Stress-induced vulnerability in cocaine-withdrawn adolescent rats involves microglial dysfunctions and excitatory-inhibitory unbalance in the hippocampus

A major challenge in the treatment of cocaine use disorder (CUD) is the high rate of relapse. In this context, stress is a critical factor that increases relapse vulnerability, mostly frequent among the early-onset users, either by directly triggering cocaine use or by interacting with drug-associated cues to intensify drug-seeking behavior. Since evidence suggests that neuroimmune adaptations might promote aberrant changes in synaptic plasticity, thus contributing to substance abuse-related behaviors such as relapse, we herein investigated how microglia dynamics modulate neuronal responses in adolescent male rats with an history of cocaine exposure and withdrawal after an acute challenge. 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), after 30 minutes to the elevated plus maze (EPM) test and 2 hours later sacrificed. Molecular analyses on microglial determinants were conducted in the dorsal (dH) and ventral (vH) hippocampus, brain subregions involved in cognitive processes and in stress responses, respectively. In the EPM, cocaine-withdrawn rats exhibited a blunted response to the acute challenge, ARS, whereas saline-exposed rats increased the time spent in the closed arms. In both dH and vH, ARS robustly upregulated the pro-inflammatory cytokine Il-1β either in saline or in cocaine-withdrawn rats. Of note, in the dH, Iba1, Trem2 and P2ry12, markers of microglial identity and function, were downregulated in cocaine-withdrawn rats, and restored after ARS. Moreover, cocaine-withdrawn rats show increased levels of the neuronal chemokine Cx3cl1, while its receptor Cx3cr1 remain unaltered. After ARS in cocaine-withdrawn rats, Cx3cl1 is upregulated while the Cx3cr1 is significantly reduced, suggesting that microglia maintain a homeostatic response despite the challenge. Conversely, in the vH the combination of cocaine and ARS downregulated microglial and chemokine markers, with the only exception of Cx3cl1 which is significantly upregulated pointing to a selective neuron–microglia miscommunication, unable to respond to the challenge despite the increased neuroinflammation. Together with a dysfunctional microglial phenotype, we also observed in cocaine-withdrawn rats increased NMDA-, in the dH, and AMPA-mediated, in the vH, glutamatergic signaling, suggestive of an imbalance towards a hyperexcitation. Overall, adolescent cocaine exposure induces a long lasting neuroinflammatory remodeling of the neuro-glia communication with a region-specific pattern, highlighting differential neuroimmune vulnerabilities which may play a key role in stress-induced relapse. Supported by MUR PRIN 20227HRFPJ and Zardi-Gori Foundation