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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


Reprint requests: Mark A. Prendergast, Department of Psychology, University of Kentucky, B449 Biomedical and Biological Sciences Building, 741 South Limestone St., Lexington, KY 40536-0509; Fax: (859)323-1979; E-mail:


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 The current studies examined effects of adenosine A1 receptor manipulation on neuronal injury in EtOH-naïve and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naïve and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine (CCPA; 10 nM), the A1 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 μM) 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 A1 receptor abundance was conducted in EtOH-naïve and EtOH pretreated slice cultures at 15 DIV.

Results:  Twenty-four hour exposure to DPCPX in EtOH-naïve 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 ∼160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A1 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 These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co-exposure to either the A1 agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A1 receptors were observed in male and female EtOH-naïve 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 A1 receptor abundance, but likely reflects increased NMDA receptor-mediated signaling downstream of A1 inhibition in females.