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. Author manuscript; available in PMC: 2024 Jan 11.
Published in final edited form as: Alcohol Clin Exp Res. 2015 Apr 23;39(6):953–961. doi: 10.1111/acer.12722

Effects of acamprosate on attentional set-shifting and cellular function in the prefrontal cortex of chronic alcohol-exposed mice

Wei Hu 1, Brett Morris 1, Angelique Carrasco 1, Sven Kroener 1
PMCID: PMC10782929  NIHMSID: NIHMS1039857  PMID: 25903298

Abstract

Background:

The medial prefrontal cortex (mPFC) inhibits impulsive and compulsive behaviors that characterize drug abuse and dependence. Acamprosate is the leading medication approved for the maintenance of abstinence, shown to reduce craving and relapse in animal models and human alcoholics. Whether acamprosate can modulate executive functions that are impaired by chronic ethanol exposure is unknown. Here we explored the effects of acamprosate on an attentional set-shifting task, and tested whether these behavioral effects are correlated with modulation of glutamatergic synaptic transmission and intrinsic excitability of mPFC neurons.

Methods:

We induced alcohol dependence in mice via chronic intermittent ethanol (CIE) exposure in vapor chambers and measured changes in alcohol consumption in a limited access 2-bottle choice paradigm. Impairments of executive function were assessed in an attentional set-shifting task. Acamprosate was applied subchronically for 2 days during withdrawal before the final behavioral test. Alcohol-induced changes in cellular function of layer 5/6 pyramidal neurons, and the potential modulation of these changes by acamprosate, were measured using patch clamp recordings in brain slices.

Results:

Chronic ethanol exposure impaired cognitive flexibility in the attentional set-shifting task. Acamprosate improved overall performance and reduced perseveration. Recordings of mPFC neurons showed that chronic ethanol exposure increased use-dependent presynaptic transmitter release and enhanced postsynaptic N-methyl-D-aspartate receptor (NMDAR) function. Moreover, CIE-treatment lowered input resistance, and decreased the threshold and the afterhyperpolarization of action potentials, suggesting chronic ethanol exposure also impacted membrane excitability of mPFC neurons. However, acamprosate treatment did not reverse these ethanol-induced changes cellular function.

Conclusion:

Acamprosate improved attentional control of ethanol exposed animals, but did not alter the concurrent changes in synaptic transmission or membrane excitability of mPFC neurons, indicating that these changes are not the pharmacological targets of acamprosate in the recovery of mPFC functions affected by chronic ethanol exposure.

Keywords: acamprosate, prefrontal cortex, attentional set-shifting, alcohol addiction, patch clamp

Introduction

Alcohol addiction is characterized by inappropriate decision making and loss of control over drinking in spite of negative outcomes. Attentional control and response inhibition heavily depend on an intact PFC (Birrell and Brown, 2000; Floresco et al., 2006). Accordingly, impairments of the PFC may contribute to reduced control over impulsive drinking (Moselhy et al., 2001; Chen et al., 2013), forming a vicious cycle that contributes to alcohol abuse and dependence. Acamprosate (calcium acetylhomotaurinate) is approved for the treatment of alcohol dependence and has been shown to reduce craving following alcohol drinking and cue-induced reactivity in both animal models and human addicts. Meta-analyses of independent, double-blind clinical trials show that acamprosate significantly reduces the risk of relapse and promotes duration of abstinence (Mason and Lehert 2012; Rösner et al., 2010). Although its underlying neural mechanism is not fully understood, acamprosate is thought to reduce alcohol craving by dampening a hyper-glutamatergic state (De Witte, 2004; Burattini et al., 2008) that develops during withdrawal. However, as of yet it is unknown whether this modulation can restore normal synaptic plasticity impaired by chronic ethanol exposure.

We have previously shown that chronic alcohol exposure using chronic intermittent ethanol (CIE) as an animal model of alcohol dependence affects attentional set-shifting in mice. These changes were paralleled by CIE-induced increases in NMDAR function at layer 5/6 pyramidal neurons of the medial PFC (Kroener et al., 2012). Deep layer pyramidal neurons of the rodent mPFC integrate signals from multiple sources and send efferents to several brain areas involved in drug addiction (Groenewegen and Uylings, 2000). These cells also show cellular and synaptic specializations that make them ideally suited to maintain persistent network activity which underlies working memory and executive functions of the PFC (Wang et al., 2006, 2008), and changes in their membrane properties that contribute to compulsive drug taking (Chen et al., 2013). Here, we show that subchronic application of acamprosate can reverse the CIE-induced attentional deficits, and we explored potential physiological mechanisms of this effect. We used whole-cell patch clamp recordings to measure CIE-induced changes and the effects of acamprosate on glutamatergic synaptic transmission onto pyramidal neurons of the mPFC and their intrinsic excitability. Our results show that CIE treatment and alcohol drinking impacted both the synaptic transmission and the intrinsic excitability of mPFC pyramidal neurons, leading to increases in use-dependent presynaptic glutamate release, enhanced postsynaptic NMDAR function, and alterations in membrane excitability. Somewhat surprisingly, despite its effects on mPFC-dependent behaviors, subchronic acamprosate treatment did not reverse these CIE-induced alterations in cellular function. The results suggest that acamprosate can restore attentional control independent of changes in glutamatergic transmission onto mPFC neurons or their membrane excitability.

METHODS

Animals

Male adult (75–85 days old at the beginning of all experiments) C57BL/6 mice from our local colony were singly housed in order to measure alcohol consumption using a limited-access paradigm in the animals’ home cages. Animals were kept on a 12-hr light/dark cycle with continuous access to food and water unless otherwise stated. All procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.

Measurements of ethanol intake

A modified sucrose-fading procedure was used to train mice to drink ethanol in their home cages under a limited access (2 hrs/day) paradigm. Ethanol (10% v/v) was adulterated with sucrose (5% w/v) and over 2 daily sessions the ethanol concentration was increased to 12.5% and 15% (v/v), respectively, before sucrose was gradually faded out of the solution (5%, 2% and finally no sucrose). Mice drank 15% ethanol or water for the rest of the study. At 30 mins prior to the start of the dark period water bottles on the home cages were replaced with two 15-ml bottles, containing either ethanol or water. For control animals both bottles contained water. Fluid consumption during the 2-hr sessions was recorded for 5 consecutive days before and after each cycle of CIE exposure (see below).

Induction of alcohol dependence

Induction of alcohol dependence followed procedures previously described (Becker and Hale, 1993). In brief, mice were exposed to ethanol vapor (15–17 mg/liter air) or air (control animals) in inhalation chambers for 16 hrs/day for 4 consecutive days with 8-hr periods of withdrawal separating each 16 hrs exposure period. Ethanol intoxication was initiated by administration of 1.6 g/kg ethanol (8% w/v; intraperitoneally) together with the alcohol dehydrogenase inhibitor pyrazole (1 mM/kg). Mice in the control group received equivalent injections of pyrazole in saline. Chamber ethanol concentration was measured daily and blood samples were collected from all mice to ensure stable blood ethanol concentration (BEC) (175–225 mg/dl) throughout the exposure period. The 4 days in the vapor chambers were followed by 2 days of withdrawal in the home-cages. This cycle was repeated 3 times, with a drinking session of 5 days preceding the first CIE cycle and then following each CIE cycle (Figure 1).

Fig. 1.

Fig. 1

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Schematic representation of the study design. Adult mice (>75 days postnatal) were first trained on an egocentric response discrimination task (Response Day). Animals were then assigned to control or alcohol groups based on their performance on the response discrimination task to achieve comparable average baseline performance scores. Mice were then exposed to 15% alcohol in a 2-bottle choice limited access paradigm (daily 2-hr drinking sessions). After a baseline drinking period of 5 days, dependence was initiated by 3 cycles of chronic intermittent ethanol (CIE) exposure in vapor chambers (see text for details). Control animals were exposed to air in the chambers and only drank water for the duration of the study. Fluid consumption during limited access drinking was measured for all animals over 5 days following each CIE cycle. The last CIE cycle was followed by 3 days of withdrawal or abstinence. During the last 2 days of this period animals received twice daily injections of saline or acamprosate. Animals were then re-tested on the original response discrimination (Re-test Day) before they had to acquire a new response strategy on the following day (Shift to Visual Cue Day). Separate groups of mice were treated the same way, but they were sacrificed for in-vitro electrophysiological recordings on the day equivalent to Shift to Visual Cue Day.

Acamprosate administration

Acamprosate (200 mg/kg) was dissolved in saline and injected subcutaneously twice a day, 12 hrs apart, at a volume of 10 ml/kg for 2 consecutive days before the Retest of the Attentional Set-shifting task (see below) or electrophysiological recordings. Animals that did not receive acamprosate were injected with equivalent volumes of saline.

Attentional Set-Shifting test

Procedures followed those previously described (Kroener et al., 2012). In brief, animals were habituated over 3 days to a plus-maze and were then trained to use an egocentric response strategy (e.g., always turn left) to obtain a food reward in a t-maze configuration, where one of the arms of the plus-maze was blocked off (Response Discrimination). Animals were food-restricted (~2.5 g food/day) for two days prior to habituation and again before Retest day (c.f. Figure 1). Acquisition of this egocentric response strategy took place in the presence of a visual cue (vertical black and white stripes on a 13×10 cm plastic sheet) placed to the side of one arm opposite the stem arm. Placement of the visual cue into the right or left arm varied pseudorandomly to balance the frequency of occurrences in each arm across blocks of 12 consecutive trials. Animals were divided into 3 experimental groups (Ctrl mice that inhaled air in the chambers and only drank water; CIE mice that inhaled alcohol vapor during CIE and had access to alcohol in the 2-bottle choice paradigm; and CIE+Acamp mice that were treated identically to the CIE group, but received acamprosate injections before behavioral or electrophysiological testing) based on their performance on the Response Discrimination to eliminate any potential baseline performance bias between the groups. Animals then went through drinking and CIE treatment as described above (Figure 1). Animals were 130–140 day old at the end of their CIE treatment and the last alcohol drinking period. Three days after the last drinking day, animals were re-tested on the Response Discrimination (Retest day). The next day, animals were then trained to shift from the previously learned strategy and always enter the arm that contained the visual cue (Shift to Visual-Cue Learning day). For all response days the total number of trials to criterion was analyzed. Errors made during the set-shifting test were broken into different types of errors that measure either the ability to shift from the previously learnt strategy (perseverative errors), or the ability to maintain the new strategy (regressive or never-reinforced errors). A perseverative error occurred when a mouse made the same egocentric response as required during the Response Discrimination day, but which was opposite to the direction of the arm containing the visual cue. Six of 12 trials per block required the mouse to respond in this manner (i.e., enter the arm opposite of the previously learned turn direction). A perseverative error was scored when the mouse entered the incorrect arm on three or more trials per block of 4 trials. Errors that followed the pattern of perseverative errors, but did not occur in groups of three or more, were scored as “other errors”. Once the animal made less than three perseverative errors in a block all subsequent errors were scored as regressive errors, as animals were now following the new strategy at least half of the trials. Finally, all other errors were counted as never-reinforced errors, because the strategy that animals followed had never been rewarded before.

Electrophysiological recordings

Additional groups of animals were treated as outlined above and were sacrificed for in-vitro recordings at time points equivalent to the Shift to Visual-Cue Learning day. Mice were anesthetized with urethane (3 g/kg body weight) and decapitated. Brains were rapidly removed and placed in ice-cold oxygenated (95% O2 - 5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mM): 110 choline, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 10 dextrose, 25 NaHCO3, 1.3 Na+-ascorbate, and 2.4 Na+-pyruvate, bubbled with 95% O2 - 5% CO2. Coronal sections of the frontal cortex were cut on a vibratome (VT1000S, Leica, Nussloch). Slices were transferred to an incubation aCSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 Na2HPO4, 2 CaCl2, 4 MgCl2, 10 glucose, 25 Na2HCO3, 1.3 Na+-ascorbate, and 2.4 Na+-pyruvate, bubbled with 95% O2 - 5% CO2. Whole-cell recordings were obtained from pyramidal neurons in layer 5/6 of the mPFC at room temperature using (in mM): 126 NaCl, 2.5 KCl, 1.2 Na2HPO4, 2 CaCl2, 2 MgCl2, 10 glucose, 25 Na2HCO3, 1.3 Na+-ascorbate, and 2.4 Na+-pyruvate, bubbled with 95% O2 - 5% CO2. For voltage-clamp recordings, electrodes (3–5 MΩ) were filled with (in mM): 130 CsCl, 20 TEA, 10 HEPES, 0.5 EGTA, 2 MgCl2, 4 Na-ATP, 0.3 Na-GTP, 14 phosphocreatine, and 2 QX-314. For current-clamp recordings, electrodes were filled with (in mM): 115 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl2, 4 Na-ATP, 0.3 Na-GTP, 14 phosphocreatine. Recordings were performed using a MultiClamp 700B amplifier (Axon Instruments, Union City, CA). Data acquisition and analysis used Axograph-X software (Axograph Scientific, New South Wales, Australia). Access resistance was monitored throughout and a <20% change was deemed acceptable. Spontaneous and miniature AMPAR-mediated currents (sEPSCs and mEPSCs) were recorded as 2-second long sweeps every 10 seconds for 10 minutes at −75mV. Tetrodotoxin (TTX, 0.5μM) was bath applied to block sodium channels during mEPSC recordings. The frequency and amplitude of sEPSCs and mEPSCs were measured using MiniAnalysis (Synaptosoft, Decatur, GA). Evoked synaptic currents were elicited by focal extracellular stimulation close to basal dendrites of the recorded neuron using theta-glass pipettes (Warner Instruments, Hamden, CT). To study relative changes in NMDAR- and AMPAR-mediated currents, we calculated the NMDA/AMPA ratio as previously described (Bonci and Malenka, 1999). Briefly, a compound EPSC was first recorded at +40 mV and the AMPA component of the synaptic response was then pharmacologically isolated by bath application of the NMDAR blocker DL-2-amino-5-phosphonopentanoic acid APV (50 μM). This AMPA current was digitally subtracted from the compound response to yield the NMDAR current. The averaged peaks of the isolated NMDA and AMPA responses were then divided to yield the NMDA/AMPA ratio. To study membrane properties of mPFC neurons, the membrane potential was held at −75 mV and the voltage response to a series of hyperpolarizing and depolarizing current steps (−100 pA to 500 pA, 20 pA steps, 1 s) was recorded in current clamp. The input resistance was calculated from the average of responses to small hyperpolarizing currents (−30 pA, 100 ms) preceding the series of current injections. The properties of the action potential (AP) were measured from the first spike of each recording. For all recordings, GABAA receptor-mediated currents were blocked by bath application of picrotoxin (75 μM). All reagents were obtained from Sigma (St. Louis, MO). Acamprosate was a generous gift from Dr. Foster Olive.

Data analysis

Data were analyzed using one-way analysis of variance (ANOVA) unless otherwise noted, followed by Tukey’s post hoc tests, using SPSS Statistics 20 (IBM Corp., USA). A p-value less than 0.05 was considered significant.

Results

Induction of alcohol dependence and voluntary alcohol consumption in 2-bottle choice paradigm

Ethanol intake (g/kg) was measured in a 2-bottle choice paradigm for five consecutive days before the first CIE cycle (baseline drinking) and after each of 3 CIE cycles. No animals had to be excluded from this study because they showed adverse reactions to the CIE treatment and/or alcohol consumption. A one way ANOVA showed a main effect of treatment (F(3, 139)=4.3, p<0.01). Post hoc tests showed that ethanol intake was significantly increased after the second cycle of CIE, compared to baseline drinking levels (Fig. 2). In contrast, CIE treatment did not influence water consumption (μl/kg) in the same animals (F(3, 139)=0.11, p=0.954). Similarly, water consumption of the control animals did not change over CIE cycles (F(3, 79)=0.469, p=0.705).

Fig. 2.

Fig. 2

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CIE treatment increases ethanol intake in the 2-bottle choice paradigm. The average ethanol intake (g/kg, left axis) of CIE treated animals (n=35) was significantly increased after the second and third CIE cycle, indicating that CIE treatment induced ethanol dependence in these animals. Water consumption (ml/kg, right axis) was not influenced by CIE treatment in the same animals. Similarly, water consumption of control animals (n=20) remained stable and was not affected by air exposure in the vapor chambers. *p<0.05 compared to baseline drinking.

Attentional set-shifting task

Flexibility in directing cognitive resources is a common index of executive functions associated with the mPFC (Birrell and Brown, 2000; Bissonette et al., 2013). We used an attentional set-shifting task to evaluate changes in mPFC function induced by CIE and alcohol drinking, and to determine whether subchronic acamprosate treatment following alcohol exposure can restore cognitive flexibility. As demonstrated previously (Kroener et al., 2012), chronic alcohol exposure did not disrupt the animals’ ability to remember the original response strategy, and animals in all groups reached criterion on Retest day faster than during the initial Response day (two-way ANOVA, Treatment × Test day. Main effect: Treatment, F(2,84)=0.03, p=0.997; Test day, F(1,82)=13.04, p<0.01, Fig. 3A). The next day, mice were trained to employ a new strategy that required them to attend to the location of the visual cue. As shown in Fig. 3A, there was a main effect of treatment on the number of trials required to reach the criterion on set-shifting day (F(2,38)=5.92, p<0.01). Post hoc tests showed that the CIE group required more trials than both the control and the CIE+Acamp group. The three treatment groups did not differ in the average number of reinforcers, i.e. correct responses resulting in food rewards, obtained during either training days (Response or Retest) (Fig. 3B), suggesting that differences in attentional set-shifting were not due to the difference in the strength of the association established during training. Further analysis of the response patterns revealed that the three treatment groups also differed in the total number of errors (F(2,38)=5.635, p<0.01) and the number of perseverative errors committed (F(2,38)=7.41, p<0.01). CIE-treated animals committed a higher total number of errors and made more perseverative errors than control and acamprosate treated animals. The CIE group also made more regressive errors than the control group, and more never-reinforced errors than the acamprosate-treated animals. In contrast, control animals and acamprosate treated animals did not differ in the number of trials to criterion or the number of errors committed (Fig. 3C). Taken together, these data suggest that chronic ethanol exposure reduced cognitive flexibility and that acamprosate treatment can rescue at least some of the mPFC functions impaired by chronic ethanol exposure.

Fig. 3.

Fig. 3

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Chronic ethanol exposure impairs attentional set-shifting, which can be reversed by subchronic acamprosate treatment. (A) Chronic ethanol exposure impaired the intra-dimensional shift in an attentional set-shifting task without affecting long-term memory of the original strategy (Retest). Compared to control animals, mice in the CIE group required more trials to reach criterion during the Shift–to-Visual-Cue day, suggesting impairment of mPFC-dependent attentional control in alcohol-exposed mice. Alcohol-exposed animals that were treated subchronically with acamprosate before the behavioral tests performed on the same level as control animals. (B) Differences in attentional set-shifting task were not due to differences in association strength established during training as the number of reinforcers earned (i.e. the number of correct choices) did not differ between the three treatment groups on either Response or Retest day. (C) Error type analyses showed that alcohol-exposed animals had a higher total number of errors than mice in the control group or animals treated with acamprosate, and specifically that they made more perseverative errors that are indicative of mPFC dysfunction. The inset shows the set-up of the t-maze used for the task. Error bars indicate SEM. Abbreviations: P, perseverative; NR, never reinforced; R, regressive. *p<0.05, **p<0.01.

Synaptic transmission and membrane excitability of pyramidal neurons of the mPFC.

Chronic ethanol exposure enhances NMDAR function, and aberrant NMDAR signaling in the mPFC or hippocampus could contribute to impaired cognitive functions following chronic ethanol exposure (Fujii et al., 2008; Kroener et al., 2012). Therefore, we first measured changes in the relative contribution of NMDA and AMPA currents to the total excitatory synaptic transmission. As shown in Figure 4B, ethanol exposure increased the NMDA/AMPA ratio (F(2,19)=5.491, p<0.05). Treating animals with acamprosate did not reverse this effect, and the NMDA/AMPA ratio in acamprosate-treated animals was still elevated relative to control animals. Because CIE treatment can selectively increases the expression of NR2B subunits (Kroener et al., 2012), which impart slow kinetics to the NMDAR (Paoletti et al., 2013), we measured the decay time of NMDAR-EPSCs. However, we found no evidence for changes in NMDAR kinetics induced by CIE or acamprosate treatment (F(2, 21)=0.437, p=0.652) (Fig. 4C).

Fig. 4.

Fig. 4

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Chronic alcohol use increases the NMDA/AMPA ratio. (A) Representative traces of AMPAR-mediated (black) and NMDAR-mediated (gray) evoked EPSCs for the three treatment groups. (B) CIE treated animals and animals treated with CIE followed by acamprosate showed similarly increased NMDA/AMPA ratios relative to control animals. (C) CIE or acamprosate treatment did not change the decay time of NMDAR-mediated EPSC. Error bars indicate SEM. *p<0.05 compared to controls.

Next, we recorded both spontaneous (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). Miniature EPSCs were recorded in the presence of TTX in the bath and represent AP-independent transmitter release. In contrast, sEPSCs represent both AP-dependent and – independent transmitter release. The amplitude of mEPSCs or sEPSCs is usually considered to reflect the number and function of post-synaptic AMPARs, while the frequency of mEPSCs or sEPSCs indicates the probability of pre-synaptic neurotransmitter release (Dobrunz and Stevens, 1997). Our results showed that neither the frequency of mEPSCs (F(2,25)=0.216, p=0.807) nor sEPSCs (F(2,24)=1.74, p=0.196) was changed by CIE or acamprosate treatment. Similarly, the amplitude of mEPSCs (F(2,25)=0.074, p=0.929) or sEPSCs (F(2,24)=1.05, p=0.36) was comparable between the three treatment groups (Fig. 5A, B, E, F). In addition, the amplitude distribution of mEPSC and sEPSCs, respectively, was similar among the three treatment groups (Fig. 5C, D). These data suggest that neither alcohol exposure nor acamprosate affected pre-synaptic neurotransmitter release or post-synaptic AMPAR function of pyramidal neurons in the mPFC, and they further support the notion that CIE-induced changes in the NMDA/AMPA ratio can be attributed to selective changes in postsynaptic NMDAR function (Kroener et al., 2012).

Fig. 5.

Fig. 5

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Ethanol or acamprosate treatment does not change the properties of spontaneous glutamate release as measured by sEPSCs and mEPSCs. (A, B) Average frequencies and amplitudes of sEPSC were similar across the three treatment groups. (C, D) Bar graphs showing the amplitude distribution of sEPSCs (C) and mEPSC (D), respectively. (E, F) The frequency and amplitude of mEPSCs were not changed by ethanol or acamprosate treatment. (G) Representative traces and individual events of mEPSC from the three treatment groups. Error bars indicate SEM.

Previous studies also found evidence that chronic ethanol exposure can impact pre-synaptic transmitter release to change short-term plasticity (Nelson et al., 1999; Weiner et al., 2004). Thus we examined changes in the paired-pulse ratio (PPR) of synaptically evoked glutamatergic inputs onto pyramidal cells. Neurons of CIE-treated animals had a lower PPR than those from control animals (F(2,39)=7.40, p<0.01), indicating that chronic alcohol exposure modulates presynaptic AP-dependent release of glutamate (Fig. 6). Acamprosate did not reverse these changes and animals treated with acamprosate showed a similar PPR as the CIE group.

Fig. 6.

Fig. 6

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Chronic ethanol exposure decreases the paired pulse ratio (PPR) at glutamatergic inputs to mPFC pyramidal cells. (A) The average PPR of the three treatment groups. The control group had a higher PPR than both the CIE group and the CIE+Acamp group. The PPRs between the CIE group and CIE+Acamp group were comparable. (B) Averaged sample traces of evoked pairs of EPSCs for the three treatment groups. Error bars indicate SEM. *p<0.05, **p<0.01, compared to control group.

Compulsive drug seeking, which characterizes addictive behavior for all drugs of abuse, including alcohol, was found to be associated with decreased intrinsic excitability of PFC pyramidal neurons (Chen et al., 2013). Therefore, we compared basic membrane properties and characteristics of AP firing in response to a series of current steps in our three treatment groups (Fig. 7, table 1). Chronic ethanol exposure decreased the input resistance (F(2, 60)=6.28, CIE vs. Ctrl p<0.01; Fig. 7A), but did not significantly affect the number of spikes evoked by somatic current injection (two-way repeated measure ANOVA, F(2, 60)=1.716, p=0.188; Fig. 7C). Acamprosate treatment did not reverse these changes in input resistance (CIE vs. CIE+Acamp, p=0.79; Ctrl vs. CIE+Acamp, p<0.05) and did not alter the number of evoked spikes (p=0.188). To examine the effect of CIE or acamprosate on properties of the AP, we measured the latency, half-width, amplitude, threshold, and AHP of the first AP (Fig. 7D, table 1). CIE treatment decreased the threshold (F(2, 60)=5.9, p<0.05) and the amplitude of AHP (F(2, 60)=8.39, p<0.01), without altering the latency (F(2, 60)=0.545, p=0.58), half-width (F(2, 60)=1.39, p=0.257) or amplitude of the first spike (F(2, 60)=0.486, p=0.617). Acamprosate treatment did not significantly affect the AP threshold, or the amplitude of the AHP. These results suggest that acamprosate’s effects on attentional set-shifting were not due to the modulation of membrane excitability and AP firing of mPFC pyramidal cells.

Fig. 7.

Fig. 7

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Chronic alcohol exposure alters the intrinsic excitability of pyramidal neurons of the mPFC. (A) Input-output curves of subthreshold membrane responses to series of somatic current injections differed between the three treatment groups. Membrane potentials of cells recorded from CIE-treated and CIE+Acamp treated animals showed a shallower dose-response curve than cells from control animals. This is also reflected in the input resistance (inset) calculated in response to a single (−30pA) current step. (B) Examples of subthreshold membrane responses from neurons of the three treatment groups to series of hyperpolarizing and depolarizing current injections (−100 pA to 80 pA, 20 pA steps). (C) Changes in subthreshold input resistance did not significantly affect the number of APs evoked by somatic current injection. The top shows sample sweeps of firing patterns from mPFC pyramidal neurons of the three treatment groups when injected with 200 pA current. (D) Alcohol exposure changed the AP threshold and reduced the amplitude of the AHP. Sample sweeps showing truncated APs of cells from the three treatment groups. The effects of alcohol exposure were not reversed by acamprosate treatment. Error bars indicate SEM. *p<0.01 **p<0.05, compared to control group.

Table 1.

Action potential properties of mPFC pyramidal neurons from the three treatment groups.

n Half width (ms) Latency (ms) Amplitude (mV) Threshold (mV) AHP amplitude (mV)
Ctrl 21 1.54±0.056 162.86±14.8 80.58±1.57 −39.78±0.75 13.78±0.71
CIE 21 1.52±0.040 158.04±15 80.53±1.87 −42.98±0.84* 10.85±0.49*
CIE+Acamp 21 1.43±0.058 182.73±21.9 82.55±1.53 −44.18±1.23* 10.25±0.73*
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*p<0.05 compared to controls.

Discussion:

We used an animal model of alcohol dependence to examine the effects of chronic ethanol exposure and acamprosate on executive functions and to explore potential underlying neural mechanisms in the mPFC. Chronic ethanol exposure impaired performance on an attentional set-shifting task, which was reversed by sub-chronic application of acamprosate. Chronic ethanol exposure impacted presynaptic neurotransmitter release, post-synaptic NMDAR function, and the input resistance of layer 5/6 pyramidal cells, but did not alter current-evoked action potential firing or the properties of sEPSCs and mEPSCs onto these cells. Acamprosate treatment did not restore the neurophysiological changes induced by chronic ethanol exposure in these cells.

We used CIE exposure (Becker and Hale, 1993) in order to induce alcohol dependence and we measured changes in voluntary alcohol consumption in a 2-bottle choice limited access paradigm. As shown previously (Becker and Lopez, 2004), CIE-exposed animals progressively increased their ethanol intake without concomitant changes in water consumption, indicating that CIE induced ethanol dependence in alcohol drinking animals.

To evaluate the effects of chronic ethanol exposure on executive functions we tested animals on an attentional set-shifting task that depends heavily on the mPFC (Floresco et al., 2009; Kroener et al., 2012). CIE-treated animals required significantly more trials to reach criterion on Shift-to-Visual-Cue Day when they were required to develop a new strategy. Error type analysis indicated that CIE-treated animals committed more perseverative errors, indicating a deficit in the ability to shift focus and adapt their strategy to the task demands. Lesions of the rodent mPFC or local blockade of NMDARs in the mPFC have been shown to impair set-shifting and to produce a selective increase in perseverative errors, indicating that the mPFC plays a crucial role in suppressing the use of a previously relevant but now incorrect strategy (Birrell and Brown, 2000; Dalton et al., 2011; Floresco et al., 2006). Subchronic treatment with acamprosate using a regime that leads to robust reductions in alcohol drinking after a withdrawal period (Hölter et al., 1997) improved both overall performance and the number of perseverative errors committed. Thus we hypothesized that acamprosate can at least partially restore executive functions of the mPFC that are impaired by chronic ethanol exposure.

Compared to the large number of studies that have examined the effect of acamprosate on alcohol drinking, data on the effects of acamprosate on cognitive function are relatively scarce. Studies involving healthy human subjects or alcoholics have reported both moderate improvements and impairments on cognitive or psychomotor tasks (Schneider et al., 1998; 1999, Soyka et al., 1998). A study using acute application of acamprosate in drug-naïve rats found no effect on short-term memory (Mikolajczak et al., 2002). Although its precise mechanisms of action are still unknown (Kiefer and Mann, 2010; Spanagel et al., 2014), evidence suggests that acamprosate restores the balance between excitatory and inhibitory amino acid neurotransmission that gets disrupted by chronic alcohol consumption (Dahchour and De Witte, 2003; De Witte et al., 2004; Kiefer and Mann, 2010). Specifically, acamprosate may attenuate hyper-glutamatergic states that occur during early withdrawal by modulating transmission at both NMDA and mGluR5 glutamate receptors, and by augmenting intracellular calcium release (Al Qatari et al., 1998; Dahchour et al., 1998; Harris et al., 2002; Naassila et al., 1998). However, there is evidence that the modulatory effects of acamprosate on behavior are in fact not due to a direct interaction with NMDARs, but instead can be mimicked by elevations in calcium levels alone (Spanagel et al., 2014). Both acute and chronic ethanol administrations regulate NMDAR function and impact NMDAR-mediated synaptic plasticity, albeit in an opposing manner. Acute ethanol administration inhibits NMDAR function and the induction of long-term potentiation in several brain areas, including the PFC (Lovinger and Roberto, 2011; Weitlauf and Woodward, 2008). In contrast, chronic ethanol exposure enhances NMDAR function and facilitates synaptic potentiation (Kroener et al., 2012; Jeanes et al., 2011; Spanagel, 2009; Xia et al., 2006). Here we replicated previous data (Kroener et al., 2012) which showed that chronic passive ethanol exposure in vapor chambers selectively increases the NMDAmediated current with no significant change in the amplitude or frequency of AMPAR-mediated mEPSCs and sEPSCs, indicating that the function of postsynaptic AMPARs remained unchanged.

Previous studies found that chronic ethanol exposure can lead to a decrease in the PPR in hippocampus (Weiner et al., 2004; Siggins et al., 2005). Consistent with these findings, our CIE treatment also decreased the PPR of pyramidal neurons of the mPFC. The PPR is generally agreed to be inversely correlated with the initial probability of AP-dependent transmitter release (Mennerick and Zorumski, 1995). Thus a lower PPR in alcohol-exposed animals indicates that chronic ethanol exposure increased the probability of transmitter release. In contrast to mEPSCs, which occur independent of AP firing and which were not affected by ethanol, the PPR reflects short-term AP-dependent plasticity of transmitter release, which can be mediated by multiple mechanisms (Inchauspe et al., 2004; Ishikawa et al., 2005). For example, the activity of calcium-binding proteins regulates calcium levels at the presynaptic terminal to reduce the initial probability of neurotransmitter release thereby increasing the PPR (Neher, 1998). At least some of the activity of these calcium-binding proteins can be disrupted by chronic ethanol exposure (Siggins et al., 2005). Although spontaneous AP firing may contribute to sEPSCs, the similarities in the amplitude and frequency of sEPSCs and mEPSCs (c.f. Figure 5) suggest that this contribution is rather small in our data, which may account for the apparent dissociation of CIE-induced changes in sEPSCs and PPR. Thus chronic ethanol exposure appears to affect an AP-dependent mechanism to decrease PPR, which in drug naïve animals contributes to short-term facilitation. Subchronic acamprosate treatment, however, did not affect the PPR of the CIE treated animals, suggesting acamprosate did not restore mPFC function via a modulation of pre-synaptic short-term plasticity that was disrupted by chronic ethanol exposure.

Chronic ethanol exposure can also enhance neuron excitability in multiple brain areas by reducing the input resistance (Scott and Edwards, 1981; Marty and Spigelman, 2012) or by decreasing spike threshold and the amplitude of the AHP (Ripley, 1996; Scott and Edwards, 1981), leading to higher spike rates or bursting probability (Hopf et al., 2007; Marty and Spigelman, 2012). We replicated most of these findings, but saw no significant increase in the number of somatically evoked spikes. This discrepancy could reflect the fact that all of our recordings were performed in the presence of the GABAA receptor blocker picrotoxin. Because chronic ethanol exposure inhibits GABA receptors function (Nevo and Hamon, 1995; De Witte, 2004), application of picrotoxin may therefore have obscured the disinhibition of pyramidal cells that results from chronic alcohol exposure.

In summary, chronic ethanol exposure increased AP-dependent transmitter release, enhanced NMDAR function, and reduced input resistance at mPFC pyramidal neurons. Either of these changes or a combination of them may contribute to the alcohol-induced impairment of PFC functions. Surprisingly, we found that the same acamprosate treatment which significantly improved the performance of alcohol-exposed animals in our PFC-dependent task had no discernible effect on these alcohol-induced neurophysiological changes. The reasons for this discrepancy are not clear. Acamprosate might affect PFC-dependent behavior via layer- or cell-type specific effects that do not involve the deep layer pyramidal neurons we focused on here.

Alternatively, acamprosate’s effects on attentional-set shifting might results from the modulation of other brain areas that contribute to attentional control and decision making. The hippocampus and the anterior cingulate cortex contribute different aspects of rule-learning and conflict-monitoring, respectively, which affect performance in attentional set-shifting tasks (Bissonette et al., 2013).The anterior cingulate cortex is especially important for tasks involving intra-dimensional rule shifts like the one we used in our study (reviewed in Bissonette et al., 2013). However, whether and how acamprosate might modulate cognitive processes in these brain regions in still unknown. Finally, the effects of acamprosate on behavior might reflect the modulation of networks involved in attention and decision making, while the physiological effects of CIE treatment on synaptic transmission and membrane excitability that we report here may be the result of a more general pharmacological effect of alcohol. Animals in our study self-administered alcohol during the drinking periods; however, both here and in our previous study (Kroener et al., 2012) we used passive exposure to relatively high concentrations of EtOH in vapor chambers to induce dependence. Therefore it remains unclear to what extent the changes in PFC physiology reflect the involuntarily induced dependence and associated increases in “relief drinking” that result from CIE. Thus future studies should examine acamprosate’s modulation of cellular function in operant self-administration paradigms where animals respond for alcohol as a reward in order to assess the importance of motivation and other factors that influence extinction learning, which govern drug seeking and relapse.

Acknowledgments

Sources of support: 1R21AA017527-01A2

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