Astrocytes in the ventral pallidum extinguish heroin seeking through GAT-3 upregulation and morphological plasticity at D1-MSN terminals.

GABAergic projections from the nucleus accumbens core to the dorsolateral ventral pallidum are necessary for drug-conditioned cues to initiate relapse-like drug seeking. Astrocytes in the ventral pallidum are situated perisynaptically and regulate GABA transmission through expression of GABA uptake transporters, but whether they are involved in regulating drug seeking is unknown. To determine the contribution of ventral pallidal astrocytes to heroin seeking, we labeled astrocytes in male and female rats with a membrane-bound fluorescent tag and used confocal microscopy to quantify astroglial expression of the GABA transporter GAT-3 and astrocyte synaptic proximity after withdrawal from heroin self-administration and during 15 min of cued heroin seeking. We found that GAT-3 was upregulated in rats that had extinguished heroin seeking, but not in animals that were withdrawn from heroin without extinction training or in rats that extinguished sucrose seeking. When GAT-3 upregulation was reversed using a vivo-morpholino oligo, heroin seeking was restored in the extinguished context and extinction of cued heroin seeking was disrupted compared to control animals. Although astrocyte synaptic proximity was not altered overall after heroin withdrawal, examination of astrocyte proximity to accumbens D1- or D2-expressing afferents revealed a selective increase in astrocyte proximity with D1-expressing terminals during extinction of heroin self-administration. Experimentally-induced reduction of astrocyte synaptic proximity through knockdown of the astrocyte-selective actin-binding protein ezrin also markedly disrupted extinction of heroin seeking. Notably, GAT-3 or ezrin knockdown had no impact on context- or cue-induced seeking in sucrose-trained animals. These data show that astrocytes in the ventral pallidum undergo plasticity after extinction of heroin use that reduces seeking and highlight the importance of astrocyte-neuron interactions in shaping behaviors associated with opioid use disorder.

Accumbens D2-MSN hyperactivity drives antipsychotic-induced behavioral supersensitivity.

Antipsychotic-induced dopamine supersensitivity, or behavioral supersensitivity, is a problematic consequence of long-term antipsychotic treatment characterized by the emergence of motor abnormalities, refractory symptoms, and rebound psychosis. The underlying mechanisms are unclear and no approaches exist to prevent or reverse these unwanted effects of antipsychotic treatment. Here we demonstrate that behavioral supersensitivity stems from long-lasting pre, post and perisynaptic plasticity, including insertion of Ca2+-permeable AMPA receptors and loss of D2 receptor-dependent inhibitory postsynaptic currents (IPSCs) in D2 receptor-expressing medium spiny neurons (D2-MSNs) in the nucleus accumbens core (NAcore). The resulting hyperexcitability, prominent in a subpopulation of D2-MSNs (21%), caused locomotor sensitization to cocaine and was associated with behavioral endophenotypes of antipsychotic treatment resistance and substance use disorder, including disrupted extinction learning and augmented cue-induced cocaine-seeking behavior. Chemogenetic restoration of IPSCs in D2-MSNs in the NAcore was sufficient to prevent antipsychotic-induced supersensitivity, pointing to an entirely novel therapeutic direction for overcoming this condition.

Central Amygdala Astrocyte Plasticity Underlies GABAergic Dysregulation in Ethanol Dependence.

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure underlying the synaptic and behavioral consequences of ethanol dependence. While accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, there is a limited understanding of the role astrocytes play in ethanol dependence. The present study used a combination of viral labeling, super resolution confocal microscopy, 3D image analysis, and slice electrophysiology to determine the effects of chronic intermittent ethanol (CIE) exposure on astrocyte plasticity in the CeA. During withdrawal from CIE exposure, we observed increased GABA transmission, an upregulation in astrocytic GAT3 levels, and an increased proximity of astrocyte processes near CeA synapses. Furthermore, GAT3 levels and synaptic proximity were positively associated with voluntary ethanol drinking in dependent rats. Slice electrophysiology confirmed that the upregulation in astrocytic GAT3 levels was functional, as CIE exposure unmasked a GAT3-sensitive tonic GABA current in the CeA. A causal role for astrocytic GAT3 in ethanol dependence was assessed using viral-mediated GAT3 overexpression and knockdown approaches. However, GAT3 knockdown or overexpression had no effect on somatic withdrawal symptoms, dependence-escalated ethanol intake, aversion-resistant drinking, or post-dependent ethanol drinking in male or female rats. Moreover, intra-CeA pharmacological inhibition of GAT3 also did not alter dependent ethanol drinking. Together, these findings indicate that ethanol dependence induces GABAergic dysregulation and astrocyte plasticity in the CeA. However, astrocytic GAT3 does not appear necessary for the drinking related phenotypes associated with dependence.

Astrocyte regulation of synaptic signaling in psychiatric disorders.

Over the last 15 years, the field of neuroscience has evolved toward recognizing the critical role of astroglia in shaping neuronal synaptic activity and along with the pre- and postsynapse is now considered an equal partner in tripartite synaptic transmission and plasticity. The relative youth of this recognition and a corresponding deficit in reagents and technologies for quantifying and manipulating astroglia relative to neurons continues to hamper advances in understanding tripartite synaptic physiology. Nonetheless, substantial advances have been made and are reviewed herein. We review the role of astroglia in synaptic function and regulation of behavior with an eye on how tripartite synapses figure into brain pathologies underlying behavioral impairments in psychiatric disorders, both from the perspective of measures in postmortem human brains and more subtle influences on tripartite synaptic regulation of behavior in animal models of psychiatric symptoms. Our goal is to provide the reader a well-referenced state-of-the-art understanding of current knowledge and predict what we may discover with deeper investigation of tripartite synapses using reagents and technologies not yet available.

Astrocyte Heterogeneity in Regulation of Synaptic Activity.

Our awareness of the number of synapse regulatory functions performed by astroglia is rapidly expanding, raising interesting questions regarding astrocyte heterogeneity and specialization across brain regions. Whether all astrocytes are poised to signal in a multitude of ways, or are instead tuned to surrounding synapses and how astroglial signaling is altered in psychiatric and cognitive disorders are fundamental questions for the field. In recent years, molecular and morphological characterization of astroglial types has broadened our ability to design studies to better analyze and manipulate specific functions of astroglia. Recent data emerging from these studies will be discussed in depth in this review. I also highlight remaining questions emerging from new techniques recently applied toward understanding the roles of astrocytes in synapse regulation in the adult brain.

Extrasynaptic therapeutic targets in substance use and stress disorders.

Treatments for substance use and stress disorders are based on ameliorating behavioral symptoms, not on reversing the synaptic pathology that has the potential to cure disorders. This failing arises in part from a research focus on how pre- and postsynaptic physiology is changed even though key neuropathology exists in the perisynaptic neuropil that homeostatically regulates synaptic transmission. We explore recent findings from the substance use and stress disorder literature pointing to a key role for perisynaptic astroglia and signaling in the extracellular matrix (ECM) in regulating synaptic pathology. We conclude that drugs and stress initiate long-lasting changes in brain synapses via enduring neuroadaptations in astroglia and the ECM, and that modulating extrasynaptic regulators may be therapeutically useful.

Plasticity in astrocyte subpopulations regulates heroin relapse.

Opioid use disorder (OUD) produces detrimental personal and societal consequences. Astrocytes are a major cell group in the brain that receives little attention in mediating OUD. We determined how astrocytes and the astroglial glutamate transporter, GLT-1, in the nucleus accumbens core adapt and contribute to heroin seeking in rats. Seeking heroin, but not sucrose, produced two transient forms of plasticity in different astroglial subpopulations. Increased morphological proximity to synapses occurred in one subpopulation and increased extrasynaptic GLT-1 expression in another. Augmented synapse proximity by astroglia occurred selectively at D2-dopamine receptor-expressing dendrites, while changes in GLT-1 were not neuron subtype specific. mRNA-targeted antisense inhibition of either morphological or GLT-1 plasticity promoted cue-induced heroin seeking. Thus, we show that heroin cues induce two distinct forms of transient plasticity in separate astroglial subpopulations that dampen heroin relapse.

Automated detection of GFAP-labeled astrocytes in micrographs using YOLOv5.

Astrocytes, a subtype of glial cells with a complex morphological structure, are active players in many aspects of the physiology of the central nervous system (CNS). However, due to their highly involved interaction with other cells in the CNS, made possible by their morphological complexity, the precise mechanisms regulating astrocyte function within the CNS are still poorly understood. This knowledge gap is also due to the current limitations of existing quantitative image analysis tools that are unable to detect and analyze images of astrocyte with sufficient accuracy and efficiency. To address this need, we introduce a new deep learning framework for the automated detection of GFAP-immunolabeled astrocytes in brightfield or fluorescent micrographs. A major novelty of our approach is the applications of YOLOv5, a sophisticated deep learning platform designed for object detection, that we customized to derive optimized classification models for the task of astrocyte detection. Extensive numerical experiments using multiple image datasets show that our method performs very competitively against both conventional and state-of-the-art methods, including the case of images where astrocytes are very dense. In the spirit of reproducible research, our numerical code and annotated data are released open source and freely available to the scientific community.

Cocaine administration protects gut mucosa barrier and reduces plasma level of TNF-α.

Background: Cocaine affects not only the central nervous system, but also systemic immunity. The role of cocaine in gut mucosal integrity is not fully understood. Methods: Here we evaluated the effect of cocaine use on gut endothelial permeability and system inflammation in rats that self-administered cocaine or saline and in humans using immunohistochemistry, qPCR, ELISA, and Transepithelial/transendothelial electrical resistance (TEER). Results: Cocaine administration maintained intact and undisturbed intestinal mucosal structures, increased tight junction claudin 1 and 2 mRNA expression, and decreased plasma TNF-α levels, compared to the control group, at the end of study in rats. Further, cocaine treatment decreased gut endothelial permeability in a dose-dependent manner in human epithelial Caco-2 cells in vitro. Consistently, chronic cocaine users exhibited decreased plasma levels of TNF-α compared with non-drug users in vivo. However, plasma IL-6 levels were similar between cocaine use and control groups both in humans and rats in vivo. Conclusions: Our results from both human and rat studies in vivo and in vitro suggest that cocaine use may exert a protective effect on the integrity of gut mucosa and suppresses plasma TNF-α levels. This study may provide information on some beneficial effects of cocaine use on gut endothelial cells integrity and systemic inflammation.

Astrocytes as cellular mediators of cue reactivity in addiction.

Relapse to addictive drug use remains a major medical problem worldwide. In rodents, glutamate release in the nucleus accumbens core triggers reinstated drug seeking in response to stress, and drug-associated cues and contexts. Glutamatergic dysregulation in addiction results in part from long-lasting adaptations in accumbens astroglia, including downregulation of the glutamate transporter GLT-1 and retraction from synapses after withdrawal from psychostimulants and opioids. While their capacity to clear glutamate is disrupted by drug use and withdrawal, accumbens astrocytes undergo rapid, transient plasticity in response to drug-associated cues that reinstate seeking. Cued reinstatement of heroin seeking, for example, restores synaptic proximity of astrocyte processes through ezrin phosphorylation, and enhances GLT-1 surface expression. These adaptations limit drug seeking behavior and largely occur on non-overlapping populations of astroglia. Here we review the growing literature supporting a critical role for accumbens astrocytes in modulating glutamate transmission during drug seeking in rodent models of relapse.

Astrocytes in Addictive Disorders.

Astroglia are key regulators of synaptic function, playing central roles in homeostatic ion buffering, energy dynamics, transmitter uptake, maintenance of neurotransmitter pools, and regulation of synaptic plasticity through release of neuroactive chemicals. Given the myriad of crucial homeostatic and signaling functions attributed to astrocytes and the variety of neurotransmitter receptors expressed by astroglia, they serve as prime cellular candidates for establishing maladaptive synaptic plasticity following drug exposure. Initial studies on astroglia and addiction have placed drug-mediated disruptions in the homeostatic regulation of glutamate as a central aspect of relapse vulnerability. However, the generation of sophisticated tools to study and manipulate astroglia have proven that the interaction between addictive substances, astroglia, and relapse-relevant synaptic plasticity extends far beyond the homeostatic regulation of glutamate. Here we present astroglial systems impacted by drug exposure and discuss how changes in astroglial biology contribute to addiction biology.

Behavioral and accumbens synaptic plasticity induced by cues associated with restraint stress.

Exposure to acute stress can increase vulnerability to develop or express many psychiatric disorders, including post-traumatic stress disorder. We hypothesized that stress-induced psychiatric vulnerability is associated with enduring neuroplasticity in the nucleus accumbens core because stress exposure can alter drug addiction-related behaviors that are associated with accumbens synaptic plasticity. We used a single 2-h stress session and 3 weeks later exposed male and female rats to stress-conditioned odors in a modified defensive burying task, and quantified both active and avoidant coping strategies. We measured corticosterone, dendritic spine and astrocyte morphology in accumbens, and examined reward sensitivity using a sucrose two-bottle choice and operant sucrose self-administration. Exposure to stress odor increased burying (active coping) and immobility (avoidant coping) in the defensive burying task in female and male rats. Systemic corticosterone was transiently increased by both ongoing acute restraint stress and stress-conditioned odors. Three weeks after administering acute restraint stress, we observed increased dendritic spine density and head diameter, and decreased synaptic association with astroglia and the astroglial glutamate transporter, GLT-1. Exposure to conditioned stress further increased head diameter without affecting spine density or astroglial morphology, and this increase by conditioned stress was correlated with burying behavior. Finally, we found that stress-exposed females have a preference for sweet solutions and higher motivation to seek sucrose than stressed male rats. We conclude that acute stress produced enduring plasticity in accumbens postsynapses and associated astroglia. Moreover, conditioned stress odors induced active behavioral coping strategies that were correlated with dendritic spine morphology.

Heroin Seeking and Extinction From Seeking Activate Matrix Metalloproteinases at Synapses on Distinct Subpopulations of Accumbens Cells.

BACKGROUND: Seeking addictive drugs is regulated by synaptic plasticity in the nucleus accumbens core and involves distinct plasticity in D1 and D2 receptor-expressing medium spiny neurons (D1/2-MSNs). However, it is unknown how differential plasticity between the two cell types is coordinated. Synaptic plasticity and seeking behavior induced by drug-paired cues depends not only on plasticity in the canonical pre- and postsynapse, but also on cue-induced changes in astrocytes and the extracellular matrix adjacent to the synapse. Drug cue-induced signaling in the extracellular matrix is regulated by catalytic activity of matrix metalloproteinases MMP-2,9. We hypothesized that the cell type-specific synaptic plasticity is associated with parallel cell-specific activity of MMP-2 and MMP-9. METHODS: Transgenic rats were trained on a heroin self-administration protocol in which a light/tone cue was paired with heroin delivery, followed by 2 weeks of drug withdrawal, and then reinstated to heroin-conditioned cues. Confocal microscopy was used to make morphological measurements in membrane reporter-transduced D1- and D2-MSNs and astrocytes, and MMP-2,9 gelatinase activity adjacent to cell surfaces was quantified using in vivo zymography. RESULTS: Presenting heroin-paired cues transiently increased MMP-9 activity around D1-MSN dendritic spines and synapse-proximal astroglial processes. Conversely, extinction training induced long-lasting increases in MMP-2 activity adjacent to D2-MSN synapses. Moreover, heroin-paired cues increased tissue inhibitor of metalloproteinases TIMP-1,2, which caused transient inhibition of MMP-2 activity around D2-MSNs during cue-induced heroin seeking. CONCLUSIONS: The differential regulation of heroin seeking and extinguished seeking by different MMP subtypes on distinct cell populations poses MMP-2,9 activity as an important mediator and contributor in heroin-induced cell-specific synaptic plasticity.

The Opioid-Addicted Tetrapartite Synapse.

Opioid administration in preclinical models induces long-lasting adaptations in reward and habit circuitry. The latest research demonstrates that in the nucleus accumbens, opioid-induced excitatory synaptic plasticity involves presynaptic and postsynaptic elements as well as adjacent astroglial processes and the perisynaptic extracellular matrix. We outline opioid-induced modifications within each component of the tetrapartite synapse and provide a neurobiological perspective on how these adaptations converge to produce addiction-related behaviors in rodent models. By incorporating changes observed at each of the excitatory synaptic compartments into a unified framework of opioid-induced glutamate dysregulation, we highlight new avenues for restoring synaptic homeostasis that might limit opioid craving and relapse vulnerability.

Mutations in the Drosophila mitochondrial tRNA amidotransferase, bene/gatA, cause growth defects in mitotic and endoreplicating tissues.

Rapid larval growth is essential in the development of most metazoans. In this article, we show that bene, a gene previously identified on the basis of its oogenesis defects, is also required for larval growth and viability. We show that all bene alleles disrupt gatA, which encodes the Drosophila homolog of glutamyl-tRNA(Gln) amidotransferase subunit A (GatA). bene alleles are now referred to as gatA. GatA proteins are highly conserved throughout eukaryotes and many prokaryotes. These enzymes are required for proper translation of the proteins encoded by the mitochondrial genome and by many eubacterial genomes. Mitotic and endoreplicating tissues in Drosophila gatA loss-of-function mutants grow slowly and never achieve wild-type size, and gatA larvae die before pupariation. gatA mutant eye clones exhibit growth and differentiation defects, indicating that gatA expression is required cell autonomously for normal growth. The gatA gene is widely expressed in mitotic and endoreplicating tissues.

Hypofunctional Dopamine Uptake and Antipsychotic Treatment-Resistant Schizophrenia.

Antipsychotic treatment resistance in schizophrenia remains a major issue in psychiatry. Nearly 30% of patients with schizophrenia do not respond to antipsychotic treatment, yet the underlying neurobiological causes are unknown. All effective antipsychotic medications are thought to achieve their efficacy by targeting the dopaminergic system. Here we review early literature describing the fundamental mechanisms of antipsychotic drug efficacy, highlighting mechanistic concepts that have persisted over time. We then reconsider the original framework for understanding antipsychotic efficacy in light of recent advances in our scientific understanding of the dopaminergic effects of antipsychotics. Based on these new insights, we describe a role for the dopamine transporter in the genesis of both antipsychotic therapeutic response and primary resistance. We believe that this discussion will help delineate the dopaminergic nature of antipsychotic treatment-resistant schizophrenia.

Post-translational S-glutathionylation of cofilin increases actin cycling during cocaine seeking.

Neuronal defense against oxidative damage is mediated primarily by the glutathione redox system. Traditionally considered a mechanism to protect proteins from irreversible oxidation, mounting evidence supports a role for protein S-glutathionylation in cell signaling in response to changes in intracellular redox status. Here we determined the specific sites on the actin binding protein cofilin that undergo S-glutathionylation. In addition, we show that S-glutathionylation of cofilin reduces its capacity to depolymerize F-actin. We further describe an assay to determine the S-glutathionylation of target proteins in brain tissue from behaving rodents. Using this technique, we show that cofilin in the rat nucleus accumbens undergoes S-glutathionylation during 15-minutes of cued cocaine seeking in the absence of cocaine. Our findings demonstrate that cofilin S-glutathionylation is increased in response to cocaine-associated cues and that increased cofilin S-glutathionylation reduces cofilin-dependent depolymerization of F-actin. Thus, S-glutathionylation of cofilin may serve to regulate actin cycling in response to drug-conditioned cues.

Heroin Cue-Evoked Astrocytic Structural Plasticity at Nucleus Accumbens Synapses Inhibits Heroin Seeking.

BACKGROUND: Opioid addiction is a critical medical and societal problem characterized by vulnerability to relapse. Glutamatergic synapses in the nucleus accumbens regulate the motivation to relapse to opioid use, and downregulation of glutamate transporters on astroglial processes adjacent to accumbens synapses contributes to heroin seeking induced by cues. However, it is not known how astroglial processes themselves respond to heroin cues or if changes in astroglial morphology are necessary for heroin seeking. METHODS: Male Sprague Dawley rats (n = 62) were trained to self-administer heroin or sucrose and were reinstated by heroin-conditioned or sucrose-conditioned cues. Astroglial proximity to accumbens synapses was estimated using a confocal-based strategy, and the association between digitally isolated astroglia and the presynaptic marker synapsin I was quantified. To determine the functional consequence of astroglial morphological plasticity on cued heroin seeking, a morpholino antisense strategy was used to knock down expression of the actin binding protein ezrin, which is expressed almost exclusively in peripheral astroglial processes in the adult rat brain. RESULTS: After heroin extinction, there was an enduring reduction in synaptic proximity by astroglia. Synaptic proximity was restored during 15 minutes of cued heroin seeking but returned to extinction levels by 120 minutes. Extinction from sucrose self-administration and reinstated sucrose seeking induced no changes in astroglial synaptic association. Ezrin knockdown reduced astroglial association with synapses and potentiated cued heroin seeking. CONCLUSIONS: Cue-induced heroin seeking transiently increased synaptic proximity of accumbens astrocytes. Surprisingly, the reassociation of astroglia with synapses was compensatory, and preventing cue-induced morphological plasticity in astrocytes potentiated heroin seeking.

Long-Term Dabigatran Treatment Delays Alzheimer's Disease Pathogenesis in the TgCRND8 Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with important vascular and hemostatic alterations that should be taken into account during diagnosis and treatment. OBJECTIVES: This study evaluates whether anticoagulation with dabigatran, a clinically approved oral direct thrombin inhibitor with a low risk of intracerebral hemorrhage, ameliorates AD pathogenesis in a transgenic mouse model of AD. METHODS: TgCRND8 AD mice and their wild-type littermates were treated for 1 year with dabigatran etexilate or placebo. Cognition was evaluated using the Barnes maze, and cerebral perfusion was examined by arterial spin labeling. At the molecular level, Western blot and histochemical analyses were performed to analyze fibrin content, amyloid burden, neuroinflammatory activity, and blood-brain barrier (BBB) integrity. RESULTS: Anticoagulation with dabigatran prevented memory decline, cerebral hypoperfusion, and toxic fibrin deposition in the AD mouse brain. In addition, long-term dabigatran treatment significantly reduced the extent of amyloid plaques, oligomers, phagocytic microglia, and infiltrated T cells by 23.7%, 51.8%, 31.3%, and 32.2%, respectively. Dabigatran anticoagulation also prevented AD-related astrogliosis and pericyte alterations, and maintained expression of the water channel aquaporin-4 at astrocytic perivascular endfeet of the BBB. CONCLUSIONS: Long-term anticoagulation with dabigatran inhibited thrombin and the formation of occlusive thrombi in AD; preserved cognition, cerebral perfusion, and BBB function; and ameliorated neuroinflammation and amyloid deposition in AD mice. Our results open a field for future investigation on whether the use of direct oral anticoagulants might be of therapeutic value in AD.