Sex differences in the neurotoxic effects of adenosine A1 receptor antagonism during ethanol withdrawal: reversal with an A1 receptor agonist or an NMDA receptor antagonist

Abstract

Background: Neuronal adaptations that occur during chronic ethanol (EtOH) exposure have been observed to sensitize the brain to excitotoxic insult during withdrawal. The adenosine receptor system warrants further examination in this regard, as recent evidence has implicated adenosine receptor involvement in the behavioral effects of both EtOH exposure and withdrawal.

Methods: The current studies examined effects of adenosine A(1) receptor manipulation on neuronal injury in EtOH-naive and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naive and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A(1) receptor agonist 2-Chloro-N(6)-cyclopentyladenosine (CCPA; 10 nM), the A(1) receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX;10 nM), or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L,-2-amino-5-phosphovalerate (APV; 20 microM) at 15 days in vitro (DIV). Cytotoxicity was measured in the primary neuronal layers of the dentate gyrus, CA3 and CA1 hippocampal regions by quantification of propidium iodide (PI) fluorescence after 24 hours. Immunohistochemical analysis of A(1) receptor abundance was conducted in EtOH-naive and EtOH pretreated slice cultures at 15 DIV.

Results: Twenty-four hour exposure to DPCPX in EtOH-naive slice cultures did not produced neurotoxicity in any region of slice cultures. Though withdrawal from 10 day EtOH exposure produced no toxicity in either male or female slice cultures, exposure to DPCPX during 24 hours of EtOH withdrawal produced a marked increase in PI uptake in all hippocampal culture subregions in female cultures (to approximately 160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A(1) antagonist during withdrawal was greater than that observed in male cultures. These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co-exposure to either the A(1) agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A(1) receptors were observed in male and female EtOH-naive or EtOH pretreated cultures.

Conclusions: The current findings suggest that the female hippocampus possesses an innate sensitivity to effects of EtOH exposure and withdrawal on neuronal excitability that is independent of hormonal influences. Further, this sex difference is not related to effects of EtOH exposure on A(1) receptor abundance, but likely reflects increased NMDA receptor-mediated signaling downstream of A(1) inhibition in females.

Fig. 1

Effects of exposure to the A1 antagonist DPCPX or co-exposure to DPCPX and the A₁ agonist CCPA on neurotoxicity in male and female organotypic hippocampal slice cultures during 24 hours of withdrawal from 10 days of ethanol exposure (43.1 to 26.9 mM). Exposure to DPCPX during ethanol withdrawal (EWD) produced significant sex-dependent toxicity in CA1 pyramidal cells of female cultures and sex-independent toxicity CA3 pyramidal cells and dentate granule cells, effects that were prevented by co-exposure to CCPA. *p < 0.01 versus female controls and ethanol withdrawn slices; **p < 0.01 versus all other groups, including male cultures treated with DPCPX during ethanol withdrawal; #p < 0.05 versus females cultures exposed to DPCPX during ethanol withdrawal. ##p < 0.05 versus EWD+DPCPX.

Fig. 2

Effects of exposure to the A₁ antagonist DPCPX and the NMDA receptor antagonist APV on neurotoxicity in male and female organotypic hippocampal slice cultures during 24 hours of withdrawal (EWD) from 10 days of ethanol exposure (43.1 to 26.9 mM). Exposure to DPCPX during ethanol withdrawal produced significant sex-dependent toxicity in the CA1 pyramidal cells, an effect that was prevented by co-exposure to APV. *p < 0.01 versus controls and ethanol withdrawn slices; **p < 0.05 versus all other groups including male cultures exposed to DPCPX during withdrawal. #p < 0.05 versus females cultures exposed to DPCPX during ethanol withdrawal.

Fig. 3

Representative images of propidium iodide fluorescence in ethanol-naïve and ethanol-withdrawn organotypic hippocampal slice cultures exposed to CCPA and/or DPCPX. (A) Female control; (B) female ethanol withdrawn; (C) female ethanol withdrawn + DPCPX; (D) female ethanol withdrawn + CCPA + DPCPX; (E) female ethanol withdrawn + CCPA + APV.