The ventral striatum is crucially involved in reward processing. The present study investigates the behavioral effects of astrocyte-specific calcium extrusion virus "CalEx" on perseverative responses in the operant five-choice serial reaction time task and ethanol-conditioned place preference. Mice were injected with CalEx via the GfaABC1D promoter to extrude cytosolic calcium from astrocytes within the ventral striatum. We found that CalEx transfection in the ventral striatum reduced evoked response duration, the maximum amplitude, and the response frequency to 500 μM ATP as measured by ΔF/F fluorescence intensity of the genetically encoded calcium indicator targeting astrocytes GCaMP6f. During the five-choice serial reaction time task, CalEx mice persisted in perseverative responses compared to their counterparts. Additionally, during ethanol-conditioned place preference, CalEx mice showed increased place preference for a low ethanol concentration compared to control group. Furthermore, we found that accumbal astrocytic calcium extrusion increased quinine adulterated ethanol drinking. Our findings suggest that diminishing ventral striatum astrocyte calcium activity contributes to compulsive behaviors, ethanol drinking, and enhanced ethanol drug reward.

At cellular and circuit levels, drug addiction is considered a dysregulation of synaptic plasticity. In addition, dysfunction of the glutamate transporter 1 (GLT-1) in the nucleus accumbens (NAc) has also been proposed as a mechanism underlying drug addiction. However, the cellular and synaptic impact of GLT-1 alterations in the NAc remain unclear. Here we show in the NAc that 10 days withdraw after 5 days treatment with cocaine or amphetamine decreases GLT-1 expression in astrocytes, which results in the prolongation of the excitatory postsynaptic potential (EPSP) decay kinetics in D1 receptor-containing medium spiny neurons (D1R-MSNs). Using the spike timing dependent plasticity (STDP) paradigm, we found that enlargement of EPSP duration results in switching the LTP elicited in control animals to LTD in psychostimulant-treated mice. In contrast to D1-MSNs, D2-MSNs did not display changes in EPSP kinetics and synaptic plasticity. Notably, the psychostimulant-induced synaptic transmission and synaptic plasticity effects were absent in IP3R2-/- mice, which lack astrocyte calcium signal, but were mimicked by the selective astrocytes stimulation with DREADDs. Finally, ceftriaxone, which upregulates GLT-1, restored normal GLT-1 function, EPSP kinetics, and synaptic plasticity in psychostimulant-treated mice. Therefore, we propose that cocaine and amphetamine increase dopaminergic levels in the NAc, which stimulates astrocytes and downregulates the GLT-1. The decreased GLT-1 function prolonged the EPSP kinetics, leading to the modulation of the STDP, transforming the LTP observed in control animals into LTD in psychostimulant-treated mice. Present work reveals a novel mechanism underlying the synaptic plasticity changes induced by these drugs of abuse.

The development of compulsive cue-controlled-incentive drug-seeking habits is a hallmark of substance use disorder that is predicated on an intrastriatal shift in the locus of control over behaviour from a nucleus accumbens (Nac) core-dorsomedial striatum network to a Nac core-anterior dorsolateral striatum (aDLS) network. This shift is paralleled by drug-induced (including cocaine) dopamine transporter (DAT) alterations originating in the ventral striatum that spread eventually to encompass the aDLS. Having recently shown that heroin self-administration results in a pan-striatal reduction in astrocytic DAT that precedes the development of aDLS dopamine-dependent incentive heroin-seeking habits, we tested the hypothesis that similar adaptations occur following cocaine exposure. We compared DAT protein levels in whole tissue homogenates, and in astrocytes cultured from ventral and dorsal striatal territories of drug-naïve male Sprague-Dawley rats to those of rats with a history of cocaine taking or an aDLS dopamine-dependent incentive cocaine-seeking habit. Cocaine exposure resulted in a decrease in whole tissue and astrocytic DAT across all territories of the striatum. We further demonstrated that compulsive (i.e., punishment-resistant) incentive cocaine-seeking habits were associated with a reduction in DAT mRNA levels in the Nac shell, but not the Nac core-aDLS incentive habit system. Together with the recent evidence of heroin-induced downregulation of striatal astrocytic DAT, these findings suggest that alterations in astrocytic DAT may represent a common mechanism underlying the development of compulsive incentive drug-seeking habits across drug classes.

Nonneuronal cells mediate neurotransmission and drug addiction. However, the role of oligodendrocytes in stress-induced cocaine relapses remains unclear. In the present study, we investigated the role of the oligodendrocyte-abundant molecule crystallin alpha B (CRYAB) in cocaine-induced conditioned place preference (CPP) relapsed by restraint stress. RNA sequencing (RNA-seq) was performed to identify oligodendrocytes and stress-associated molecules in the nucleus accumbens (NAcc) of both drug users and cocaine-treated animals. Further, we studied which cell subtypes in the brain express CRYAB. The effects of stress hormones and cocaine on CRYAB expression were evaluated in vitro in human oligodendrocytes. CRYAB is upregulated in the NAcc of both cocaine-treated animals and drug users. CRYAB levels in the NAcc of mice increased during CPP development but decreased following stress-induced relapse. Interestingly, CRYAB is expressed in oligodendrocytes in the NAcc of mice. Extracellular CRYAB levels are regulated by cocaine and stress hormone treatments in oligodendrocyte cultures. Dopamine levels in the NAcc and CPP development of CPP are significantly increased by cocaine in CRYAB knockout (KO) mice. Further, we demonstrated that CRYAB binds to the excitatory amino acid transporter 2 (EAAT2) in the NAcc of mice treated with cocaine. We suggest that oligodendrocyte-derived CRYAB regulates dopamine neurotransmission and stress-evoked cocaine reward behaviour via the modulation of EAAT2 in the NAcc.

Excitotoxicity is a common hallmark of epilepsy and other neurological diseases associated with elevated extracellular glutamate levels. Thus, here, we studied the protective effects of (R)-AS-1, a positive allosteric modulator (PAM) of glutamate uptake in epilepsy models.

(R)-AS-1 was evaluated in a range of acute and chronic seizure models, while its adverse effect profile was assessed in a panel of standard tests in rodents. The effect of (R)-AS-1 on glutamate uptake was assessed in COS-7 cells expressing the transporter. WAY 213613, a selective competitive EAAT2 inhibitor, was used to probe the reversal of the enhanced glutamate uptake in the same transporter expression system. Confocal microscopy and Western blotting analyses were used to study a potential influence of (R)-AS-1 on GLT-1 expression in mice.

(R)-AS-1 showed robust protection in a panel of animal models of seizures and epilepsy, including the maximal electroshock- and 6 Hz-induced seizures, corneal kindling, mesial temporal lobe epilepsy, lamotrigine-resistant amygdala kindling, as well as seizures induced by pilocarpine or Theiler's murine encephalomyelitis virus. Importantly, (R)-AS-1 displayed a favorable adverse effect profile in the rotarod, the minimal motor impairment, and the Irwin tests. (R)-AS-1 enhanced glutamate uptake in vitro and this effect was abolished by WAY 213613, while no influence on GLT-1 expression in vivo was observed after repeated treatment.

Collectively, our results show that (R)-AS-1 has favorable tolerability and provides robust preclinical efficacy against seizures. Thus, allosteric enhancement of EAAT2 function could offer a novel therapeutic strategy for treatment of epilepsy and potentially other neurological disorders associated with glutamate excitotoxicity. ANN NEUROL 2025;97:344-357.

Cocaine use disorder is an intersecting issue in populations with HIV-1, further exacerbating the clinical course of the disease and contributing to neurotoxicity and neuroinflammation. Cocaine and HIV neurotoxins play roles in neuronal damage during neuroHIV progression by disrupting glutamate homeostasis in the brain. Even with combined antiretroviral therapy (cART), HIV-1 Nef, an early viral protein expressed in approximately 1% of infected astrocytes, remains a key neurotoxin. This study investigates the relationship among Nef, glutamate homeostasis, and cocaine in the nucleus accumbens (NAc), a critical brain region associated with drug motivation and reward.

Male and female Sprague Dawley rats were used to compare the effects of astrocytic Nef and cocaine by molecular analysis of glutamate transporters, GLT-1 and the cysteine glutamate exchanger (xCT), in the NAc. Behavioral assessments for cocaine self-administration were used to evaluate cocaine-seeking behavior.

The findings indicate that both cocaine and Nef independently decrease the expression of the glutamate transporter GLT-1 in the NAc. Additionally, rats with astrocytic Nef expression exhibited increased cocaine-seeking behavior but demonstrated sex-dependent molecular differences after the behavioral paradigm.

The results suggest that the expression of Nef intensifies cocaine-induced alterations in glutamate homeostasis in the NAc, potentially underlying increased cocaine-seeking behavior. Understanding these interactions better may inform therapeutic strategies for managing cocaine use disorder in HIV-infected individuals.

The glutamatergic system, located throughout the brain including the prefrontal cortex and nucleus accumbens, plays a critical role in reward and reinforcement processing, and mediates the psychotropic effects of addictive drugs such as cocaine. Glutamate transporters, including EAAT2/GLT-1, are responsible for removing glutamate from the synaptic cleft. Reduced expression of GLT-1 following chronic cocaine use and abstinence has been reported. Here, we demonstrate that targeting GLT-1 with a novel positive allosteric modulator (PAM), NA-014, results in reduction of cocaine-associated behaviors in rats. Pharmacokinetic analysis demonstrated that NA-014 is brain-penetrant and suitable for in vivo studies.We found that 15 and 30 mg/kg NA-014 significantly reduced cocaine-induced locomotion in males. Only the 15 mg/kg dose was effective in females and 60 mg/kg was ineffective in both sexes. Furthermore, 30 and 60 mg/kg NA-014 reduced expression of cocaine conditioned place preference (CPP) in males. 30 mg/kg NA-014 reduced expression of cocaine CPP in females and 15 mg/kg did not affect cocaine CPP in either sex, suggesting GLT-1 influences cocaine-associated behaviors in a sex-dependent manner. NA-014 did not elicit rewarding behavior, nor alter baseline locomotion. Twice daily/7-day administration of 100 mg/kg of NA-014 did not alter GLT-1 or GLAST expression in either sex in the prefrontal cortex (PFC). Collectively, these studies demonstrated that NA-014 reduced the locomotor stimulant and rewarding effects of cocaine in male and female rats. In the context of psychostimulant use disorders, our study suggests studying GLT-1 PAMs as alternatives to β-lactam compounds that increase GLT-1 protein levels.

N-acetylcysteine (NAC) has established use as an antidote for acetaminophen overdose and treatment for pulmonary conditions and nephropathy. It plays a role in regulating oxidative stress and interacting with various cytokines including IL-1β, TNFα, IL-8, IL-6, IL-10, and NF-κB p65. The overexpression of reactive oxygen species (ROS) is believed to contribute to chronic pain states by inducing inflammation and accelerating disease progression, favoring pain persistence in neuropathic and musculoskeletal pain conditions. Through a comprehensive review, we aim to explore the mechanisms and inflammatory pathways through which NAC may manage neuropathic and musculoskeletal pain. Evidence suggests NAC can attenuate neuropathic and musculoskeletal pain through mechanisms such as inhibiting matrix metalloproteinases (MMPs), reducing reactive oxygen species (ROS), and enhancing glutamate transport. Additionally, NAC may synergize with opioids and other pain medications, potentially reducing opioid consumption and enhancing overall pain management. Further research is needed to fully elucidate its therapeutic potential and optimize its use in pain management. As an adjuvant therapy, NAC shows potential for chronic pain management, offering significant benefits for public health.

Cocaine use disorder is an intersecting issue in populations with HIV-1, further exacerbating the clinical course of the disease, contributing to neurotoxicity and neuroinflammation. Cocaine and HIV neurotoxins play roles in neuronal damage during neuroHIV progression by disrupting glutamate homeostasis in the brain. Even with cART, HIV-1 Nef, an early viral protein expressed in approximately 1% of infected astrocytes, remains a key neurotoxin. This study investigates the relationship that exists between Nef, glutamate homeostasis, and cocaine in the NAc, a critical brain region associated with drug motivation and reward. Using a rat model, we compared the effects of astrocytic Nef and cocaine by molecular analysis of glutamate transporters in the NAc. We further conducted behavioral assessments for cocaine self-administration to evaluate cocaine-seeking behavior. Our findings indicate that both cocaine and Nef independently decrease the expression of the glutamate transporter GLT-1 in the NAc. Additionally, rats with astrocytic Nef expression exhibited increased cocaine-seeking behavior but demonstrated sex dependent molecular differences after behavioral paradigm. In conclusion, our results suggest the expression of Nef intensifies cocaine-induced alterations in glutamate homeostasis in the NAc, potentially underlying increased cocaine-seeking. Understanding these interactions better may inform therapeutic strategies for managing cocaine use disorder in HIV-infected individuals.

Mood disorders and substance use disorder (SUD) are of immense medical and social concern. Although significant progress on neuronal involvement in mood and reward circuitries has been achieved, it is only relatively recently that the role of glia in these disorders has attracted attention. Detailed understanding of the glial functions in these devastating diseases could offer novel interventions. Here, following a brief review of circuitries involved in mood regulation and reward perception, the specific contributions of neurotrophic factors, neuroinflammation, and gut microbiota to these diseases are highlighted. In this context, the role of specific glial cells (e.g., microglia, astroglia, oligodendrocytes, and synantocytes) on phenotypic manifestation of mood disorders or SUD are emphasized. In addition, use of this knowledge in the potential development of novel therapeutics is touched upon.

The emergence of compulsive drug-seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatal control over behaviour. This process involves the dopamine-dependent functional coupling of the anterior dorsolateral striatum (aDLS) with the nucleus accumbens core, but the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), the level of which is altered in individuals with heroin use disorder. Astrocytes are therefore uniquely placed functionally to bridge dopamine-dependent mechanisms across the striatum. Here we tested the hypothesis that exposure to heroin influences the expression of DAT in striatal astrocytes across the striatum before the development of DLS-dependent incentive heroin seeking habits. Using Western-blot, qPCR, and RNAscope™, we measured DAT protein and mRNA levels in whole tissue, culture and in situ astrocytes from striatal territories of rats with a well-established cue-controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior aDLS that was preceded by a heroin-induced reduction in DAT mRNA and protein in astrocytes across the striatum. Striatal astrocytes were also shown to be susceptible to direct dopamine- and opioid-induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits.

Cocaine use disorder (CUD) remains a severe health problem with no effective pharmacological therapy. One of the potential pharmacological strategies for CUD pharmacotherapy includes manipulations of the brain glutamatergic (Glu) system which is particularly involved in drug withdrawal and relapse. Previous research indicated a pivotal role of ionotropic N-methyl-D-aspartate (NMDA) receptors or metabotropic receptors' type 5 (mGlu5) receptors in controlling the reinstatement of cocaine. Stimulation of the above molecules results in the activation of the downstream signaling targets such as neuronal nitric oxide synthase (nNOS) and the release of nitric oxide.

In this paper, we investigated the molecular changes in nNOS in the prefrontal cortex and nucleus accumbens following 3 and 10 days of cocaine abstinence as well as the effectiveness of nNOS blockade with the selective enzyme inhibitor N-ω-propyl-L-arginine hydrochloride (L-NPA) on cocaine seeking in male rats. The effect of L-NPA on locomotor activity in drug-naïve animals was investigated.

Ten-day (but not 3-day) cocaine abstinence from cocaine self-administration increased nNOS gene and protein expression in the nucleus accumbens, but not in the prefrontal cortex. L-NPA (0.5-5 mg/kg) administered peripherally did not change locomotor activity but attenuated the reinstatement induced with cocaine priming or the drug-associated conditioned cue.

Our findings support accumbal nNOS as an important molecular player for cocaine seeking while its inhibitors could be considered as anti-cocaine pharmacological tools in male rats.

Engagement of astrocytes within the brain's reward circuitry has been apparent for approximately 30 years, when noncontingent drug administration was observed to lead to cytological markers of reactive astrocytes. Since that time, advanced approaches in rodent behavior and astrocyte monitoring have revealed complex interactions between astrocytes with drug type, animal sex, brain region, and dose and duration of drug administration. A number of studies now collectively reveal that rodent drug self-administration followed by prolonged abstinence results in decreased features of structure and synaptic colocalization of astrocytes. In addition, stimulation of astrocytes in the nucleus accumbens with DREADD receptors or pharmacological compounds opposes drug-seeking behavior. These findings provide a clear path for ongoing investigation into astrocytes as mediators of drug action in the brain and underscore the potential therapeutic utility of astrocytes in the regulation of drug craving and relapse vulnerability.

Craving is a core symptom of cocaine use disorder and a major factor for relapse risk. To date, there is no pharmacological therapy to treat this disease or at least to alleviate cocaine craving as a core symptom. In animal models, impaired prefrontal-striatal signalling leading to altered glutamate release in the nucleus accumbens appear to be the prerequisite for cocaine-seeking. Thus, those network and metabolic changes may constitute the underlying mechanisms for cocaine craving and provide a potential treatment target. In humans, there is recent evidence for corresponding glutamatergic alterations in the nucleus accumbens, however, the underlying network disturbances that lead to this glutamate imbalance remain unknown. In this state-dependent randomized, placebo-controlled, double-blinded, cross-over multimodal study, resting state functional magnetic resonance imaging in combination with small-voxel proton magnetic resonance spectroscopy (voxel size: 9.4 × 18.8 × 8.4 mm3) was applied to assess network-level and associated neurometabolic changes during a non-craving and a craving state, induced by a custom-made cocaine-cue film, in 18 individuals with cocaine use disorder and 23 healthy individuals. Additionally, we assessed the potential impact of a short-term challenge of N-acetylcysteine, known to normalize disturbed glutamate homeostasis and to thereby reduce cocaine-seeking in animal models of addiction, compared to a placebo. We found increased functional connectivity between the nucleus accumbens and the dorsolateral prefrontal cortex during the cue-induced craving state. However, those changes were not linked to alterations in accumbal glutamate levels. Whereas we additionally found increased functional connectivity between the nucleus accumbens and a midline part of the thalamus during the cue-induced craving state. Furthermore, obsessive thinking about cocaine and the actual intensity of cocaine use were predictive of cue-induced functional connectivity changes between the nucleus accumbens and the thalamus. Finally, the increase in accumbal-thalamic connectivity was also coupled with craving-related glutamate rise in the nucleus accumbens. Yet, N-acetylcysteine had no impact on craving-related changes in functional connectivity. Together, these results suggest that connectivity changes within the fronto-accumbal-thalamic loop, in conjunction with impaired glutamatergic transmission, underlie cocaine craving and related clinical symptoms, pinpointing the thalamus as a crucial hub for cocaine craving in humans.

The shift from drug abuse to addiction is considered to arise from the transition between goal-directed and habitual control over drug behavior. Habitual responding for appetitive and skill-based behaviors is mediated by potentiated glutamate signaling in the dorsolateral striatum (DLS), but the state of the DLS glutamate system in the context of habitual drug-behavior remains undefined. Evidence from the nucleus accumbens of cocaine-experienced rats suggests that decreased transporter-mediated glutamate clearance and enhanced synaptic glutamate release contribute to the potentiated glutamate signaling that underlies the enduring vulnerability to relapse. Preliminary evidence from the dorsal striatum of cocaine-experienced rats suggests that this region exhibits similar alterations to glutamate clearance and release, but it is not known whether these glutamate dynamics are associated with goal-directed or habitual control over cocaine-seeking behavior. Therefore, we trained rats to self-administer cocaine in a chained cocaine-seeking and -taking paradigm, which yielded goal-directed, intermediate, and habitual cocaine-seeking rats. We then assessed glutamate clearance and release dynamics in the DLS of these rats using two different methods: synaptic transporter current (STC) recordings of patch-clamped astrocytes and the intensity-based glutamate sensing fluorescent reporter (iGluSnFr). While we observed a decreased rate of glutamate clearance in STCs evoked with single-pulse stimulation in cocaine-experienced rats, we did not observe any cocaine-induced differences in glutamate clearance rates from STCs evoked with high frequency stimulation (HFS) or iGluSnFr responses evoked with either double-pulse stimulation or HFS. Furthermore, GLT-1 protein expression in the DLS was unchanged in cocaine-experienced rats, regardless of their mode of control over cocaine-seeking behavior. Lastly, there were no differences in metrics of glutamate release between cocaine-experienced rats and yoked-saline controls in either assay. Together, these results suggest that glutamate clearance and release dynamics in the DLS are largely unaltered by a history of cocaine self-administration on this established cocaine seeking-taking paradigm, regardless of whether the control over the cocaine seeking behavior was habitual or goal directed.

Psychostimulant exposure and withdrawal cause neuroimmune dysregulation and anxiety that contributes to dependence and relapse. Here, we tested the hypothesis that withdrawal from the synthetic cathinone MDPV (methylenedioxypyrovalerone) produces anxiety-like effects and enhanced levels of mesocorticolimbic cytokines that are inhibited by cyanidin, an anti-inflammatory flavonoid and nonselective blocker of IL-17A signaling. For comparison, we tested effects on glutamate transporter systems that are also dysregulated during psychostimulant free period. Rats injected for 9 d with MDPV (1 mg/kg, IP) or saline were pretreated daily with cyanidin (0.5 mg/kg, IP) or saline, followed by behavioral testing on the elevated zero maze (EZM) 72 h after the last MDPV injection. MDPV withdrawal caused a reduction in time spent on the open arm of the EZM that was prevented by cyanidin. Cyanidin itself did not affect locomotor activity or time spent on the open arm, or cause aversive or rewarding effects in place preference experiments. MDPV withdrawal caused enhancement of cytokine levels (IL-17A, IL-1β, IL-6, TNF=α, IL-10, and CCL2) in the ventral tegmental area, but not amygdala, nucleus accumbens, or prefrontal cortex, that was prevented by cyanidin. During MDPV withdrawal, mRNA levels of glutamate aspartate transporter (GLAST) and glutamate transporter subtype 1 (GLT-1) in the amygdala were also elevated but normalized by cyanidin treatment. These results show that MDPV withdrawal induced anxiety, and brain-region specific dysregulation of cytokine and glutamate systems, that are both prevented by cyanidin, thus identifying cyanidin for further investigation in the context of psychostimulant dependence and relapse.

After a history of intermittent cocaine intake, rats develop patterns of drug use characteristic of substance use disorder. The dorsal striatum is involved in the increased pursuit of cocaine after intermittent drug self-administration experience. Within the dorsal striatum, chronic cocaine use changes metabotropic glutamate type II receptor (mGlu_2/3) density and function.

We examined the extent to which activity at Glu_2/3 receptors mediates responding for cocaine after intermittent cocaine use.

Male (n = 11) and female (n = 10) Wistar rats self-administered 0.25 mg/kg/infusion cocaine during 10 daily intermittent access (IntA) sessions (5 min ON/25 min OFF, for 5 h/session). We then examined the effects of microinjections of the mGlu_2/3 receptor agonist LY379268 (0, 1, and 3 µg/hemisphere) into the ventrolateral part of the dorsal striatum on cocaine self-administration under a progressive ratio schedule of reinforcement.

Across 10 IntA sessions, the sexes showed similar levels of cocaine intake. In females only, locomotion significantly increased over sessions, suggesting that female rats developed psychomotor sensitization to self-administered cocaine. After 10 IntA sessions, intra-dorsal striatum LY379268 significantly reduced breakpoints achieved for cocaine, active lever presses, and cocaine infusions earned under progressive ratio. LY379268 had no effects on locomotion or inactive lever presses, indicating no motor effects.

These results suggest that mGlu_2/3 receptor activation in the ventrolateral dorsal striatum suppresses incentive motivation for cocaine, and this holds promise for new treatments to manage substance use disorder.

The present study investigates the possible therapeutic effects of human mesenchymal stem cell-derived secretome on morphine dependence and relapse. This was studied in a new model of chronic voluntary morphine intake in Wistar rats which shows classic signs of morphine intoxication and a severe naloxone-induced withdrawal syndrome. A single intranasal-systemic administration of MSCs secretome fully inhibited (>95%; p < 0.001) voluntary morphine intake and reduced the post-deprivation relapse intake by 50% (p < 0.02). Since several studies suggest a significant genetic contribution to the chronic use of many addictive drugs, the effect of MSCs secretome on morphine self-administration was further studied in rats bred as high alcohol consumers (UChB rats). Sub-chronic intraperitoneal administration of morphine before access to increasing concentrations of morphine solutions and water were available to the animals, led UChB rats to prefer ingesting morphine solutions over water, attaining levels of oral morphine intake in the range of those in the Wistar model. Intranasally administered MSCs secretome to UChB rats dose-dependently inhibited morphine self-administration by 72% (p < 0.001); while a single intranasal dose of MSC-secretome administered during a morphine deprivation period imposed on chronic morphine consumer UChB rats inhibited re-access morphine relapse intake by 80 to 85% (p < 0.0001). Both in the Wistar and the UChB rat models, MSCs-secretome administration reversed the morphine-induced increases in brain oxidative stress and neuroinflammation, considered as key engines perpetuating drug relapse. Overall, present preclinical studies suggest that products secreted by human mesenchymal stem cells may be of value in the treatment of opioid addiction.

Neuropathic pain, a disease of the somatosensory nervous system, afflicts many individuals and adequate management with current pharmacotherapies remains elusive. The glutamatergic system of neurons, receptors and transporters are intimately involved in pain but, to date, there have been few drugs developed that therapeutically modulate this system. Glutamate transporters, or excitatory amino acid transporters (EAATs), remove excess glutamate around pain transmitting neurons to decrease nociception suggesting that the modulation of glutamate transporters may represent a novel approach to the treatment of pain. This review highlights and summarizes (1) the physiology of the glutamatergic system in neuropathic pain, (2) the preclinical evidence for dysregulation of glutamate transport in animal pain models, and (3) emerging novel therapies that modulate glutamate transporters. Successful drug discovery requires continuous focus on basic and translational methods to fully elucidate the etiologies of this disease to enable the development of targeted therapies. Increasing the efficacy of astrocytic EAATs may serve as a new way to successfully treat those suffering from this devastating disease.

Glutamate transporters facilitate the removal of this excitatory neurotransmitter from the synapse. Increasing evidence indicates that this process is linked to intrinsic chloride channel activity that is thermodynamically uncoupled from substrate transport. A recent cryo-EM structure of Glt_Ph - an archaeal homolog of the glutamate transporters - in an open channel state has shed light on the structural basis for channel opening formed at the interface of two domains within the transporter which is gated by two clusters of hydrophobic residues. These transporters cycle through several conformational states during the transport process, including the chloride conducting state, which appears to be stabilised by protein-membrane interactions and membrane deformation. Several point mutations that perturb the chloride conductance can have detrimental effects and are linked to the pathogenesis of the neurological disorder, episodic ataxia type 6.

Evidence that glial cells influence behavior has been gaining a steady foothold in scientific literature. Out of the five main subtypes of glial cells in the brain, astrocytes and microglia have received an outsized share of attention with regard to shaping a wide spectrum of behavioral phenomena and there is growing appreciation that the signals intrinsic to these cells as well as their interactions with surrounding neurons reflect behavioral history in a brain region-specific manner. Considerable regional diversity of glial cell phenotypes is beginning to be recognized and may contribute to behavioral outcomes arising from circuit-specific computations within and across discrete brain nuclei. Here, we summarize current knowledge on the impact of astrocyte and microglia activity on behavioral outcomes, with a specific focus on brain areas relevant to higher cognitive control, reward-seeking, and circadian regulation.

Drug addiction is associated with disruption of a multitude of biomarkers in various brain regions, particularly in the reward center. The most pronounced are dopaminergic and glutamatergic biomarkers, which are affected at various levels. Neuropathological changes in biomarkers alter the homeostasis of the glutamatergic and dopaminergic nervous systems and promote addiction-associated characteristics such as repeated intake, maintenance, withdrawal, reinstatement, and relapse. Exercise has been shown to have a buffering effect on such biomarkers and reverse the effects of addictive substances.

A review of the literature searched in PubMed, examining drug addiction and physical exercise in relation to dopaminergic and glutamatergic systems at any of the three biomarker levels (i.e., neurotransmitter, receptor, or transporter).

We review the collective impact of addictive substances on the dopaminergic and glutamatergic systems and the beneficial effect of exercise in terms of reversing the damage to these systems. We propose future directions, including implications of exercise as an add-on therapy, substance use disorder (SUD) prognosis and diagnosis and designing of optimized exercise and pharmaceutical regimens based on the aforementioned biomarkers.

Exercise is beneficial for all types of drug addiction at all stages, by reversing molecular damages caused to dopaminergic and glutamatergic systems.

N-acetylcysteine (NAC) is an antioxidant with some demonstrated efficacy in a range of neuropsychiatric disorders. NAC has shown anticonvulsant effects in animal models. NAC effects on absence seizures are still not uncovered, and considering its clinical use as a mucolytic in patients with lung diseases, people with epilepsy are also likely to be exposed to the drug. Therefore, we aimed to study the effects of NAC on absence seizures in the WAG/Rij rat model of absence epilepsy with neuropsychiatric comorbidities. The effects of NAC chronic treatment in WAG/Rij rats were evaluated on: absence seizures at 15 and 30 days by EEG recordings and animal behaviour at 30 days on neuropsychiatric comorbidities. Furthermore, the mechanism of action of NAC was evaluated by analysing brain expression levels of some possible key targets: the excitatory amino acid transporter 2, cystine-glutamate antiporter, metabotropic glutamate receptor 2, the mechanistic target of rapamycin and p70S6K as well as levels of total glutathione. Our results demonstrate that in WAG/Rij rats, NAC treatment significantly increased the number and duration of SWDs, aggravating absence epilepsy while ameliorating neuropsychiatric comorbidities. NAC treatment was linked to an increase in brain mGlu2 receptor expression with this being likely responsible for the observed absence seizure-promoting effects. In conclusion, while confirming the positive effects on animal behaviour induced by NAC also in epileptic animals, we report the aggravating effects of NAC on absence seizures which could have some serious consequences for epilepsy patients with the possible wider use of NAC in clinical therapeutics.

Pharmacological manipulations of glutamatergic ionotropic receptors have been suggested as a promising target for addiction treatment. Antagonists of AMPA/kainate receptors were shown to reduce alcohol intake or alcohol-seeking in various animal models. In this study, we evaluated the effect of NBQX, an AMPA/kainate receptor antagonist, on methamphetamine (METH) and nicotine self-administration in rats.

Male Wistar rats were trained to self-administer METH (0.08 mg/kg per infusion, session of 90 min) and nicotine (0.03 mg/kg per infusion, session of 60 min) under the fixed ratio 1 schedule of reinforcement. The maintenance training was 2 weeks. During the second week, NBQX was injected subcutaneously at doses of 5 or 10 mg/kg 20 min before the session or intravenously (IV) at doses of 1 and 5 mg/kg 10 min before the session. Following the maintenance training, rats were subjected to forced abstinence for 2 weeks and 1 day of the drug-free relapse-like session with IV NBQX treatment performed as before.

Although NBQX did not affect nicotine maintenance, it significantly suppressed the drug-paired responding in the relapse session. Regarding METH, NBQX did not exert a significant effect at either phase of the study.

These findings suggest selective involvement of AMPA/kainate receptors in the relapse of nicotine seeking after a period of forced abstinence.

Opiates produce a strong rewarding effect, but abstinence from opiate use emerges with severe negative emotions. Depression is one of the most frequent emotion disorders associated with opiate abstinence, which is thought to be a main cause for relapse. However, neurobiological bases of such an aversive emotion processing are poorly understood. Here, we find that morphine abstinence activates κ-opioid receptors (KORs) by increasing endogenous KOR ligand dynorphin expression in the amygdala, which in turn facilitates glutamate transporter 1 (GLT1) expression by activation of p38 mitogen-activated protein kinase (MAPK). Upregulation of GLT1 expression contributes to opiate-abstinence-elicited depressive-like behaviors through modulating amygdalar glutamatergic inputs to the nucleus accumbens (NAc). Intra-amygdala injection of GLT1 inhibitor DHK or knockdown of GLT1 expression in the amygdala significantly suppresses morphine-abstinence-induced depressive-like behaviors. Pharmacological and pharmacogenetic activation of amygdala-NAc projections prevents morphine-abstinence-induced behaviors. Overall, our study provides key molecular and circuit insights into the mechanisms of depression associated with opiate abstinence.

There is robust preclinical literature and preliminary clinical findings supporting the use of N-Acetylcysteine (NAC) to treat substance use disorders, including tobacco use disorder (TUD). However, randomized controlled trials have yielded mixed results and NAC's efficacy for TUD has not been established. The goals of this study were to assess the efficacy of NAC in promoting early and end-of-treatment abstinence and preventing relapse among adult smokers.

This randomized, double-blinded clinical trial enrolled adult, daily smokers (N = 114; ages 23-64; 51 % female; 65 % White; 29 % Black/African American; 7% Hispanic/Latinx), who were randomized 1:1 to receive NAC (n = 59) or placebo (n = 55) (1200 mg b.i.d.) for eight weeks. Participants received brief cessation counseling and incentives for abstinence during the first three days of the quit attempt. Primary outcomes: (i) carbon monoxide (CO)-confirmed abstinence during the first three days of the quit attempt.

(ii) time to relapse; (iii) biologically confirmed abstinence at Week 8.

No differences were found between NAC and placebo groups on measures of early abstinence (3-day quit attempt; 11 % for NAC vs. 15 % for placebo; all p > 0.11), time to relapse (p = 0.19), and end-of-treatment abstinence (7% for NAC vs. 11 % for placebo; all p > 0.40].

Results indicate that NAC is a well-tolerated pharmacotherapy but is unlikely to be efficacious as a monotherapy for TUD in adults. Considered in the collective context of other research, NAC may potentially be more useful in a younger population, as a combination pharmacotherapy, or in the presence of more intensive psychosocial treatment.

Relapse to drug use during abstinence is a defining feature of addiction. During the last several decades, this clinical scenario has been studied at the preclinical level using classic relapse/reinstatement models in which drug seeking is assessed after experimenter-imposed home-cage forced abstinence or extinction of the drug-reinforced responding in the self-administration chambers. To date, however, results from studies using rat relapse/reinstatement models have yet to result in Food and Drug Administration-approved medications for relapse prevention. The reasons for this state of affairs are complex and multifaceted, but one potential reason is that, in humans, abstinence is often self-imposed or voluntary and occurs either because the negative consequences of drug use outweigh the drug's rewarding effects or because of the availability of nondrug alternative rewards that are chosen over the drug. Based on these considerations, we and others have recently developed rat models of relapse after voluntary abstinence, achieved either by introducing adverse consequences to drug taking (punishment) or seeking (electric barrier) or by providing mutually exclusive choices between the self-administered drug and nondrug rewards (palatable food or social interaction). In this review, we provide an overview of these translationally relevant relapse models and discuss recent neuropharmacological findings from studies using these models. We also discuss sex as a biological variable, future directions, and clinical implications of results from relapse studies using voluntary abstinence models. Our main conclusion is that the neuropharmacological mechanisms controlling relapse to drug seeking after voluntary abstinence are often different from the mechanisms controlling relapse after home-cage forced abstinence or reinstatement after extinction. SIGNIFICANCE STATEMENT: This review describes recently developed rat models of relapse after voluntary abstinence, achieved either by introducing adverse consequences to drug taking or seeking or by providing mutually exclusive choices between the self-administered drug and nondrug rewards. This review discusses recent neuropharmacological findings from studies using these models and discusses future directions and clinical implications.

Depression has drawn increasing attention from the public around the world in recent years. Studies have shown that liver injury caused by chronic stress is relevant to depression and neurotransmitter changes. It is essential to clarify the relationship between neurotransmitter changes and hepatic gene expression in depression. In this study, we used the chronic unpredictable mild stress (CUMS) model combined with UHPLC-MS to explore the changes of neurotransmitters in serum and hippocampus and to decipher the differential gene expression in the liver by using the RNA-Seq combined with multivariate statistical analysis. Compared with the control group, the levels of neurotransmitters including 5-hydroxytryptamine (5-HT), acetylcholine, glutamate (Glu), and dopamine (DA) in the hippocampus and 5-HT, norepinephrine, γ-aminobutyric acid (GABA), and 5-hydroxyindoleacetic acid in serum were significantly changed in the CUMS rats. The results of liver transcriptomic analysis and correlation analysis showed that the Glu, DA, 5-HT, and GABA were impacted by 68 liver genes which were mainly enriched in three pathways including circadian rhythm, serotonergic synapse, and p53 signaling pathway. The expressive levels of clock genes and serotonergic synapse genes were validated by using q-PCR, and the diurnal rhythms of neurotransmitters were validated by in vivo hippocampus microdialysis. The CUMS stressors might cause phase advance of Glu and GABA by adjusting clock genes. The transcriptomic technique combined with correlation analysis and in vivo microdialysis could be used to discover comprehensive pathways of depression. It provides a new strategy for the rational assessment of the mechanism of disease.

Cocaine, amphetamine, and methamphetamine abuse disorders are serious worldwide health problems. To date, there are no FDA-approved medications for the treatment of these disorders. Elucidation of the biochemical underpinnings contributing to psychostimulant addiction is critical for the development of effective therapies. Excitatory signaling and glutamate homeostasis are well known pathophysiological substrates underlying addiction-related behaviors spanning multiple types of psychostimulants. To alleviate relapse behavior to psychostimulants, considerable interest has focused on GLT-1, the major glutamate transporter in the brain. While many brain regions are implicated in addiction behavior, this review focuses on two regions well known for their role in mediating the effects of cocaine and amphetamines, namely the nucleus accumbens (NAc) and the ventral tegmental area (VTA). In addition, because many investigators have utilized Cre-driver lines to selectively control gene expression in defined cell populations relevant for psychostimulant addiction, we discuss potential off-target effects of Cre-recombinase that should be considered in the design and interpretation of such experiments.

Oxidative stress, which results in the damage of diverse biological molecules, is a ubiquitous cellular process implicated in the etiology of many illnesses. The sulfhydryl-containing tripeptide glutathione (GSH), which is synthesized and maintained at high concentrations in all cells, is one of the mechanisms by which cells protect themselves from oxidative stress. N-acetylcysteine (NAC), a synthetic derivative of the endogenous amino acid L-cysteine and a precursor of GSH, has been used for several decades as a mucolytic and as an antidote to acetaminophen (paracetamol) poisoning. As a mucolytic, NAC breaks the disulfide bonds of heavily cross-linked mucins, thereby reducing mucus viscosity. In vitro, NAC has antifibrotic effects on lung fibroblasts. As an antidote to acetaminophen poisoning, NAC restores the hepatic GSH pool depleted in the drug detoxification process. More recently, improved knowledge of the mechanisms by which NAC acts has expanded its clinical applications. In particular, the discovery that NAC can modulate the homeostasis of glutamate has prompted studies of NAC in neuropsychiatric diseases characterized by impaired glutamate homeostasis. This narrative review provides an overview of the most relevant and recent evidence on the clinical application of NAC, with a focus on respiratory diseases, acetaminophen poisoning, disorders of the central nervous system (chronic neuropathic pain, depression, schizophrenia, bipolar disorder, and addiction), cardiovascular disease, contrast-induced nephropathy, and ophthalmology (retinitis pigmentosa).

Large number of promising preclinical psychiatric studies in rodents later fail in clinical trials, raising concerns about the efficacy of this approach to generate novel pharmacological interventions. In this mini-review we argue that over-reliance on behavioral tests that are brief and highly sensitive to external factors play a critical role in this failure and propose that automated home-cage monitoring offers several advantages that will increase the translational utility of preclinical psychiatric research in rodents. We describe three of the most commonly used approaches for automated home cage monitoring in rodents [e.g., operant wall systems (OWS), computerized visual systems (CVS), and automatic motion sensors (AMS)] and review several commercially available systems that integrate the different approaches. Specific examples that demonstrate the advantages of automated home-cage monitoring over traditional tests of anxiety, depression, cognition, and addiction-like behaviors are highlighted. We conclude with recommendations on how to further expand this promising line of preclinical research.

Smoking remains the leading cause of morbidity and mortality in the United States, with less than 5% of smokers attempting to quit succeeding. This low smoking cessation success rate is thought to be due to the long-term adaptations and alterations in synaptic plasticity that occur following chronic nicotine exposure and withdrawal. Glial cells have recently emerged as active players in the development of dependence phenotypes due to their roles in modulating neuronal functions and synaptic plasticity. Fundamental studies have demonstrated that microglia and astrocytes are crucial for synapse formation and elimination in the developing brain, likely contributing to why glial dysfunction is implicated in numerous neurological and psychiatric disorders. Recently, there is increasing evidence for the involvement of glial cells in drug dependence and its associated behavioral manifestations. This review summarizes the newly evaluated role of microglia and astrocytes as molecular drivers of nicotine dependence and withdrawal phenotypes. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology.

Converging epidemiological studies show that a life-threatening event increases the incidence of posttraumatic stress disorder (PTSD), which carries 30% to 50% comorbidity with substance use disorders (SUDs). Such comorbidity results in greater drug use and poorer treatment outcomes. There is overlap between the enduring synaptic neuroadaptations produced in nucleus accumbens core (NAcore) by acute restraint stress and cocaine self-administration. Because of these coincident neuroadaptations, we hypothesized that an odor paired with acute restraint stress would reinstate drug seeking and chose two mechanistically distinct drugs of abuse to test this hypothesis: alcohol and cocaine. Rats were trained to self-administer either drug beginning 3 weeks after odor pairing with acute stress or sham, and acute restraint stress increased alcohol consumption. Following context extinction training, the stress-paired odor reinstated both alcohol and cocaine seeking, while an unpaired odor had no effect. N-Acetylcysteine (NAC) restores drug and stress-induced reductions in glial glutamate transporter-1 and has proven effective at reducing cue-induced reinstatement of drug seeking. We administered NAC for 5 days prior to reinstatement testing and abolished the capacity of the stress-paired odor to increase alcohol and cocaine seeking. Importantly, daily NAC given during or just following experiencing acute restraint stress also prevented the capacity of stress-paired odors to reinstate alcohol and cocaine seeking and prevented stress-induced deficits in behavioral flexibility. These data support using daily NAC treatment during or immediately after experiencing a strong acute stress to prevent subsequent conditioned stress responding, in particular relapse and cognitive deficits induced by stress-conditioned stimuli.

Alcohol use disorder has multiple characteristics including excessive ethanol consumption, impaired control over drinking behaviors, craving and withdrawal symptoms, compulsive seeking behaviors, and is considered a chronic condition. Relapse is common. Determining the neurobiological targets of ethanol and the adaptations induced by chronic ethanol exposure is critical to understanding the clinical manifestation of alcohol use disorders, the mechanisms underlying the various features of the disorder, and for informing medication development. In the present review, we discuss ethanol's interactions with a variety of neurotransmitter systems, summarizing findings from preclinical and translational studies to highlight recent progress in the field. We then describe animal models of ethanol self-administration, emphasizing the value, limitations, and validity of commonly used models. Lastly, we summarize the behavioral changes induced by chronic ethanol self-administration, with an emphasis on cue-elicited behavior, the role of ethanol-related memories, and the emergence of habitual ethanol seeking behavior.

Alcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models. But none of these studies have systematically investigated expression within the unique cell types present in the brain. We utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16 000 nuclei isolated from the prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes and microglia. To our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species and the first such analysis in humans for any addictive substance. These findings greatly advance the understanding of transcriptomic changes in the brain of alcohol-dependent individuals.

Gambling disorder is a common condition that was previously listed as an impulse control disorder, but is now considered a substance-related and addictive disorder. Gambling disorder has been associated with various untoward long-term outcomes including impaired quality of life, relationship break-ups, debt and mortgage foreclosure, and elevated risk of suicidality. This paper provides a concise primer on gambling disorder, with a special focus on its parallels with substance use disorders. We consider clinical presentations, comorbid expression, heritability, and treatment approaches (psychological and pharmacological). Lastly, we highlight new treatment directions suggested by the literature.

Alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD) are two prevalent psychiatric conditions in the U.S. The co-occurrence of AUD and PTSD is also common, and associated with a more severe clinical presentation and worse treatment outcomes across the biopsychosocial spectrum (e.g., social and vocational functioning, physical health) as compared to either disorder alone. Despite the high co-occurrence and negative outcomes, research on effective medications for AUD/PTSD is sparse and there is little empirical evidence to guide treatment decisions. The study described in this paper addresses this knowledge gap by testing the efficacy of N-acetylcysteine (NAC) in reducing alcohol use and PTSD symptoms. Animal studies and prior clinical research suggest a role for NAC in the treatment of substance use disorders and PTSD via glutamate modulation. NAC is a cysteine pro-drug that stimulates the cystine-glutamate exchanger, normalizes glial glutamate transporters, and restores glutamatergic tone on presynaptic receptors in reward regions of the brain. Moreover, NAC is available over-the-counter, has a long-established safety record, and does not require titration to achieve the target dose. This paper describes the rationale, study design, and methodology of a 12-week, randomized, double-blind, placebo-controlled trial of NAC (2400 mg/day) among adults with co-occurring AUD and PTSD. Functional magnetic resonance imaging (fMRI) and proton magnetic resonance spectroscopy (1H-MRS) are utilized to investigate the neural circuitry and neurochemistry underlying comorbid AUD/PTSD and identify predictors of treatment outcome. This study is designed to determine the efficacy of NAC in the treatment of co-occurring AUD/PTSD and provide new information regarding mechanisms of action implicated in co-occurring AUD/PTSD.

Several studies strongly support the role of the dopamine D2-like and glutamate mGlu5 receptors in psychostimulant reward and relapse.

The present study employed cocaine or MDMA self-administration with yoked-triad procedure in rats to explore whether extinction training affects the drug-seeking behavior and the D2-like and mGlu5 receptor Bmax and Kd values in several regions of the animal brain.

Both cocaine and MDMA rats developed maintenance of self-administration, but MDMA evoked lower response rates and speed of self-administration acquisition. During reinstatement tests, cocaine or MDMA seeking behavior was produced by either exposure to the drug-associated cues or drug-priming injections. The extinction training after cocaine self-administration did not alter significantly D2-like receptor expression the in the limbic and subcortical brain areas, while MDMA yoked rats showed a decrease of the D2-like binding density in the nucleus accumbens and increase in the hippocampus and a rise of affinity in the striatum and hippocampus. Interestingly, in the prefrontal cortex a reduction in the mGlu5 receptor density in cocaine- or MDMA-abstinent rats was demonstrated, with significant effects being observed after previous MDMA exposure. Moreover, rats self-administered cocaine showed a rise in the density of mGlu5 receptor for the nucleus accumbens.

This study first time shows that abstinence followed extinction training after cocaine or MDMA self- or passive-injections changes the D2-like and mGlu5 density and affinity. The observed changes in the expression of both receptors are brain-region specific and related to either pharmacological and/or motivational features of cocaine or MDMA.