition by Cmpd101 produces region-specific molecular effects and reshapes protein-behavior relationships without significantly altering locomotor sensitization. These findings support a model in which GRKs act as context-dependent modulators of dopaminergic signaling rather than direct drivers of behavioral output.

Craving - the powerful urge to seek and consume alcohol in response to alcohol-associated cues does not diminish after drinking cessation but rather is magnified throughout abstinence. This phenomenon, termed "incubation of craving", contributes to the relapsing nature of alcohol use disorder. Despite its occurrence in human populations and being well-studied in rodent models of psychostimulant drug relapse, the underlying neural mechanisms and potential treatments remain largely unexplored for alcohol-related incubation of craving. Our research seeks to meet this gap, and this particular study investigated the neural correlates of the incubation of craving for alcohol-associated cues and assessed whether exercise could prevent increased relapse propensity in rats. Male Long Evans rats were trained to lever press for an alcohol reward delivered with simultaneous presentation of a discrete cue. This response was then extinguished and reinstated by presenting the discrete cue alone when rats pressed the lever. Cue-induced reinstatement occurred either on day 1 following extinction (No Abstinence) or on day 29 (Abstinence). A third group was tested on day 29 and had 4-hour daily voluntary running wheel access throughout this abstinence period (Exercise). All rats were perfused 90 minutes following test, and relative activation across the brain was estimated by quantifying c-Fos protein immunoreactivity. The brain-wide coordination of neural activity was also mapped. We found a robust incubation of craving effect for alcohol-associated cues, which was mitigated by exercise. Immunohistochemistry revealed that the Abstinence group demonstrated higher c-Fos immunoreactivity compared to the No Abstinence group in multiple reinstatement-related brain regions. This effect was reversed in the Exercise group. Brain-wide neural mapping demonstrated that the Abstinence group had decreased modularity (groups of coordinating brain regions) compared to the no-abstinence group. Although network connectivity profile in the exercise group was different from no abstinence, we found that overall neural activation returned to a similar modularity profile of clustered regions as this condition, indicating that exercise does not attenuate the incubation of craving effect by reversing all the neural effects of abstinence. Rather, exercise may be acting upon select brain regions or pathways to exert relapse protective effects by restoring widespread interconnectivity. This is the first study to investigate neural activation in incubated alcohol-seeking, and provides supporting evidence for promoting voluntary exercise as an adjunctive treatment for relapse prevention in alcohol-use disorder.

Neuroimmune signaling is increased in postmortem brain tissue from individuals with alcohol use disorder (AUD), and growing evidence suggests that it contributes to persistent alcohol-related neuroadaptations. Interferon regulatory factor 7 (IRF7), a transcription factor downstream of endosomal Toll-like receptor signaling, is induced in alcohol-relevant brain regions and may contribute to escalated drinking. Here, we tested whether chronic intermittent ethanol (CIE) vapor exposure engages IRF7 signaling during subsequent alcohol self-administration and whether this is associated with altered molecular E/I balance in the aIC and altered functional E/I balance in aICnucleus accumbens projection neurons. Female Wistar rats (n=30) were trained to self-administer alcohol (15% v/v; FR2 vs inactive lever) during 30-minute sessions. After establishing baseline drinking, rats underwent 1-3 cycles of CIE, which increased alcohol self-administration at the 72 h post vapor test. This increase positively correlated with IRF7 levels in the anterior insular cortex (aIC) and nucleus accumbens, while molecular, and immunofluorescence showed that CIE shifted aIC excitatory/inhibitory (E/I) balance toward reduced excitation. Electrophysiological recordings further showed reduced functional E/I balance in aIC neurons projecting to the nucleus accumbens. Knockdown of IRF7 in the aIC attenuated CIE induced escalation of alcohol self-administration, supporting a role for insular IRF7 signaling in alcohol related neuroadaptations that promote escalated drinking.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

BACKGROUNDThe proliferation of the potent synthetic opioid fentanyl has exacerbated the ongoing crisis of substance use disorder and associated overdose deaths, yet the neurobiological mechanisms that underlie individual vulnerability to addiction and relapse remain poorly understood, particularly in the context of fentanyl use. The prefrontal cortex (PFC) has been identified as a key brain structure important for cognitive functions impacted in addiction, including inhibitory control of behavior and association of drug experience with specific cues, contexts, or actions. Although the heterogenous neuronal composition of the PFC complicates attribution of addiction-related behavioral regulation to specific cortical cell types and circuits, application of cell-type-specific methods in translationally relevant rodent models have begun to elucidate the key neural substrates of opioid addiction. METHODSWe used an intermittent access fentanyl self-administration (IntA SA) model to characterize individual variation and sex differences in addiction vulnerability in male and female rats. Longitudinal wireless fiber photometry recording was used to track calcium activity patterns in intratelencephalic (IT) neurons of the prelimbic cortex across acquisition of self-administration, escalation of fentanyl intake, extinction training, and cue-induced reinstatement of fentanyl seeking. RESULTSWe found that our fentanyl IntA SA paradigm produces distinct low- and high-risk addiction severity phenotypes and that female rats exhibited a greater propensity for high-risk classification, which was characterized by abundant and consistent fentanyl intake, robust responsiveness to conditioned and discriminative fentanyl-associated cues, and high levels of fentanyl-seeking during periods of drug unavailability, extinction training, and a cue-induced reinstatement test. Fiber photometry recordings revealed dynamic encoding of fentanyl-associated stimuli by prelimbic IT neurons across the IntA SA paradigm with event-related calcium transients observed in association with lever presses, fentanyl infusions, and presentation of conditioned and discriminative cues. CONCLUSIONSOur data indicate that fentanyl IntA SA is a translationally relevant paradigm that enables investigation of phenotypic diversity and the role of sex in fentanyl addiction. Longitudinal cell-type-selective calcium recordings revealed dynamic representation of fentanyl-associated stimuli by IT neurons of the prelimbic cortex consistent with a role for this cortical subpopulation in addiction-related behaviors.

The Reln gene encodes the extracellular glycoprotein Reelin that regulates synaptic plasticity and activity-dependent gene expression with implications in several neuropsychiatric disorders, including substance use disorder. While reduced Reln expression alters responses to psychostimulants and cannabinoid, its role in opioid-related behaviors remains unknown. Here, we examined whether Reln haploinsufficiency modifies behavioral and molecular responses to the synthetic opioid fentanyl. Heterozygous Reeler (Reln+/-) mice and wild-type littermates were assessed using using complementary contingent and non-contingent models of fentanyl exposure, including multi-phase fentanyl intravenous self-administration paradigm, conditioned place preference paradigm, locomotor assay, and dorsal striatal immediate early gene expression. Reln haploinsufficiency did not alter acquisition, extinction, or cue-induced reinstatement during self-administration, indicating stable opioid reinforcement and relapse-like behavior. Progressive ratio testing revealed a sex-dependent effect in which male Reln+/- mice showed reduced motivation for fentanyl compared to male wild-type mice. In contrast, following passive fentanyl exposure, Reln+/- mice exhibited enhanced fentanyl-induced locomotion and increased Fos immunoreactivity in the dorsal striatum, while CPP remained unchanged. Together, these findings demonstrate that Reln haploinsufficiency does not substantially modify opioid reinforcement or cue-driven drug seeking but enhances acute pharmacological sensitivity to fentanyl. These results identify Reln as a modulatory factor in opioid-responsive neural circuits that preferentially influences acute drug-evoked neuronal activation rather than the associative learning processes underlying opioid reinforcement.

Acute COVID-19 outcomes are exacerbated by substance use, however, the impact of substance use on Long-COVID is unknown. Here, we investigated the impact of chronic cocaine administration on spike-induced Long-COVID-like outcomes in the rat. Rats received intermittent chronic cocaine administration and a single intravenous injection of the SARS-CoV-2 spike protein. Two months following spike administration, Long-COVID-like outcomes were assessed. Exposure to spike protein in the presence of cocaine produced a persistent reduction in weight gain as compared with controls or spike protein alone. Further, cocaine-treated rats exposed to spike had lower withdrawal thresholds compared to control animals as well as their own baseline, suggesting increased pain sensitivity. Spike and/or cocaine increased the ratio of interleukin-6 (IL-6) to interleukin-10 (IL-10) levels in the hippocampus, indicating a shift towards a proinflammatory state. Paw withdrawal thresholds were positively correlated with IL-10 levels in the hippocampus and prefrontal cortex. Regarding olfaction, rats exposed to spike spent less time sniffing an odor attractant. Cocaine produced an anxiolytic-like phenotype during the elevated plus maze test. Further analysis of behaviors on the maze revealed that the latency to enter the open arms was shorter in rats exposed to spike or cocaine, suggesting a possible impulsive-like phenotype in these animals. These findings demonstrate the negative impact of cocaine on Long-COVID-like outcomes suggesting a need for increased clinical observations of people with co-occurring Long-COVID and cocaine use disorder. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/729575v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@12fdc19org.highwire.dtl.DTLVardef@11b1b0dorg.highwire.dtl.DTLVardef@8d1e21org.highwire.dtl.DTLVardef@b53d20_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundRelapse remains a central obstacle in the treatment of cocaine use disorder (CUD), for which no medications have received approval from the U.S. Food and Drug Administration. Transcranial magnetic stimulation (TMS) has shown promise as a potential therapeutic intervention. However, current clinical trials often rely on a "trial-and-error" approach in target selection and experimental design. We previously developed a novel TMS platform and high-density theta burst stimulation (hdTBS) technology, enabling precise, focal stimulation of the rat medial prefrontal cortex (mPFC), including the prelimbic and anterior cingulate cortices. MethodsWe applied hdTBS intervention to a well-established rat model of cocaine relapse and craving after cessation of extended access intravenous drug self-administration and assessed brain response using resting-state functional magnetic resonance imaging (fMRI). ResultsAs expected, we observed robust time-dependent increases in cocaine seeking (incubation of cocaine craving) in control rats receiving sham stimulation over 3 weeks of abstinence accompanied by a reduction in prefrontal functional connectivity. In contrast, daily sessions of hdTBS for 7 days delivered on abstinence days 14-20 prevented the emergence of the incubation effect and restored prefrontal network functional connectivity. ConclusionsThis study provides strong preclinical evidence demonstrating that precise circuit modulation of medial prefrontal subregions causally reverses both behavioral and network-level adaptations associated with relapse vulnerability. Given the clinical accessibility and established safety profile of TMS, this work provides a mechanistically grounded framework for target selection and supports the translation of focal TMS of the mPFC for relapse prevention in CUD patient. One Sentence SummaryFocal transcranial magnetic stimulation (TMS) of the mPFC using the hdTBS procedure prevents incubation of cocaine craving and restores functional connectivity.

Endocannabinoid (eCB) signaling is a key regulator of reward-related dopaminergic signaling, particularly in response to drugs of abuse, such as cocaine. To date, our understanding of this mechanism has primarily been limited to male subjects. Prior work establishes that female cocaine users have more adverse outcomes, and female rats show greater sensitivity to cannabinoid type 1 receptor (CB1R) regulation of cocaine self-administration. Therefore, we hypothesize that female rats exhibit enhanced eCB regulation of cocaine-evoked dopamine (DA). We used in vivo fiber photometry recording of the dopamine biosensor, dLight 1.3b, in the nucleus accumbens medial shell (NAcms) in response to cocaine in male and female rats. Rats were pretreated with cannabinoid-targeting drugs to investigate the effects of CB1R inactivation or augmentation of the eCB 2-AG on cocaine-evoked DA. Our results revealed that CB1R inactivation attenuates cocaine-evoked DA in male and female rats, but females showed enhanced sensitivity for CB1R regulation of cocaine-evoked DA. Cocaine-evoked DA was enhanced by augmenting 2-AG levels, and females again showed increased sensitivity to this manipulation. Finally, females show greater cocaine-evoked DA when in a non-estrous cycle compared to estrous, reinforcing that estrous cycle is a determinant of cocaine-evoked DA. These data indicate that females show enhanced eCB regulation of cocaine-evoked DA signaling, underscoring the importance of sex as a biological variable in our understanding of endocannabinoid regulation of drug reward. HighlightsO_LICB1R inactivation attenuates cocaine-evoked DA in NAcms, preferentially in females C_LIO_LI2-AG augmentation via MAGL inhibition enhances cocaine-evoked DA, with female bias C_LIO_LIEstrous phase modulates the dopamine response to a high dose of cocaine in females C_LIO_LIMale and female rats show similar baseline DA and locomotor responses to cocaine C_LI

Craving is a hallmark feature of substance use disorders (SUDs) and a major risk factor for relapse, yet reliable biomarkers that enable individual-level prediction remain scarce. Here, we applied connectome-based predictive modeling (CPM) to resting-state functional magnetic resonance imaging (fMRI) data in a transdiagnostic sample of individuals with cannabis, opioid, or tobacco use disorder (n = 78). Using CPM, we identified a distributed functional brain network that reliably predicted self-reported craving. Computational lesion analyses revealed key contributions from the right medial orbitofrontal cortex, right dorsal posterior cingulate cortex, and left lateral medial frontal gyrus. Importantly, the craving network generalized across two independent datasets. In alcohol-dependent patients (n = 41), the identified craving network, along with its positive and negative subnetworks, predicted distinct cognitive and motivational components of craving. In a second external dataset of smokers (n = 28), the craving network predicted both nicotine craving after abstinence as well as intra-individual changes in craving between sated and craving states. Together, these findings provide evidence for a robust, transdiagnostic craving signature in SUDs. Future work should assess the networks predictive utility for longitudinal outcomes such as relapse risk and treatment response.

Substance Use Disorders (SUDs) constitute a major and rising public health concern. In addition, there is a growing appreciation that different classes of addictive substances are likely to lead to qualitatively different types of SUDs requiring differing treatment and relapse prevention strategies to be most effectively managed. Biological temperament, particularly on the internalizing - externalizing axis, is well established to influence addiction susceptibility. Externalizing behavior has long been understood to predispose individuals to addiction through novelty-seeking, sensation-seeking and impulsivity, while internalizing behavior provides an alternate pathway into addiction via increased occurrence of comorbid disorders (anxiety, depression). Here, we utilize a selectively bred rat model of internalizing vs externalizing temperament (bred High Responders, representing genetically mediated externalizing behavior and bred Low Responders, representing internalizing behavior) to examine differences in the acquisition of self-administration of the prototypical psychostimulant cocaine and the prototypical opioid heroin (diacetylmorphine). We found that, as predicted, cocaine and heroin drove different patterns of acquisition in the two different bred lines of rats. Further, this was influenced by temperament in complex ways. Notably, in females the "telescoping effect" for opioid addiction-like behavior was primarily specific to externalizing temperament. These findings highlight the impact and interaction of many factors, including drug class, temperament, and sex, on the acquisition of drug-taking behavior. Additionally, these findings indicate that sex differences in addiction vulnerability may be influenced in part by biological temperament.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.