Dose mediates the protracted effects of adolescent THC exposure on reward and stress reactivity in males relevant to perturbation of the basolateral amygdala transcriptome.

Despite the belief that cannabis is relatively harmless, exposure during adolescence is associated with increased risk of developing several psychopathologies in adulthood. In addition to the high levels of use amongst teenagers, the potency of ∆-9-tetrahydrocannabinol (THC) has increased more than fourfold compared to even twenty years ago, and it is unclear whether potency influences the presentation of THC-induced behaviors. Expanded knowledge about the impact of adolescent THC exposure, especially high dose, is important to delineating neural networks and molecular mechanisms underlying psychiatric risk. Here, we observed that repeated exposure to low (1.5 mg/kg) and high (5 mg/kg) doses of THC during adolescence in male rats produced divergent effects on behavior in adulthood. Whereas low dose rats showed greater sensitivity to reward devaluation and also self-administered more heroin, high dose animals were significantly more reactive to social isolation stress. RNA sequencing of the basolateral amygdala, a region linked to reward processing and stress, revealed significant perturbations in transcripts and gene networks related to synaptic plasticity and HPA axis that were distinct to THC dose as well as stress. In silico single-cell deconvolution of the RNAseq data revealed a significant reduction of astrocyte-specific genes related to glutamate regulation in stressed high dose animals, a result paired anatomically with greater astrocyte-to-neuron ratios and hypotrophic astrocytes. These findings emphasize the importance of dose and behavioral state on the presentation of THC-related behavioral phenotypes in adulthood and dysregulation of astrocytes as an interface for the protracted effects of high dose THC and subsequent stress sensitivity.

Granulocyte-Colony Stimulating Factor Reduces Cocaine-Seeking and Downregulates Glutamatergic Synaptic Proteins in Medial Prefrontal Cortex.

Psychostimulant use disorder is a major public health issue, and despite the scope of the problem there are currently no Food and Drug Administration (FDA)-approved treatments. There would be tremendous utility in development of a treatment that could help patients both achieve and maintain abstinence. Previous work from our group has identified granulocyte-colony stimulating factor (G-CSF) as a neuroactive cytokine that alters behavioral response to cocaine, increases synaptic dopamine release, and enhances cognitive flexibility. Here, we investigate the role of G-CSF in affecting extinction and reinstatement of cocaine-seeking and perform detailed characterization of its proteomic effects in multiple limbic substructures. Male Sprague Dawley rats were injected with PBS or G-CSF during (1) extinction or (2) abstinence from cocaine self-administration, and drug seeking behavior was measured. Quantitative assessment of changes in the proteomic landscape in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were performed via data-independent acquisition (DIA) mass spectrometry analysis. Administration of G-CSF during extinction accelerated the rate of extinction, and administration during abstinence attenuated cue-induced cocaine-seeking. Analysis of global protein expression demonstrated that G-CSF regulated proteins primarily in mPFC that are critical to glutamate signaling and synapse maintenance. Taken together, these findings support G-CSF as a viable translational research target with the potential to reduce drug craving or seeking behaviors. Importantly, recombinant G-CSF exists as an FDA-approved medication which may facilitate rapid clinical translation. Additionally, using cutting-edge multiregion discovery proteomics analyses, these studies identify a novel mechanism underlying G-CSF effects on behavioral plasticity.SIGNIFICANCE STATEMENT Pharmacological treatments for psychostimulant use disorder are desperately needed, especially given the disease's chronic, relapsing nature. However, there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies. Emerging evidence suggests that targeting the immune system may be a viable translational research strategy; preclinical studies have found that the neuroactive cytokine granulocyte-colony stimulating factor (G-CSF) alters cocaine reward and reinforcement and can enhance cognitive flexibility. Given this basis of evidence we studied the effects of G-CSF treatment on extinction and reinstatement of cocaine seeking. We find that administration of G-CSF accelerates extinction and reduces cue-induced drug seeking after cocaine self-administration. In addition, G-CSF leads to downregulation of synaptic glutamatergic proteins in medial prefrontal cortex (mPFC), suggesting that G-CSF influences drug seeking via glutamatergic mechanisms.

Chromatin accessibility mapping of the striatum identifies tyrosine kinase FYN as a therapeutic target for heroin use disorder.

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.

Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity.

Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.

Synaptic Microtubule-Associated Protein EB3 and SRC Phosphorylation Mediate Structural and Behavioral Adaptations During Withdrawal From Cocaine Self-Administration.

Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.

Granulocyte-colony stimulating factor controls neural and behavioral plasticity in response to cocaine.

Cocaine addiction is characterized by dysfunction in reward-related brain circuits, leading to maladaptive motivation to seek and take the drug. There are currently no clinically available pharmacotherapies to treat cocaine addiction. Through a broad screen of innate immune mediators, we identify granulocyte-colony stimulating factor (G-CSF) as a potent mediator of cocaine-induced adaptations. Here we report that G-CSF potentiates cocaine-induced increases in neural activity in the nucleus accumbens (NAc) and prefrontal cortex. In addition, G-CSF injections potentiate cocaine place preference and enhance motivation to self-administer cocaine, while not affecting responses to natural rewards. Infusion of G-CSF neutralizing antibody into NAc blocks the ability of G-CSF to modulate cocaine's behavioral effects, providing a direct link between central G-CSF action in NAc and cocaine reward. These results demonstrate that manipulating G-CSF is sufficient to alter the motivation for cocaine, but not natural rewards, providing a pharmacotherapeutic avenue to manipulate addictive behaviors without abuse potential.

Histone arginine methylation in cocaine action in the nucleus accumbens.

Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.

Genome-Wide DNA Methylation Profiling Reveals Epigenetic Changes in the Rat Nucleus Accumbens Associated With Cross-Generational Effects of Adolescent THC Exposure.

Drug exposure during critical periods of development is known to have lasting effects, increasing one's risk for developing mental health disorders. Emerging evidence has also indicated the possibility for drug exposure to even impact subsequent generations. Our previous work demonstrated that adolescent exposure to Δ(9)-tetrahydrocannabinol (THC), the main psychoactive component of marijuana (Cannabis sativa), in a Long-Evans rat model affects reward-related behavior and gene regulation in the subsequent (F1) generation unexposed to the drug. Questions, however, remained regarding potential epigenetic consequences. In the current study, using the same rat model, we employed Enhanced Reduced Representation Bisulfite Sequencing to interrogate the epigenome of the nucleus accumbens, a key brain area involved in reward processing. This analysis compared 16 animals with parental THC exposure and 16 without to characterize relevant systems-level changes in DNA methylation. We identified 1027 differentially methylated regions (DMRs) associated with parental THC exposure in F1 adults, each represented by multiple CpGs. These DMRs fell predominantly within introns, exons, and intergenic intervals, while showing a significant depletion in gene promoters. From these, we identified a network of DMR-associated genes involved in glutamatergic synaptic regulation, which also exhibited altered mRNA expression in the nucleus accumbens. These data provide novel insight into drug-related cross-generational epigenetic effects, and serve as a useful resource for investigators to explore novel neurobiological systems underlying drug abuse vulnerability.