The ethanol metabolite acetic acid activates mouse nucleus accumbens shell medium spiny neurons

Abstract

Although ethanol consumption leads to an array of neurophysiological alterations involving the neural circuits for reward, the underlying mechanisms remain unclear. Acetic acid is a major metabolite of ethanol with high bioactivity and potentially significant pharmacological importance in regulating brain function. Yet, the impact of acetic acid on reward circuit function has not been well explored. Given the rewarding properties associated with ethanol consumption, we investigated the acute effects of ethanol and/or acetic acid on the neurophysiological function of medium spiny neurons of the nucleus accumbens shell, a key node in the mammalian reward circuit. We find that acetic acid, but not ethanol, provided a rapid and robust boost in neuronal excitability at physiologically relevant concentrations, whereas both compounds enhanced glutamatergic synaptic activity. These effects were consistent across both sexes in C57BL/6J mice. Overall, our data suggest acetic acid is a promising candidate mediator for ethanol effects on mood and motivation that deserves further investigation.

NEW & NOTEWORTHY Ethanol consumption disrupts many neurophysiological processes leading to alterations in behavior and physiological function. The possible involvement of acetic acid, produced via ethanol metabolism, has been insufficiently explored. Here, we demonstrate that acetic acid contributes to rapid neurophysiological alterations in the accumbens shell. These findings raise the interesting possibility that ethanol may serve as a prodrug—generating acetic acid as a metabolite—that may influence ethanol consumption-associated behaviors and physiological responses by altering neurophysiological function.

INTRODUCTION

Ethanol, like many addictive drugs, can impart profound alterations to the brain in both humans and rodents. Known for its anxiolytic, euphoric, and rewarding properties (1, 2), ethanol is one of the most used and abused compounds in the United States (3). It is generally thought that ethanol is directly responsible for the rewarding properties of alcohol consumption. However, the end metabolite of ethanol, acetic acid, is also bioactive. The possibility that acetic acid might be a primary driver of changes in neuronal activity has yet to be thoroughly explored.

Although the mechanism by which ethanol consumption elicits rewarding-like physiological responses remains unclear, neurophysiological alterations within the mammalian reward circuit likely play a key role (4). A key node in the mammalian reward circuit is the nucleus accumbens (NAc), composed of the NAc shell (NAcSh) and the NAc core to influence reward-like behavior. When comparing these two subregions, rats preferentially self-administer ethanol into the NAcSh (5); hence, we chose to directly explore the pharmacological effects of ethanol and acetic acid on medium spiny neurons (MSNs) of the NAcSh. We used whole cell recordings from NAcSh MSNs in male and female mice to determine the effects of ethanol, acetic acid, and the combination on intrinsic excitability and glutamatergic synaptic strength—fundamental parameters that influence the firing of these reward-related neurons.

MATERIALS AND METHODS

Animals

Animal procedures were performed at the University of Minnesota 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 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/6J mice were obtained from The Jackson Laboratory, Bar Harbor, ME, group housed, and kept on a 12:12 light:dark cycle with food and water ad libitum.

Chemicals

All chemicals were obtained from Sigma-Aldrich (St Louis, MO).

Whole Cell Recordings

Mice (8–14 wk 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, pH 7.3–7.4, continuously gassed with 95:5 O₂:CO₂ to maintain pH and pO₂. A brain block was cut including the NAcSh region and affixed to a vibrating microtome (Leica VT 1000S; Leica, Nussloch, Germany). Sagittal sections of 240-µm thickness were cut, and the slices transferred to a holding container of artificial cerebral spinal fluid (ACSF) maintained at 30°C, continuously gassed with 95:5 O₂:CO₂, 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 (osmolality: 295–302 mosmol · L⁻¹; pH 7.3–7.4) (6–8) and allowed to recover for 1 h. Following recovery, slices were transferred to a glass-bottomed recording chamber and viewed through an upright microscope (Olympus) equipped with DIC optics, infrared (IR) filter, and an IR-sensitive video camera (DAGE-MTI).

Slices transferred to the glass-bottomed recording chamber were continuously perfused with ACSF, gassed with 95:5 O₂:CO₂, maintained at room temperature, and circulated at a flow of 2 mL · min⁻¹. Patch electrodes were pulled (Flaming/Brown P-97, Sutter Instrument, Novato, CA) 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 (osmolality: 280–285 mosmol · L⁻¹; pH 7.3) (9–11). MSNs were identified under IR-DIC based on morphology and their hyperpolarizing membrane potential (−70 to −80 mV) and were voltage clamped at −80 mV using a Multiclamp 700B amplifier (Molecular Devices), currents 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 allowed to stabilize, which was determined by monitoring capacitance, membrane resistance, access resistance, and resting membrane potential (V_m) (9, 10, 12). 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 V_m negative to −68 mV, and <20% change in series resistance during the recording.

To measure NAcSh MSN neuronal excitability, V_m was adjusted to −80 mV by continuous negative current injection, and a series of square-wave current injections were delivered in steps of +20 pA, each for a duration of 800 ms. To determine the action potential voltage threshold (Vt), ramp current injections (0.437 pA/ms, 800 ms) were made from a potential of −80 mV. Square-wave and ramp current injections were made in the same neurons.

For miniature excitatory postsynaptic current recordings (mEPSC), slices transferred to the glass-bottomed recording chamber were continuously perfused with ACSF containing lidocaine (0.7 mM) to block voltage-gated sodium channels and picrotoxin (100 μM) to block GABAR and was continuously gassed with 95:5 O₂:CO₂, maintained at room temperature and circulated at a flow of 2  mL · min⁻¹. Patch electrodes were pulled (Flaming/Brown P-97, Sutter Instrument, Novato, CA) from borosilicate glass capillaries with a tip resistance of 5–10 MΩ and whole cell recordings made. Electrodes were filled with a cesium methanesulfonate (CsMeSO₄) solution containing (in mM): 120 CsMeSO₄, 15 CsCl, 10 TEA-Cl, 10 HEPES, 0.4 EGTA, 8.0 NaCl, 2.0 Na₂ATP, and 0.3 Na₂GTP (osmolality: 280–285 mosmol · L⁻¹; pH 7.3). MSNs were identified under IR-DIC based on morphology and their hyperpolarizing membrane potential (−70 to −80 mV) and were voltage clamped at −80 mV using A Multiclamp 700B amplifier (Molecular Devices), currents filtered at 2 kHz and digitized at 10 kHz. Holding potentials were not corrected for the liquid junction potential. mEPSCs were recorded for 2 min and analyzed offline using Mini Analysis software (synaptosoft) with an amplitude threshold set at three times the noise level.

Statistical Analysis

Data values were reported as mean ± SE. Depending on the experiments, group means were compared using a paired Student’s t test, a one-way or a 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. Outliers were identified at 2× the standard deviation and included 5 data points within the voltage threshold to action potential analysis only. All statistical analyses were performed with a commercially available statistical package (GraphPad Prism, v. 8.0).

RESULTS

To study the acute physiological impact of ethanol and/or acetic acid on a key node in mammalian reward circuitry, we prepared sagittal slices containing NAcSh from 8- to 14-wk-old C57BL/6J male and female mice for electrophysiological recording. This strain of mice is known for its susceptibility to consumption of high levels of ethanol (13, 14). While using whole cell methods to record activity from NAcSh MSNs, we bath applied physiologically relevant concentrations of ethanol [44 mM, equivalent to ∼0.2% blood alcohol (15)], acetic acid [4 mM, reported ranges of 2–5 mM (16–18)], or a combination. We analyzed each data set separately based on sex and found statistically indistinguishable responses in all parameters; thus, data were pooled for display. Passive membrane properties are displayed in Table 1.

Table 1.

Passive NAcSh MSN membrane properties

	Capacitance (pF)	Vm (mV)	Rm (MΩ)
Male (n = 28)	80.5 ± 2.9	−75.2 ± 0.9	115.9 ± 3.8
Female (n = 28)	94.1 ± 2.6**	−77.0 ± 0.7	100.6 ± 3.4**
P value	0.001	0.11	0.0043

MSN, medium spiny neuron; NAcSh, nucleus accumbens shell; R_m, membrane resistance; V_m, resting membrane potential; ** denotes significance between male and female, unpaired t test.

Bath application (5 min) of acetic acid [F_(8,126) = 9.71, P < 0.0001, two-way ANOVA repeated measures] alone or in combination with ethanol [F_(8,117) = 15.00, P < 0.0001, two-way ANOVA repeated measures] produced a rapid and robust leftward shift in the stimulus response curve (Fig. 1, F and N). Surprisingly, bath application of ethanol [F_(8,108) = 1.54, P = 0.15, two-way ANOVA repeated measures] or time course control [F_(8,117) = 0.92, P = 0.51, two-way ANOVA repeated measures] produced no apparent changes to the overall excitability of MSNs (Fig. 1, B and J). Voltage threshold to firing an action potential was unaltered in every treatment group: time course control (P = 0.53, paired t test; Fig. 1D), acetic acid (P = 0.41, paired t test; Fig. 1H), ethanol (P = 0.67, paired t test; Fig. 1L), and ethanol and acetic acid (P = 0.79, paired t test; Fig. 1P).

Figure 1.

Neurophysiological responsiveness of NAcSh MSNs to time, ethanol (EtOH), and acetic acid (HOAc). A: representative raw traces at 160 pA and 220 pA at baseline (black) and 5-min postbaseline recording (blue). B: current injection response at baseline (black) and 5-min postbaseline recording (blue). C: representative raw traces from ramp tests. D: voltage threshold (Vt) for action potential was unaltered between baseline and 5-min postbaseline recordings. E: representative raw traces at 160 pA and 220 pA at baseline (black) and 5-min post-HOAc bath application (blue). F: current injection response at baseline (black) and 5-min post-HOAc bath application (blue). G: representative raw traces from ramp tests. H: voltage threshold (Vt) for action potential was unaltered between baseline and 5-min post-HOAc bath application. I: representative raw traces at 160 pA and 220 pA at baseline (black) and 5-min post-EtOH bath application (blue). J: current injection response at baseline (black) and 5-min post-EtOH bath application (blue). K: representative raw traces from ramp tests. L: voltage threshold (Vt) for action potential was unaltered between baseline and 5-min post-EtOH bath application. M: representative raw traces at 160 pA and 220 pA at baseline (black) and 5-min post-EtOH and HOAc bath application (blue). N: current injection response at baseline (black) and 5-min post-EtOH and HOAc bath application (blue). O: representative raw traces from ramp tests. P: voltage threshold (Vt) for action potential was unaltered between baseline and 5-min post-EtOH and HOAc bath application. N.S., not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NAcSh, nucleus accumbens shell; MSNs, medium spiny neurons.

To determine whether acetic acid changed the pH of our artificial cerebrospinal fluid (ACSF) outside physiological range (7.30–7.40), we added acetic acid (4 mM) into a premade ACSF (pH = 7.39) and measured the resultant pH change with continuous O₂:CO₂ gassing as in our recordings. As expected, adding acetic acid slightly reduced the pH of ACSF (to 7.34 ± 0.01 from 7.39 in standard ACSF, n = 3), but the pH values remained within physiological range.

Many addictive drugs impact the strength of excitatory synaptic transmission within the NAc (19–21), altering mEPSC frequency and/or amplitudes of glutamatergic responses in MSNs—effects that have been postulated to drive addiction-related behaviors (19, 22–24). Thus, we explored the possible effects of acute ethanol and/or acetic acid exposure on these measures in the NAcSh. We found that bath application of both compounds or their combination increased mEPSC frequency (Fig. 2, E, H, and K) compared with baseline (5 min after the start of bath application), acetic acid (P < 0.0001, paired t test), ethanol (P < 0.0001, paired t test), and ethanol and acetic acid (P < 0.001, paired t test). Amplitude of mEPSCs was only affected in the ethanol treatment group (P = 0.0026, paired t test; Fig. 2I). As expected, time course controls revealed no significant differences in mEPSC frequencies (P = 0.22, paired t test) or amplitudes (P = 0.56, paired t test; Fig. 2, B and C).

Figure 2.

mEPSC responsiveness to time, HOAc, EtOH, and EtOH and HOAc. A: representative time control mEPSC traces. B: mEPSC frequency to time control. C: mEPSC amplitude to time control. D: representative HOAc (4 mM) mEPSC traces. E: mEPSC frequency to HOAc (4 mM). F: mEPSC amplitude to HOAc (4 mM). G: representative EtOH (44 mM) mEPSC traces. H: mEPSC frequency to EtOH (44 mM). I: mEPSC amplitude to EtOH (44 mM). J: representative EtOH (44 mM) and HOAc (4 mM) mEPSC traces. K: mEPSC frequency to EtOH (44 mM) and HOAc (4 mM). L: mEPSC amplitude to EtOH (44 mM) and HOAc (4 mM). N.S., not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. EtOH, ethanol; HOAc, acetic acid; mEPSC, miniature excitatory postsynaptic current.

DISCUSSION

In this study, we tested the effects of physiologically relevant concentrations of ethanol (44 mM, blood alcohol ∼0.2%) and acetic acid (4 mM) on neurophysiological properties in the NAcSh—a key node in the mammalian reward circuit. Our choice of doses was guided by several factors. Following ethanol administration in rodents, blood ethanol concentrations up to 66 mM have been reported (25), with 44 mM a commonly used precedent for brain slice studies (26, 27). Of note, blood acetate concentrations in humans and rodents were originally reported in the range of ∼1–2 mM following a moderate dose of ethanol (28, 29). However, use of enzymatic colorimetric assays or derivatization reactions to volatile esters is now known to underestimate the actual blood acetate concentration as: 1) acetate is a common water contaminant that offsets the negative controls and calibration curve in the colorimetric assay; and 2) ester derivatizations to volatile esters with organic extractions result in side reactions and transfer loss. Recently, using a more accurate ion chromatography method (30) following ethanol administration (2 g/kg intraperitoneal), we measured peak serum acetate concentrations of 3–4 mM (31). Thus, we selected 4 mM acetic acid for the current study.

We examined intrinsic excitability and glutamatergic synaptic physiology in NAcSh MSNs—two factors known to be modulated by exposure to a variety of drugs of abuse, and we found that acetic acid produces a rapid increase in MSN excitability whereas ethanol is without effect. At the same time, both ethanol and acetic acid are capable of increasing the frequency of mEPSCs on NAcSh MSNs—consistent with the possibility that these treatments increase presynaptic glutamate release onto these neurons. We also found that these effects on intrinsic excitability and glutamatergic synapses were consistent across sexes. Although our data do not explicitly rule out a connection between the effects on mEPSCs and intrinsic excitability, a straightforward linkage between increased glutamate release and increased firing seems unlikely given our observed dissociation between the effects of ethanol and acetic acid on these properties.

As of yet, there is no consensus on the neural mechanisms for the rewarding effects of ethanol consumption. Administering ethanol to an organism produces broad actions on many neurotransmitter systems and ion channels, eliciting both excitatory and inhibitory actions dependent on the brain region and preparation (32). The degree to which ethanol metabolites might be responsible for some of these effects is largely unknown (33). Although it is reasonable to hypothesize that ethanol itself is the key signaling compound, the possibility that active metabolites might play important roles is intriguing and not without precedent for addictive compounds. For example, heroin undergoes a deacetylation reaction in the brain to produce its more potent metabolite, 6-monoacetylmorphine (34). Our results suggest that acetic acid may be another such active metabolite that can modulate reward circuitry.

Intrinsic excitability and glutamatergic synaptic function in NAc MSNs are emerging as prime targets for plasticity induced by acute or chronic exposure to addictive substances including psychostimulants, opioids, tetrahydrocannabinol, and ethanol (35–39). Whether the mechanisms for these forms of drug-induced plasticity are specific to drug class or due to engagement of a common signaling pathway (e.g., rises in extracellular dopamine) is not yet clear. In the case of the acetic acid effects observed here, there are several possible candidate mechanisms that may contribute to direct modulation of MSN excitability. For example, acid sensing ion channels (ASICs)—channels that are sensitive to decreases in pH (increase in hydronium ions) or alterations to bicarbonate buffering (i.e., fluctuations in pH)—are expressed in NAc neurons (40), and activation of ASICs under normal physiological conditions can provide modest depolarization to MSNs (41), which would increase their tendency to fire, as observed here. On the other hand, we did not observe any change in the voltage threshold for firing during acetic acid exposure—a change that might be expected under depolarizing conditions. It is possible that such an ASIC-mediated depolarization would be subtle enough to elude detection using methods used here yet still produce a significant firing boost.

Might ASICs activation also be a plausible candidate for a role in the acetic acid effects on synaptic transmission we observed? Interestingly, evidence points to a role of ASICs-mediated enhancement of glutamatergic synaptic transmission in NAc MSNs (41), even under normal conditions. Thus, an acetic acid-mediated increase in ASIC activation might be expected to further enhance synaptic function. However, the most likely version of this scenario would involve changes in postsynaptic function, which is not consistent with the pattern of changes we observe here (i.e., no changes in mEPSC amplitude but changes in mEPSC frequency). Perhaps a more possible mechanism involves acidification of the cytosol in presynaptic terminals—an effect of acetic acid/acetate that precipitates neurotransmitter release in synaptosomal preparations (42). Although the mechanism for cytosolic accumulation is not clear, one likely possibility is through the action of ubiquitous monocarboxylate transporters, which carry small organic compounds containing one carboxylic acid functional group, acetic acid among them, into neurons (43). Another potential candidate mechanism involves adenosinergic A_2A receptors that acetic acid/acetate can indirectly activate through adenosine formed during ATP-coupled conversion to acetyl-CoA. A_2A receptors are expressed in the nucleus accumbens (44) and modulate cocaine seeking (45, 46), whereas A_2A activation in the cerebral cortex was found to stimulate glutamate release (47). Taken together, it is possible the acetic acid-induced increase in excitability and presynaptic glutamate release may be indirectly the result of A_2A activation, although any activation of presynaptic A₁ receptors might be expected to counteract this effect (48). Thus, although some candidate mechanisms exist for the effects observed here, more work is needed.

Scant research has addressed the potential impact of acetic acid on behavior and cognition following ethanol consumption. One complicating factor is the complexity of ethanol metabolism. The chief metabolic pathway of alcohols (Fig. 3A) involves alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (33). ADH converts ethanol to an intermediate acetaldehyde, which is then rapidly converted to the short-chain fatty acid, acetic acid, via ALDH (Fig. 3A). However, alternative metabolism of alcohols occurs through catalase (major metabolic pathway in the brain) (18) and cytochrome P450 (33), and thus interruption of the chief (i.e., ADH/ALDH) metabolic pathway—which could otherwise be used as a diagnostic tool for lack of metabolite action—is not conclusive. Thus, to thoroughly dissect the mechanisms for ethanol’s physiological actions will require interruption of multiple metabolic pathways. For example, future studies, even with a brain-slice approach as used here, would benefit from using enzymatic inhibitors of metabolic pathways to distinguish ethanol’s actions from those of its metabolites.

Figure 3.

Graphical summary. A: major metabolic pathway for ethanol metabolism. B: experimental findings: 1) basal conditions of NAcSh MSN intrinsic excitability and incoming glutamatergic synapses; 2) HOAc enhanced NAcSh MSN excitability and glutamatergic synapses. C: proposed circuit-level effects of acetic acid in the NAcSh. Enhanced MSN excitability including increased presynaptic glutamate and likely dopamine release. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; CYP450, cytochrome P450; HOAc, acetic acid; MSN, medium spiny neuron; NAcSh, nucleus accumbens shell; VTA, ventral tegmental area.

Taking these complications into account, recent studies have begun to shed light on some of the bioactive properties of acetic acid/acetate (16, 18, 49, 50). For example, we recently demonstrated that ethanol microinjection into the central nucleus of the amygdala (CeA) elicited sympathoexcitatory responses driven primarily through activation of N-methyl-d-aspartate receptors (NMDARs) (51). This was unexpected, given the considerable evidence that ethanol inhibits NMDARs in neurons from other brain regions (27, 52, 53). Hypothesizing that local brain metabolism of ethanol was responsible, we microinjected acetate into the CeA and found similar NMDAR-dependent sympathoexcitatory responses. Among other examples of acetate bioactivity, a recent epigenetics study found that ethanol metabolism to acetic acid/acetate—a feedstock for acetylation reactions—increased brain histone acetylation, which led to modulation of transcriptional programs linked to alcohol-associated behaviors (54). These studies demonstrate the importance of further investigating the impact of acetic acid/acetate on neurophysiological function, especially with regard to ethanol action in the CNS.

In conclusion, our data suggest rapid and robust neurophysiological alterations from the short-chain fatty acid, acetic acid—a chief metabolite of ethanol—in NAcSh MSNs. These data raise the intriguing possibility that ethanol can operate as a prodrug, with its metabolite producing more neuroactive effects than ethanol itself. Future studies will need to determine whether acetic acid alone can elicit rewarding and/or aversive behavioral responses and how chronic ethanol and/or withdrawal alters behavioral and neurophysiological sensitivity to acetic acid.

GRANTS

This study was supported by NIH R01DA041808 and MnDRIVE Neuromodulation Fellowship (A.D.C.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.D.C. conceived and designed research; A.D.C. performed experiments; A.D.C. analyzed data; A.D.C. and M.J.T. interpreted results of experiments; A.D.C., P.G.M., and M.J.T. prepared figures; A.D.C., P.G.M., and M.J.T. drafted manuscript; A.D.C., P.G.M., and M.J.T. edited and revised manuscript; A.D.C., P.G.M., and M.J.T. approved final version of manuscript.