Astrocytes provide support for neurons, regulate metabolic processes, and influence neuronal communication in a variety of ways, including through the homeostatic regulation of glutamate. Following 2-hour cocaine or methamphetamine self-administration (SA) and extinction, rodents display decreased levels of basal glutamate in the nucleus accumbens core (NAcore), which transitions to elevated glutamate levels during drug seeking. We hypothesized that, like cocaine, this glutamate 'overflow' during methamphetamine seeking arises via decreased expression of the astroglial glutamate transporter GLT-1, and withdrawal of perisynaptic astroglial processes (PAPs) from synapses. As expected, methamphetamine self-administration and extinction decreased the level of contact made by PAPs in the NAcore, yet did not impact glutamate uptake, GLT-1 expression, or the general structural characteristics of astrocytes. Interestingly, systemic administration of N-acetylcysteine (NAC), a drug that both upregulates GLT-1 and promotes glialglutamate release, reduced cued methamphetamine seeking. In order to test the impact of astrocyte activation and the induction of glial glutamate release within the NAcore, we employed astrocyte-specific expression of designer receptors exclusively activated by designer drugs (DREADDs). We show here that acute activation of Gq-coupled DREADDs in this region inhibited cued methamphetamine seeking. Taken together, these data indicate that cued methamphetamine seeking following two-hour SA is not mediated by deficient glutamate clearance in the NAcore, yet can be inhibited by engaging NAcore astrocytes.

Cocaine self-administration (SA) in rats dysregulates glutamatergic signaling in the prelimbic (PrL) cortex and glutamate release in the nucleus accumbens (NA) core, promoting cocaine seeking. PrL adaptations that affect relapse to drug seeking emerge during the first week of abstinence; switching from an early (2 hr) hypoglutamatergic state to a later (7 days) hyperglutamatergic state. Different interventions that normalize glutamatergic signaling in PrL cortex at each timepoint are necessary to suppress relapse. We hypothesized that plasticity-related proteins that regulate glutamatergic neurotransmission as well as dendritic spine morphology would be biphasically regulated during these two phases of abstinence in PrL cortical neurons projecting to the NA core (PrL-NA core). A combinatorial viral approach was used to selectively label PrL-NA core neurons with an mCherry fluorescent reporter. Male rats underwent two weeks of cocaine SA or received yoked-saline infusions and were perfused either 2 hr or 7 days after the final SA session. Confocal microscopy and 3D reconstruction analyses were performed for Fos and p-CREB immunoreactivity (IR) in the nucleus of layer V PrL-NA core neurons and GluA1-IR and GluA2-IR in apical dendritic spines of the same neurons. Here we show that cocaine SA decreased PrL-NA core spine head diameter, nuclear Fos-IR and pCREB-IR, and GluA1-IR and GluA2-IR in putative mushroom-type spines 2 hours after the end of cocaine SA whereas the opposite occurred following one week of abstinence. Our findings reveal biphasic, abstinence duration-dependent alterations in structural plasticity and relapse-related proteins in the PrL-NA core pathway after cocaine SA.

Chronic intermittent ethanol (CIE) exposure in rats induces neuroplastic adaptations in the prelimbic cortex (PL) and nucleus accumbens core (NAcore) during withdrawal, contributing to deficits in decision-making and escalation of alcohol consumption. Increased evidence has linked neuroplastic changes associated with the development of alcohol dependence and withdrawal to neuroimmune signaling and activation of microglia. Microglia exhibit a well-defined structure/function relationship, with alterations in cytokine release occurring concomitantly with reconfiguration of cellular dimensions and redistribution of fine processes. Accordingly, gaining a better understanding of ethanol-induced microglia activation will provide insight into how neuroimmune signaling may impact neural structure and function following ethanol exposure. The present study characterized CIE-induced alterations in microglia morphology in the PL and NAcore using confocal microscopy and 3D image reconstruction.Rats were sacrificed at peak withdrawal, 10 hours after discontinuation of 15 days of CIE or Air exposure. CIE-exposed rats exhibited an increase in microglia soma size in the PL and NAcore compared to Air-exposed control rats. However, the CIE-mediated increase in soma volume in the PL cortex was accompanied by a reduction in microglial complexity. While systemic LPS elicited similar increases in soma volume in the PL cortex and NAcore, the LPS-induced increase in soma size was associated with increased complexity of microglial processes in the PL cortex, an effect opposite to that of CIE. These data are consistent with the hypothesis that the microglial activation state following CIE is structurally and chemically distinct from the activation state associated with induction of the innate neuroimmune system following systemic LPS exposure.

Both clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission underlie heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce linked molecular and cellular adaptations in both astrocytes and neurons in the nucleus accumbens (NA) core that are required for cued relapse. For example, decreased expression of the glutamate transporter GLT-1 and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate overflow from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Importantly, normalizing heroin-induced GLT-1 downregulation prevents glutamate overflow, medium spiny neuron plasticity, and relapse. Surprisingly, little is known about heroin-induced alterations in cortical astroglia and their interaction with neurons. Here we show that heroin SA followed by extinction leads to increased astrocyte complexity and association with synaptic markers in the PrL cortex in male Sprague-Dawley rats. Enhanced astroglial complexity and synaptic interaction were reversed during extinction by repeated treatment with N-acetylcysteine (NAC), an antioxidant drug previously shown to inhibit heroin seeking. We also show that dendritic spines of PrL cortical neurons projecting to the NAcore are enlarged, yet the density of spines is decreased, after extinction from heroin SA. Repeated NAC treatment prevented the decrease in spine density but not dendritic spine expansion. These results reveal circuit-level adaptations in cortical dendritic spine morphology that are related to heroin-induced alterations in astrocyte complexity and association at synapses and demonstrate that NAC reverses cortical heroin-induced adaptations in multiple cell types.

Abbreviations: SA (self-administration), NO (nitric oxide), MSNs (medium spiny neurons), nNOS (neuronal nitric oxide synthase), NAcore (nucleus accumbens core), PL (prelimbic), vSub (ventral subiculum), VTA (ventral tegmental area), GLT-1 (glutamate transporter-1), BLA (basolateral amygdala), DETA (DETA-NONOate), CNO (Clozapine-n-oxide), FR (Fixed ratio), standard error of the mean (SEM), eGFP (enhanced green fluorescent protein), cGMP (cyclic GMP), sGC (soluble guanylyl cyclase), CPP (conditioned place preference), MMPs (matric metallo-proteinases), i.v. (intravenous), LOD (limit of detection), GluOx (glutamate oxidase) AbstractCue-induced reinstatement of cocaine seeking after self-administration (SA) and extinction relies on glutamate release in the nucleus accumbens core (NAcore), which in turn activates neuronal nitric oxide synthase (nNOS) interneurons. Nitric oxide (NO) is required for structural plasticity in NAcore medium spiny neurons (MSNs), as well as cued cocaine seeking. However, NO release in the NAcore during reinstatement has yet to be directly measured. Further, the temporal relationship between glutamate release, and the induction of a NO response also remains unknown. Using wireless amperometric recordings in awake behaving rats, we quantified the magnitude and temporal dynamics of novel context-and cue-induced reinstatement-evoked glutamate and NO release in the NAcore. We found that re-exposure to cocaine-conditioned stimuli following SA and extinction increased extracellular glutamate, leading to release of NO in the NAcore. In contrast, exposing drug-naïve rats to a novel context led to a lower magnitude rise in glutamate in the NAcore relative to cue-induced reinstatement. Interestingly, novel context exposure evoked a higher magnitude NO response relative to cue-induced reinstatement. Despite differences in magnitude, novel context evoked-NO release in the NAcore was also temporally delayed when compared to glutamate. These results demonstrate a dissociation between the magnitude of cocaine cue-and novel context-evoked glutamate and NO release in the NAcore, yet similarity in the temporal dynamics of their release. Together, these data contribute to a greater understanding of the relationship between glutamate and NO, two neurotransmitters implicated in encoding the valence of distinct contextual stimuli.