Sex-specific behavioral and monoaminergic network alterations following adolescent binge-like ethanol and WIN55,212-2 exposure under chronic nicotine in rats

INTRODUCTION: Recreational drug use often begins during adolescence, a critical period of brain maturation characterized by heightened vulnerability to druginduced neuroplastic changes. Although weekend consumption of alcohol and cannabis and nicotine is commonly perceived as relatively harmless, the longterm neurobiological consequences of such patterns remain poorly understood. METHODS: In this study, we investigated the behavioral and neurochemical consequences of combined adolescent exposure to moderate doses of alcohol, cannabinoid receptor agonist WIN55,212-2, and nicotine in male and female Long-Evans rats. From postnatal day (P)30 to P60, animals received daily nicotine (0.3 mg/kg, i.p.) together with intermittent ethanol (3 g/kg, intragastric) and WIN55,212-2 (1.2 mg/kg, i.p.) administered twice weekly, modeling a weekend-like binge pattern of substance use. Behavioral assessments were conducted at baseline (P30), immediately after treatment (P60), and in early adulthood (P90) using the hole-board (HB) and elevated plus maze (EPM) tests. Monoamine levels were subsequently quantified postmortem in selected cortical, limbic, and midbrain regions involved in anxiety and reward processing and correlation analysis performed to explore relationships between neurochemical across these brain regions. RESULTS: In the HB test, but not in the EPM, treated females exhibited behaviors consistent with increased anxiety-like responses and reduced exploratory drive, including reduced walking and head-dipping, increased grooming, and decreased climbing at P60, with some effects persisting at P90. In contrast, males were minimally affected. Neurochemical analyses revealed only modest changes in absolute serotonin (5-HT) levels in the prefrontal cortex (PFC), with no significant alterations in dopamine (DA) or noradrenaline (NA). However, correlation analyses uncovered a pronounced, sex-dependent reorganization of monoaminergic networks. Females showed increased functional coupling among DA, 5-HT, and NA pathways, particularly involving the PFC and amygdala, whereas males exhibited reduced dopaminergic coherence within the substantia nigra. DISCUSSION: Together, these findings demonstrate that even moderate-low levels of adolescent polydrug exposure can produce long-lasting, sex-specific behavioral alterations, most prominently in females, alongside persistent remodeling of monoaminergic circuitry in both sexes. These results highlight the vulnerability of the adolescent brain to seemingly moderate recreational drug patterns and underscore the importance of considering sex-dependent neurobiological adaptations in assessing long-term neuropsychiatric risk.