Prenatal alcohol and cannabinoid co-exposure increases ethanol-seeking in adult offspring and produces sex-dependent medial prefrontal cannabinoid receptor 1 expression.

Rouzer S.; Bowring A.; George A.; Domen M.; Labbe E.; Palacios M.; Miranda R. · bioRxiv : the preprint server for biology · 2026

**Background & Methods** This preclinical study examined combined prenatal alcohol and cannabinoid exposure effects on adult offspring using pregnant C57BL/6J mice exposed to ethanol vapor, CP-55,940 (a CB1 agonist), both substances, or neither during gestational days 12-15. Offspring behavior was assessed beginning at postnatal day 200 using home-cage ethanol drinking and operant self-administration paradigms, with medial prefrontal cortex (mPFC) and dorsomedial striatum cannabinoid receptor 1 (CNR1) and CNR1-interacting protein 1 (CNRIP1) quantified via proteomics. **Key Findings** • Prenatal co-exposure (ALC+CB) produced a sex-dependent phenotype: co-exposed males demonstrated significantly elevated ethanol-seeking across multiple measures (cumulative home-cage intake, 40% ethanol progressive-ratio consumption, extinction responding, and reinstatement intake) compared to all other male groups, whereas all exposed female groups showed increased consumption relative to controls but without co-exposure-specific enhancement. • Co-exposed males exhibited elevated mPFC CNR1 protein abundance relative to all other male groups; conversely, cannabinoid exposure (CB and ALC+CB) reduced mPFC CNR1 in females, establishing a sexually dimorphic molecular response to prenatal exposure. • Higher mPFC CNR1 abundance was significantly associated with fixed-ratio intake, 40% progressive-ratio consumption, extinction responding, and reinstatement exclusively in males, suggesting a mechanistic link between altered cannabinoid receptor expression and persistent alcohol-seeking behavior in males. **Dosage & Administration** Ethanol: vapor exposure during gestational days 12-15. Cannabinoid: CP-55,940 at 0.75 mg/kg during gestational days 12-15. Route and specific administration method for CP-55,940 not explicitly detailed. **Safety & Adverse Effects** Not reported. Study focused on behavioral and molecular outcomes rather than maternal or fetal safety parameters. **Evidence Quality** This is a preclinical animal model study with moderate-to-good internal validity (N=57-108 depending on outcome measure) but significant limitations for human translation. Key limitations include: use of rodent models that may not fully recapitulate human prenatal pharmacokinetics and placental transfer; assessment only in late adulthood (postnatal day 200+), limiting understanding of developmental trajectory; and lack of investigation into potential mechanisms beyond mPFC CNR1/CNRIP1. The preprint status indicates peer review is pending. Sex-dependent findings are notable and warrant investigation, though mechanistic understanding remains incomplete. Results justify future studies but do not establish clinical relevance without human evidence.

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