Sex differences in hippocampal and prefrontal gene expression of endocannabinoid, glutamatergic and GABAergic systems and amino acids profile underlie cocaine plus ethanol seeking incubation.

Addiction is conceptualized as a maladaptive form of learning in which drug-associated memories progressively strengthen during abstinence and increasing relapse vulnerability. This process may be particularly complex in the context of cocaine and ethanol abuse, a prevalent pattern of polyconsumption linked to greater clinical severity. To model this condition, in our previous work male and female rats were subjected to an incubation of drug-seeking paradigm. While females exhibited robust incubation of cocaine plus ethanol seeking after prolonged withdrawal, males did not (Garrido-Matilla et al., 2025). In the present study, we characterized neurobiological alterations in the same cohort by analysing gene expression of endocannabinoid, glutamatergic, and GABAergic systems, as well as neurotransmission-related amino acids in the hippocampus and PFC. In the hippocampus, females showed increased cannabinoid receptor 1 expression and reduced monoacylglycerol lipase levels, together with upregulation of GABAA receptor subunits, suggesting persistent adaptations in synaptic plasticity. In contrast, males exhibited downregulation of glutamatergic-related genes along with increased taurine and L-glutamine levels. In the PFC, females displayed increased cannabinoid receptor 1 and NMDA receptor subunit 2B expression, reduced GABAergic-related gene expression, and decreased taurine and L-glutamine levels, consistent with an altered excitatory-inhibitory balance. Moreover, prefrontal glutamate levels positively correlated with cocaine plus ethanol intake in females. Overall, these findings reveal sex-dependent neuroadaptations within the hippocampus and PFC following cocaine plus ethanol polyconsumption, with pronounced endocannabinoid and GABAergic alterations in females, and glutamatergic changes in males that may reflect compensatory mechanisms potentially limiting relapse vulnerability.