Cocaine withdrawal during adolescence induces hippocampal microglial dysfunction and excitatory–inhibitory imbalance in response to an acute stress

Stress is a major determinant of vulnerability to substance use disorder (SUD), particularly during adolescence, a developmental window marked by heightened sensitivity to environmental influences. In individuals with early-onset use, stress can directly precipitate drug-seeking behavior and potentiate responses to drug-associated cues. In this work, we investigated how an acute stress challenge affects stress-coping responses in adolescent male rats with a history of cocaine exposure and withdrawal. Interestingly, at molecular level growing evidence indicates that stress-driven neuroimmune adaptations may impair neuroplasticity, thereby sustaining substance use–related behaviors. Hence, particular attention was given to examine the molecular mechanisms of microglia–neuron interactions and their influence in the regulation of glutamatergic and GABAergic signaling pathways in the hippocampus. For this purpose, adolescent male rats were treated subcutaneously with 5 mg/kg/day of cocaine or saline from post-natal day (PND) 28 to PND42. Following 2 weeks of withdrawal, rats were exposed to 1 hour of acute restraint stress (ARS). Thirty minutes after the end of the acute stress, rats were exposed to the elevated plus maze (EPM) test and sacrificed 2 hours later. In the EPM, saline-exposed rats increased the time spent in the closed arms, thus showing an anxiety-like behavior; conversely, cocaine-withdrawn rats did not show any behavioral responses to the stress, suggesting their inability to physiologically cope with an acute challenge. At a cellular level, we observed a significant reduction in microglial activated cells in the hippocampus of cocaine-withdrawn rats, together with reduced expression of Cx3cr1. Moreover, zooming into the ventral subregion of the hippocampus, a region critically involved in stress responses, cocaine-withdrawn rats show a reduced transcriptional profile of pro-inflammatory and microglial markers, suggesting a dampening of microglial reactivity that may represent a transient, compensatory neuroimmune state. In parallel, we observed that cocaine-withdrawn rats have reduced levels of GAD67 and vGAT together with reduced PSD95, suggesting a dysregulation of the excitatory–inhibitory synaptic balance that may compromise hippocampal network stability and enhance vulnerability to stress-induced maladaptive behavioral responses. Indeed, ARS exposure significantly increased microglial activated cells, upregulated Cx3cl1, and the expression of GABAergic and glutamatergic markers in cocaine-withdrawn rats, an effect that may reflect a stress-induced engagement of microglia-mediated synaptic remodeling processes. Altogether these findings suggest that, in male rats undergoing repeated cocaine exposure and prolonged withdrawal during adolescence, stress-induced alterations in microglial activity and synaptic composition within the ventral hippocampus may contribute to the maladaptive processing of anxiogenic stimuli observed in the EPM, thereby potentially increasing susceptibility to relapse.