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. 2023 Oct 16;49(5):885–892. doi: 10.1038/s41386-023-01752-8

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

Andrew D Chapp 1,2,#, Chinonso A Nwakama 1,2,3,#, Andréa R Collins 4, Paul G Mermelstein 1,2,5,✉, Mark J Thomas 1,2,5,✉
PMCID: PMC10948831  PMID: 37845488

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.

Subject terms: Excitability, Reward, Biological techniques

Introduction

Acetic acid is a short-chain fatty acid and a major metabolite of ethanol [1]. Research has identified acetic acid as a novel candidate mediator in the behavioral [2–5], electrophysiological [4, 6, 7] and epigenetic changes involved in alcohol use [8]; however, potential mechanisms of action remain scarce. There are relatively limited studies investigating inherent sex differences in neurophysiological mechanisms of action of acetic acid. In addition, there have been no reports of sex-specific production of acetic acid from ethanol metabolism. However, a growing body of evidence suggests that the prevalence of alcohol use disorder in females continues to increase, resulting in a narrowing sex difference for alcohol use between males and females [9, 10]. Moreover, females demonstrate higher sensitivity to alcohol and are more likely to develop alcohol-related neuropathologies [9, 10]. Thus, identifying potential neuroactive compounds and mechanisms are crucial to understanding alcohol use disorder [11] and its associated sex disparities.

One of the most underestimated but highly neuroactive compounds from ethanol metabolism is acetic acid, produced primarily via a two-step oxidation reaction. Notably, ethanol is metabolized (Fig. 1A) to an intermediate, acetaldehyde, primarily through the enzyme alcohol dehydrogenase. Secondary alcohol metabolizing enzymes such as catalase [1] and cytochrome P450 [1] are also capable of converting ethanol to acetaldehyde. The volatile acetaldehyde is then rapidly converted to acetic acid via aldehyde dehydrogenase. Acetic acid loses its acidic hydrogen primarily via the bicarbonate buffering system to form acetate, which can be measured with ion chromatography (IC) [12]. This metabolic pathway is often neglected in research where ethanol is administered to subjects, as the underlying assumption is that ethanol is the compound driving behavioral and neurophysiological responses. This potential oversight has left a gap in the research field, as acetic acid is a reactive and bioactive metabolite capable of disrupting pH homeostasis and influencing neuronal signaling [5, 8, 13–16].

Fig. 1. Serum acetate after ethanol exposure.

Fig. 1

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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.

The nucleus accumbens is a key node in the mammalian reward circuit and is involved in motivational and rewarding behaviors, including drug addictive-associated behaviors [17]. The nucleus accumbens is separated into the core and shell (NAcSh) subregions, where the shell appears to have a robust and longer-sustained response to drugs of abuse [18–20]. Furthermore, ethanol related studies of core vs shell specific self-infusions have revealed shell specificity over core [21], suggesting ethanol has a direct and rewarding effect in this subregion of the nucleus accumbens. As such, we chose to focus our attention on NAcSh medium spiny neuron (MSN) neurophysiology in response to acetic acid.

First, we used ion chromatography (IC) [12] to quantify serum acetic acid/acetate production and investigate any potential sex differences following acute ethanol exposure (1, 2, and 4 g/kg). After quantifying physiological acetic acid concentrations from ethanol metabolism, these concentrations were then used to guide electrophysiology studies to examine the physiological effects of acetic acid on NAcSh MSN neurophysiology. We also explored the potential mechanism by which acetic acid impacted NAcSh excitability, and if there were any inherent sex differences in the acetic acid-induced response.

Materials and methods

Animals

Animal procedures were performed at the University of Minnesota Twin Cities in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and in accordance with protocols approved by the University of Minnesota Twin Cities Institutional Animal Care and Use Committee (IACUC), as well as the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA), group-housed, and kept on a 10:14 dark:light cycle with food and water ad libitum.

Chemicals

All chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) except TTX (Abcam, Boston, MA, USA), D-2-amino-5-phosphonovalerate (D-AP5) (Cayman), memantine HCL (Cayman), KOH IC cartridge (ThermoFisher), ethanol 200 proof USP (United States Pharmacopeia) (Decon Labs, PA) and glacial acetic acid USP (Spectrum Chemicals).

Serum sample collection

Male and female C57BL/6 J mice (>8 weeks old) were weighed and administered a single intraperitoneal (i.p.) injection of 200 proof USP standard ethanol at a dose of 1, 2, or 4 g/kg body weight or an equivalent saline volume. We used 200 proof USP ethanol because it is pharmaceutical grade, devoid of any water for accurate dosing and free from methanol and other harmful additives. Prior to sample collection, female mice were vaginally lavaged for cytology to determine estrous cycle stage. At different time points (5, 15, 30, or 60 min) post ethanol administration, mice were anesthetized with an i.p. injection of pentobarbital (50 mg/kg) and a thoracotomy was performed to obtain venous blood from a right ventricle draw [12], followed by rapid decapitation. Blood samples were deposited into a 1.5 mL centrifuge tube and centrifuged at 10,000 RPM for 5 min. The serum was removed and added to a new 1.5 mL centrifuge tube. All serum samples were placed in a −20 °C freezer until ion chromatography (IC) analysis was performed.

For serum acetate analysis, serum samples were diluted 700-fold by adding 10 μL liquid sample to 6.990 mL of ddH2O (>18 MΩ) in sterile 10 mL centrifuge tubes. The samples were then transferred to IC vials (5 mL, Thermofisher) and analyzed for acetate via IC (Dionex Integrion, Thermofisher), as previously described [12].

Quantification of acetate using ion chromatography (IC)

Diluted samples were loaded into a Dionex AS-DV autosampler (Thermofisher) connected to a Dionex Integrion RFIC system (Thermofisher) by high pressure tubing. 1 mL of sample was injected from the poly vial, which was passed to the IC system. The Dionex Integrion was equipped with a Dionex EGC potassium hydroxide (KOH) RFIC, eluent generator cartridge (Thermofisher) and an AS17-C 4 mm analytical and guard column set. Water (>18 MΩ) used for generating the eluent was auto degassed within the IC system. The sample was eluted with KOH using the following method:

  1. −5–0 min: Equilibration at 1 mmol/L KOH.

  2. 0–10 min: Isocratic at 1 mmol/L KOH.

  3. 10-17 min: Isocratic, 40 mmol/L KOH.

To determine the acetate concentration of the samples, a standard curve was constructed for known concentrations of acetate and linear regression was obtained from the areas under the curve for the known concentrations of acetate. Unknown sample acetate concentrations were determined based on the linear fit and then back-calculated based on the 700-fold dilution. Blanks that were subjected to the same preparation as samples were used during each batch run to assess for any detectable background acetate contamination and there was none.

Whole-cell recordings

In separate experiments from serum acetate analysis, mice (8–14 weeks old) were anesthetized with isoflurane (3% in O2) and decapitated. The brain was rapidly removed and chilled in ice cold cutting solution, containing (in mM): 228 sucrose, 2.5 KCl, 7 MgSO4, 1.0 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 11 d-glucose, continuously gassed with 95:5 O2:CO2 to maintain pH (7.3–7.4) and oxygen saturation (~95%). The extracted brain was trimmed into the shape of a cube and affixed to a vibrating microtome (Leica VT 1000 S; Leica, Nussloch, Germany). Sagittal sections of 240 µm thickness were cut, and the slices were allowed to recover from the brain slicing process for 1 hr in a modified bicarbonate/HEPES ACSF to extend slice longevity [22], continuously gassed with 95:5 O2:CO2, containing (in mM): 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 Na-ascorbate, 2 CaCl2, and 2 MgSO4, pH 7.25–7.3. Following recovery, slices were transferred to a glass-bottomed recording chamber circulated at a rate of 2 ml min−1 with standard ACSF, continuously gassed with 95:5 O2:CO2, and containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 26.2 NaHCO3, 2.5 CaCl2, 11 d-glucose, and 1.0 ascorbic acid (osmolarity: 295–302 mosmol L−1; pH 7.3–7.4). Slices were viewed through an upright microscope (Olympus) equipped with DIC optics, an infrared (IR) filter and an IR-sensitive video camera (DAGE-MTI).

Patch electrodes (Flaming/Brown P-97, Sutter Instrument, Novato, CA) were pulled from borosilicate glass capillaries with a tip resistance of 5–10 MΩ. Electrodes were filled with a solution containing (in mM) 135 K-gluconate, 10 HEPES, 0.1 EGTA, 1.0 MgCl2, 1.0 NaCl, 2.0 Na2ATP, and 0.5 Na2GTP (osmolarity: 280–285 mosmol L−1; pH 7.3) [23–25]. MSNs were identified under IR-DIC based on their morphology and hyperpolarizing membrane potential (−70 to −80 mV). MSNs were voltage clamped at −80 mV using a Multiclamp 700B amplifier (Molecular Devices), and the currents were filtered at 2 kHz and digitized at 10 kHz. Holding potentials were not corrected for the liquid junction potential. Once a GΩ seal was obtained, slight suction was applied to break into whole-cell configuration and the cell was allowed to stabilize. Stability was determined by monitoring capacitance, membrane resistance, access resistance and resting membrane potential (Vm) [23, 24, 26]. Records were not corrected for a liquid junction potential of −15 mV. Cells that met the following criteria were included in the analysis: action potential amplitude ≥50 mV from threshold to peak, resting Vm negative to −64 mV, and <20% change in series resistance during the recording.

To measure NAcSh MSN neuronal excitability, Vm was adjusted to −80 mV by continuous negative current injection. A series of square-wave current injections was delivered in steps of +20 pA, each for a duration of 800 ms. Working concentrations of NMDAR antagonists, AP5 (60 µM) or memantine (30 µM) were diluted from stock solutions, made in ddH2O and were bath applied as a cocktail with acetic acid (4 mM) for excitability studies.

To examine acetic acid-induced inward currents and the role of NMDAR in NAcSh MSNs, brain slices were continuously perfused with modified Mg2+-free ACSF containing (in mM): 121 NaCl, 2.5 KCl, 1.0 NaH2PO4, 26.2 NaHCO3, 2.5 CaCl2, 11 d-glucose, and 1.0 ascorbic acid (osmolarity: 295–302 mosmol L−1; pH 7.3–7.4) at a flow of 2 mL min−1. Slices were gassed with 95:5 O2:CO2. Tetrodotoxin (TTX, voltage gated sodium channel blocker, 0.5 µM) and picrotoxin (GABA-A blocker, 100 µM) were added into the circulating extracellular Mg2+-free ACSF [7]. Cells were voltage clamped at Vm = −80 mV and allowed to stabilize by monitoring baseline current [7]. Once cells were stable and a baseline recording in the absence of acetic acid was observed (~3.0 min), acetic acid (4 mM) was added to the circulating bath and the cells were recorded for 9 min. At this point, washout was observed with no recovery of any neurons tested to baseline holding currents. In separate groups of neurons, acetic acid (4 mM) and memantine (30 µM) were added to the circulating bath and the NMDAR current was observed. To measure total inward current, the difference was taken between the end of drug application and baseline. Note that current-clamp and voltage-clamp experiments were performed in different NAcSh neurons.

Statistical analysis

Data values were reported as mean ± SEM. Depending on the experiments, group means were compared using an unpaired Student’s t-test, a one-way, or two-way ANOVA with repeated measures. Differences between means were considered significant at p < 0.05. Where differences were found, Bonferroni post hoc tests were used for multiple pair-wise comparisons. All statistical analyses were performed with a commercially available statistical package (GraphPad Prism, version 9.3).

Results

Measurement of acetic acid/acetate in serum following acute ethanol exposure

C57BL/6J mice were injected with either saline or ethanol (1, 2, or 4 g/kg). Serum acetate concentrations were quantified 5, 15, 30, and 60 min post injection, using previously developed ion chromatography methodology [12]. Standard curves for acetate were linear and well fit (r2 = 0.9992; Fig. 1B), and acetate peaks displayed good resolution and consistent retention times (~5.05 min) in both sexes (Fig. 1C–F). Female estrous cycle was determined via vaginal cytology; no estrous-dependent effects in acetate production were observed, and thus female data were pooled.

Baseline serum acetate (males, 0.62 ± 0.04 mM and females, 0.56 ± 0.06 mM) was determined from saline treated animals only (15–30 min after saline injection), which was not time locked to ethanol treated animals. Five minutes following injection of 1 g/kg ethanol, males exhibited greater acetate production than females (two-way ANOVA with Bonferroni posthoc test; 1.46 mM ± 0.09 vs 1.00 mM ± 0.043, p = 0.004; Fig. 1G). At other time points and at higher doses of ethanol, no additional sex differences were observed. Ethanol increased serum acetate concentrations relative to saline at all time points tested; males (1, 2, and 4 g/kg; one-way ANOVA, F(4,27) = 27.58, p < 0.0001, one-way ANOVA, F(4,33) = 17.55, p < 0.0001, one-way ANOVA, F(4,28) = 36.71, p < 0.0001), females (1, 2, and 4 g/kg; one-way ANOVA, F(4,24) = 17.50, p < 0.0001, one-way ANOVA, F(4,27) = 10.24, p < 0.0001, one-way ANOVA, F(4,26) = 14.13, p < 0.0001). Increasing the dose of ethanol shifted the apparent time to peak for serum acetate, ~30 min for 2 g/kg and ~60 min for 4 g/kg. It also increased the peak serum acetate concentration (Fig. 1G). The maximum mean serum acetate concentration was produced at the 4 g/kg dose 60 min after ethanol administration, in both males and females; (3.06 ± 0.35 mM and 3.03 ± 0.53 mM). The absolute maximum concentration of serum acetate measured in males was 3.96 mM and females was 4.55 mM (Fig. 1G).

Acetic acid increases excitability of NAcSh MSNs

Since our experimental design was a within group treatment (pre vs post measurements) and also involved comparisons between males and females, we conducted a time-course control as well as monitored estrous cycle and performed recordings in diestrus and estrus [6, 27]. Additionally, since we were unsure of the long-term effects or washout capability of acetic acid, each successful neuronal recording involving acetic acid was from one neuron per slice (e.g., 9 neuronal recordings were obtained from 9 individual slices across 3–6 animals at a minimum). We found no differences in neuronal excitability (effect of time) in both males (two-way ANOVA, F(1,9) = 0.8353, p = 0.3846; Fig. S1A, B) and females (two-way ANOVA, F(1,8) = 2.977, p = 0.1228; Fig. S1C, D). Similarly, we observed no changes in neuronal excitability during different phases of the estrous cycle (two-way ANOVA, F(1,15) = 0.016, p = 0.9012; Fig. S1E, F).

To explore the pharmacologic impact of physiologically relevant concentrations of acetic acid guided by our in vivo acute experiments (Fig. 1), we first assessed for a dose-dependent response to acetic acid (2 mM and 4 mM) in both males and females. We found that a 5-min bath application of both concentrations of acetic acid significantly increased the excitability of NAcSh MSNs in both sexes: males 2 mM (two-way ANOVA, F(1,10) = 11.12, p = 0.0076) (Fig. 2A, B), females 2 mM (two-way ANOVA, F(1,9) = 11.53, p = 0.0079) (Fig. 2C, D). Acetic acid (4 mM) in males and females also increased excitability, (two-way ANOVA, F(1,13) = 25.67, p = 0.0002) (Fig. 2E, F), (two-way ANOVA, F(1,13) = 26.14, p = 0.0002) (Fig. 2G, H). Passive membrane properties between male and female NAcSh MSNs did not differ (Table 1).

Fig. 2. Acetic acid increases NAcSh MSN excitability.

Fig. 2

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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).

Table 1.

Passive NAcSh MSN membrane properties.

Capacitance (pF) Vm (mV)
Male (n = 54) 83.7 ± 2.4 −76.3 ± 0.6
Female (n = 53) 84.6 ± 1.8 −77.4 ± 0.5
P value 0.76 0.18
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Vm = Resting membrane potential, Rm = membrane resistance.

NMDAR antagonists attenuate acetic acid induced increases in NAcSh MSN excitability

As opposed to some previous excitability studies where synaptic blockers were added (picrotoxin and kynurenic acid) [28, 29], here we assessed NAcSh MSN excitability with excitatory synaptic transmission intact as we have done previously [6, 24]. Thus, it remained possible that excitatory neurotransmission, perhaps via NMDARs [30], could play a role in the excitability enhancement by acetic acid. To test this, we used two NMDAR antagonists: AP5, the classical competitive antagonist and memantine, an NMDAR pore blocking antagonist. In males, the co-application of AP5 (60 µM) with acetic acid (4 mM) attenuated the acetic acid-induced increase in excitability across the stimulus response curve (two-way ANOVA, F(1,9) = 2.825, p = 0.1271) (Fig. S2A, B). In females, this treatment drastically blunted the effects of acetic acid across the stimulus response curve but was still significantly different from baseline (two-way ANOVA, F(1,10) = 6.98, p = 0.0246) (Fig. S2C, D). Co-application of memantine (30 µM) with acetic acid (4 mM) was able to abolish the acetic acid-induced increase in excitability across the stimulus response curve in both males (two-way ANOVA, F(1,8) = 2.441, p = 0.1568) (Fig. 3A, B) and females (two-way ANOVA, F(1,8) = 3.048, p = 0.119) (Fig. 3C, D).

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

Fig. 3

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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).

Acetic acid induces greater NMDAR-mediated inward currents in females than males and is not estrous cycle dependent

Given the apparent influence of NMDAR activation on the acetic acid-induced increase in excitability, we pharmacologically isolated NMDAR currents in NAcSh neurons to test for direct effects. Recording in voltage clamp mode (−80mV) in Mg2+-free ACSF to prevent voltage-dependent Mg2+ block, we found that acetic acid (4 mM) induced significant inward currents in both male (−17.38 ± 2.77 pA) and female (−41.74 ± 6.69 pA) NAcSh MSNs compared to their baseline holding current (Fig. 4). Given the larger variability seen in females, we assessed for estrous cycle effects and found no difference in acetic acid-induced NMDAR inward currents between estrus and diestrus (−38.6 ± 9.3 pA vs −32.0 ± 7.8 pA, unpaired t-test, p = 0.59) (Fig. S3), and thus all female data was pooled. When comparing across sex, these acetic acid-induced currents were greater in females compared to males (Fig. 4E, p = 0.037, two-tailed unpaired t-test). To confirm the source of these currents, we co-applied memantine (30 µM) with acetic acid (4 mM), which significantly blunted the acetic acid-elicited current in males (−17.38 ± 2.77 pA vs −7.78 ± 3.23 pA, p = 0.0385, two-tailed unpaired t-test) and females (−41.74 ± 6.69 pA vs −6.23 ± 5.95 pA, p = 0.007, two-tailed unpaired t-test) (Fig. 4C, D), confirming its source as NMDARs.

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

Fig. 4

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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).

Discussion

Our study reveals several findings of relevance to alcohol research. First, we quantified serum acetic acid/acetate levels across sexes after acute ethanol exposure. In general, we found that females and males had similar serum acetate production following ethanol. The only exception was the earliest time point in the lowest dose (5 min, 1 g/kg) where males had higher serum acetate compared to females. This finding is somewhat counterintuitive to previous reports of sex differences in peak ethanol concentrations and clearance, where females are greater than males in both areas [31]. However, similar ALDH activity among sexes [32] may normalize rate of acetate production and account for the relatively similar serum acetate concentrations we find in mice. While these data provide a snapshot of possible brain concentrations of post-ethanol acetic acid levels, brain specific metabolism and/or sequestration kinetics of acetic acid/acetate via monocarboxylate transporters [33] still remains to be explored.

We also found that physiologically relevant concentrations of acetic acid (2 and 4 mM) produced robust boosts in excitability of medium spiny neurons (MSNs) in the NAcSh. This boost occurs within 5 min of exposure to acetic acid as previously reported [6], indicating the potential of acetic acid to rapidly modulate reward circuitry. Similar effects of acetate boosts to excitability have been reported in the central nucleus of the amygdala [7], suggesting that this response is likely not species or brain region- specific.

In addition, we determined that the effects of acetic acid on excitability were sensitive to NMDAR blockade. And while we cannot discern whether this acetic acid-NMDAR interaction is synaptic or extrasynaptic, uncovering any potential difference in future studies may provide additional insight into AUD neurophysiology. Ethanol is widely regarded as an NMDAR antagonist [34–36], whereas our current data and previous studies provide evidence for acetic acid as an NMDAR enhancer [2, 4, 7]. One previous study demonstrated that high ethanol concentrations (44 mM) had no effect on NAcSh neuronal excitability, but a combination of ethanol (44 mM) and acetic acid (4 mM) increased excitability [6]. This highlights the possibility that acetic acid can override the effects of ethanol, which may be especially important in vivo where both ethanol and acetate are in circulation simultaneously, with acetate remaining elevated long after ethanol has been cleared [37]. Future work investigating this mixed pharmacological activity of ethanol and its metabolites at NMDARs in vivo will be needed to understand the neurobiological and compound specific mechanisms engaged by alcohol use.

While our results implicate NMDARs in acetic acid’s effect on excitability, other mechanisms cannot yet be ruled out. For example, evidence from genetically modified mice [3] suggests that acetate is a likely causative agent in alcohol behavioral intoxication [3, 5], and this may be further supported by findings suggesting that intoxication is at least also partially mediated through acid sensing ion channels (ASICs) [38]. While this ethanol/ASICs mechanism was attributed to ethanol-hydrogen bonding interactions with the ion channel [38], we speculate the acidic hydrogen of acetic acid generated from ethanol metabolism may also be involved (Fig. 1). Thus, in addition to an acetic acid/NMDAR mechanism, there may be engagement of ASICs via acetic acid. Our data demonstrate that both AP5 and memantine reduced the excitatory effects of acetic acid, although memantine was more effective than AP5 in females (Fig. S2). Interestingly, memantine is also known to antagonize ASICs, specifically ASIC1A [39], the same ion channel reported to be responsible for behavioral alcohol intoxication [38]. Thus, acetic acid may activate both NMDARs and ASICs, with memantine providing more pharmacological coverage of impacted ion channels [4, 7, 13, 39, 40]. As such, we cannot rule out an acetic acid/ASICs effect within the NAcSh. However, given the significant blunting with AP5 (see Fig. S2) suggests that any acetic acid-ASICs mediated excitation may be a minor effect, especially in males.

An intriguing aspect of our data is the striking sex difference in NMDAR engagement by acetic acid (Fig. 4). On one hand, this appears to follow a trend across the literature of greater NMDAR effects in females than in males [41–44]. On the other hand, given our findings of a lack of sex difference in the boost of excitability by acetic acid (Fig. 2) and a blockade of this boost by NMDAR antagonists (Fig. 3), this robust sex difference in engagement of NMDARs seems surprising. One important note in this regard, is that our excitability experiments were conducted with both excitatory and inhibitory synaptic transmission intact. While NAcSh synaptic neurophysiology has been well mapped in male C57BL/6 J mice [18, 45–47], the same cannot be said for females. It is possible there is stronger GABAergic tone in the female NAcSh which is only unmasked by the addition of picrotoxin—present during our NMDAR experiments, but not while assessing depolarization-induced firing. Acetate has been found to activate GABAergic signaling [3] and would be anticipated to potentially reduce some of the excitatory effects of acetic acid on NMDAR in females if they had stronger GABAergic inputs. Given the complex nature of acetic acid on excitatory [2, 4, 7, 13, 15] and inhibitory ion channels [3, 15, 48], further investigation of synaptic mechanisms altered by acetic acid seems warranted.

Sex differences in the presentation and sequelae of alcohol use disorder in humans are well-described. There is evidence that females advance more quickly from first alcohol use to the development of alcohol use disorder [49]. There is also evidence that the onset of alcohol-related neurodegeneration occurs sooner in females than in males [50]. Acetic acid-NMDAR interactions may be involved in both of these clinical phenomena. Chronic alcohol use has been shown to upregulate NMDAR [51], a process that is implicated in the development of neurodegeneration [52]. In particular, acetic acid activation of NMDAR may cause excitotoxicity through increased cytosolic calcium [52, 53]. This has been identified preclinically in acute acetate treatments [13]. After chronic upregulation of NMDARs, the brain may become more vulnerable to acetic acid-driven neurodegeneration, as more receptors are available for acetate binding. The fact that females show increased vulnerability to the development of AUD and are more sensitive to the neurotoxic effects of chronic alcohol use suggests that the acetic acid-NMDAR interaction may be enhanced in females.

Patients with AUD demonstrate increased brain capacity for acetate, which is utilized as an energy source and is speculated to contribute to continued use of ethanol in AUD [37]. In AUD patients, increased acetate clearance in the brain [37] may be a compensating, neuroprotective mechanism to reduce NMDAR activation. Alternatively, NMDAR activation by acetic acid following ethanol consumption may prime the reward circuit, and subsequent acetate clearance may be aversive [54]. On/off gating of NMDARs when acetic acid is present and absent may be a mechanism driving alcohol relapse. The off-gating component during ethanol withdrawal or acetic acid clearance may create an inherent expectation for acetic acid (either for energy or reward), and this may drive drinking behavior. The fact that memantine has demonstrated efficacy in reducing alcohol cravings in patients with AUD [55, 56] may lend support to this hypothesis.

From a treatment standpoint, if acetic acid/acetate is neuroactive, potentially rewarding, and implicated in alcohol-related neurodegeneration, one might ask how we can utilize this knowledge to develop new interventions for AUD. NMDAR antagonists reduce alcohol craving but do not reduce drinking [55, 56]. Compounds that mimic the effect of acetic acid on NAcSh MSN neuronal excitability, such as apremilast, a PDE4 inhibitor [57], may represent an alternative therapeutic option to reduce drinking. In a clinical-preclinical study, apremilast reduced binge drinking in both humans and rodents, presumably by increasing the excitability of dopamine 1 receptor-expressing MSNs of the NAcSh [57]. Other compounds which similarly increase NAcSh MSN neuronal excitability may be of benefit for AUD. Likewise, investigating acetic acid actions on dopamine 1 and 2 receptor expressing MSNs in the NAcSh also seems necessary. Our study highlights a need for continued exploration of the effects of the ethanol metabolite, acetic acid, on behavior and neurophysiology to address the neurobiological underpinnings of AUD.

Supplementary information

Supplemental Material (1MB, docx)

Acknowledgements

We would like to thank Chau-Mi H. Phan and Pramit J. Jagtap for their assistance. We would also like to thank Dr. Manuel Esguerra, Dr. Timothy W. Chapp, Dr. Scott M. Chapp, Casey L. Mallo and Dr. Qing-Hui Chen for their proofreading and suggestions.

Author contributions

ADC and CAN performed experiments; ADC and CAN, analyzed data; ADC, CAN, PGM and MJT, prepared figures; ADC, CAN, ARC, PGM and MJT drafted manuscript; ADC, CAN, ARC, PGM and MJT interpreted results of experiment; ADC, CAN, ARC, PGM and MJT edited and revised manuscript.

Funding

This study was supported by NIH R01DA041808 (MJT and PGM), T32DA007234 (ADC) and an MnDRIVE fellowship (ADC).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Andrew D. Chapp, Chinonso A. Nwakama.

Contributor Information

Paul G. Mermelstein, Email: pmerm@umn.edu

Mark J. Thomas, Email: tmhomas@umn.edu

Supplementary information

The online version contains supplementary material available at 10.1038/s41386-023-01752-8.

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