Physiological acetic acid concentrations from ethanol metabolism stimulate accumbens shell medium spiny neurons via NMDAR activation in a sex-dependent manner

Abstract

Recent studies have implicated the ethanol metabolite, acetic acid, as neuroactive, perhaps even more so than ethanol itself. In this study, we investigated sex-specific metabolism of ethanol (1, 2, and 4 g/kg) to acetic acid in vivo to guide electrophysiology experiments in the accumbens shell (NAcSh), a key node in the mammalian reward circuit. There was a sex-dependent difference in serum acetate production, quantified via ion chromatography only at the lowest dose of ethanol (males > females). Ex vivo electrophysiology recordings of NAcSh medium spiny neurons (MSN) in brain slices demonstrated that physiological concentrations of acetic acid (2 mM and 4 mM) increased NAcSh MSN excitability in both sexes. N-methyl-D-aspartate receptor (NMDAR) antagonists, AP5 and memantine, robustly attenuated the acetic acid-induced increase in excitability. Acetic acid-induced NMDAR-dependent inward currents were greater in females compared to males and were not estrous cycle dependent. These findings suggest a novel NMDAR-dependent mechanism by which the ethanol metabolite, acetic acid, may influence neurophysiological effects in a key reward circuit in the brain from ethanol consumption. Furthermore, these findings also highlight a specific sex-dependent sensitivity in females to acetic acid-NMDAR interactions. This may underlie their more rapid advancement to alcohol use disorder and increased risk of alcohol related neurodegeneration compared to males.

Fig. 1. Serum acetate after ethanol exposure.

A Major metabolic pathway of ethanol. B Standard curve for acetate obtained from ion chromatography (IC). C Representative IC chromatogram for serum from a male injected with saline depicting lactate and acetate peak separation with acetate elution time ~5.05 min. D Representative IC chromatogram for serum from a female injected with saline depicting lactate and acetate peak separation with acetate elution time ~5.05 min. E Representative IC chromatogram for serum from a male injected with EtOH (4 g/kg) depicting lactate and acetate peak separation with acetate elution time ~5.05 min. F Representative IC chromatogram for serum from a female injected with EtOH (4 g/kg) depicting lactate and acetate peak separation with acetate elution time ~5.05 min. G Summary serum acetate data of time course ethanol metabolism for male and female animals (**p = 0.0046). Alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), cytochrome P450 (CYP 450). Dashed line depicts i.p. saline acetate levels.

Fig. 2. Acetic acid increases NAcSh MSN excitability.

A AP trains at 160 and 220 pA for male NAcSh neurons during acetic acid (2 mM) treatment (black, baseline; blue, 5 min after). B Summary data for current-injection response to acetic acid (2 mM) treatment for males (**p = 0.0076, ****p < 0.0001). C Representative traces of AP trains at 160 and 220 pA for female NAcSh neurons during acetic acid (2 mM) treatment (black, baseline; red, 5 min after). D Summary data for current-injection response to acetic acid (2 mM) treatment for females (**p = 0.0079, ****p < 0.0001). E AP trains at 160 and 220 pA for male NAcSh neurons during acetic acid (4 mM) treatment (black, baseline; blue, 5 min after). F Summary data for current-injection response to acetic acid (4 mM) treatment for males (***p = 0.0002, ****p < 0.0001). G Representative traces of AP trains at 160 and 220 pA for female NAcSh neurons during acetic acid (4 mM) treatment (black, baseline; red, 5 min after). H Summary data for current-injection response to acetic acid (4 mM) treatment for females (***p = 0.0002,****p < 0.0001).

Fig. 3. Impact of NMDAR antagonist on acetic acid induced increase in NAcSh MSN excitability.

A AP trains at 160 and 220 pA for male NAcSh neurons during acetic acid (4 mM) and memantine (30 µM) treatment (black, baseline; blue, 5 min after). B Summary data for current-injection response to acetic acid (4 mM) and memantine (30 µM) treatment for males. C Representative traces of AP trains at 160 and 220 pA for female NAcSh neurons during acetic acid (4 mM) and memantine (30 µM) treatment (black, baseline; red, 5 min after). D Summary data for current-injection response to acetic acid (4 mM) and memantine (30 µM) treatment for females (**p = 0.0023).

Fig. 4. Acetic acid induces NMDAR-mediated inward currents and produces a more robust response in females.

A Representative NMDAR-mediated inward current traces in male NAcSh neurons (black, acetic acid; blue, acetic acid and memantine). B Representative NMDAR-mediated inward current traces in female NAcSh neurons (black, acetic acid; red, acetic acid and memantine). C Summary data for the effects of acetic acid with and without memantine on NMDAR-mediated inward currents in male NAcSh neurons (*p = 0.0385). D Summary data for the effects of acetic acid with and without memantine on NMDAR-mediated inward currents in female NAcSh neurons (**p = 0.007). E Male vs female acetic acid-induced NMDAR currents (*p = 0.037).