Sex differences in caffeine neurotoxicity following chronic ethanol exposure and withdrawal

Aims: Caffeine is a central nervous system stimulant that produces its primary effects via antagonism of the A₁ and A_2A adenosine receptor subtypes. Previous work demonstrated a sex difference in neurotoxicity produced by specific adenosine A₁ receptor antagonism during ethanol withdrawal (EWD) in vitro that was attributable to effects downstream of A₁ receptors at NMDA receptors. The current studies were designed to examine the effect of non-specific adenosine receptor antagonism with caffeine during ethanol withdrawal on hippocampal toxicity in cultures derived from male and female rats. Methods: At 5 days in vitro (DIV), half of the male and female organotypic hippocampal slice cultures were exposed to 50 mM ethanol (EtOH) in culture media for 10 days before exposure to caffeine (5, 20 and 100 μM) for the duration of a 24 h EWD period. In keeping with this timeline, the remaining ethanol-naïve cultures were given media changes at 10 and 15 DIV and exposed to caffeine (5, 20 and 100 μM) for 24 h at 15 DIV. Cytotoxicity was assessed by fluorescent microscopy and quantification of propidium iodide (PI) uptake in the pyramidal cell layers of the CA1 and CA3 regions and the granule cell layer of the dentate gyrus (DG). A two-way (sex × treatment) ANOVA was conducted within each hippocampal region. Results: Twenty-four-hour withdrawal from 10-day exposure to 50 mM ethanol did not produce increased PI uptake in any hippocampal region. Caffeine exposure (5, 20 and 100 μM) in ethanol-naïve cultures did not produce toxicity in the DG or CA1 region, but 20 μM caffeine produced modest toxicity in the CA3 region. Exposure to 20 μM caffeine during EWD produced cytotoxicity in all hippocampal regions, though toxicity was sex-dependent in the DG and CA1 region. In the DG, both 5 and 20 μM caffeine produced significantly greater PI uptake in ethanol-exposed female cultures compared to ethanol-naïve female cultures and all male cultures. Similarly, 20 μM caffeine caused markedly greater toxicity in female cultures as compared to male cultures in the CA1 region. Conclusions: Twenty-four-hour exposure to caffeine during EWD produced significant toxicity in the pyramidal cell layer of the CA3 region in male and female cultures, though toxicity in the granule cell layer of the DG and pyramidal cell layer of the CA1 region was observed only in female cultures. Greater sensitivity of the female slice cultures to toxicity upon caffeine exposure after prolonged ethanol exposure is consistent with previous studies of effects of a specific A₁ receptor antagonism during EWD on toxicity and indicate that this effect is independent of the hormonal milieu. Together, these data suggest that the A₁ receptor subtype is predominant in mediating caffeine's neurotoxic effects during EWD. These findings demonstrate the importance of considering gender/sex when examining neuroadaptive changes in response to ethanol exposure and withdrawal.