Hippocampal microglial dysfunction and excitatory–inhibitory imbalance underlie stress vulnerability in cocaine-withdrawn adolescent rats

Stress is a key factor in vulnerability to cocaine use disorder (CUD), especially during adolescence, a period of heightened sensitivity to environmental challenges. In early-onset users, stress can directly trigger cocaine-seeking or enhance responses to drug-associated cues. Emerging evidence suggests that stress-induced neuroimmune adaptations may disrupt synaptic plasticity, promoting persistent substance use–related behaviors. Here, we investigated how microglial dynamics influence neuronal responses to acute stress in adolescent male rats with a history of cocaine exposure and withdrawal, focusing on their regulation of glutamatergic and GABAergic systems. 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rats increased the time spent in the closed arms, thus showing an anxiety- like behavior; conversely, cocaine-withdrawn rats did not show any behavioral responses to the stress, suggesting their inability to physiologically cope with an acute challenge. At a cellular level, we observed a significant reduction in microglial activated cells in the hippocampus of cocaine-withdrawn rats. 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zooming into the ventral subregion of the hippocampus, a region critically involved in stress responses, cocaine-withdrawn rats show a reduced transcriptional profile of pro-inflammatory and microglial markers, suggesting a dampening of microglial reactivity that may represent a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 In parallel, we observed that cocaine- withdrawn rats have reduced levels of GAD67 and vGAT together with reduced PSD95, suggesting a dysregulation of the excitatory–inhibitory synaptic balance that may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microglial activated cells, upregulated Cx3cl1, and the expression of GABAergic and glutamatergic markers in cocaine-withdrawn rats, an effect that may reflect a stress- induced engagement of microglia-mediated synaptic remodeling processes. Together, these findings indicate that, in adolescent cocaine-withdrawn rats, stress-induced microglial and synaptic adaptations in the ventral hippocampus may underlie the maladaptive processing of anxiogenic stimuli observed in the EPM, potentially priming vulnerability to relapse.