Chronic intermittent ethanol exposure enhances NMDA-receptor-mediated synaptic responses and NMDA receptor expression in hippocampal CA1 region

Brain Research 1048 (2005) 69 – 79 www.elsevier.com/locate/brainres

Research report

Chronic intermittent ethanol exposure enhances NMDA-receptor-mediated synaptic responses and NMDA receptor expression in hippocampal CA1 region T.E. Nelson, C.L. Ur, D.L. Gruol* Department of Neuropharmacology, CVN-11, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Accepted 15 April 2005 Available online 24 May 2005

Abstract In previous studies, we found that chronic intermittent ethanol (CIE) treatment – a model of ethanol consumption in which animals are exposed to and withdrawn from intoxicating levels of ethanol on a daily basis – produces neuroadaptive changes in hippocampal area CA1 excitatory synaptic transmission and plasticity. Synaptic responses mediated by N-methyl-d-aspartate (NMDA) receptors are known to be sensitive to ethanol and could play an important role in the neuroadaptive changes induced by CIE treatment. To address this issue, we compared electrophysiological recordings of pharmacologically isolated NMDA-receptor-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of hippocampal slices prepared from control rats and rats exposed to 2 weeks of CIE treatment administered by vapor inhalation. We found that fEPSPs induced by NMDA receptor activation were unaltered in slices prepared shortly after cessation of CIE treatment (i.e., 1 day of withdrawal from CIE). However, following 7 days of withdrawal from CIE treatment, NMDA-receptor-mediated fEPSPs were augmented relative to age-matched controls. Western blot analysis of NMDA receptor subunit expression showed that, at 7 days of withdrawal, the level of protein for NR2A and NR2B subunits was elevated in the CA1 region of hippocampal slices from CIE-treated animals compared with slices from age-matched controls. These results are consistent with an involvement of NMDA-receptor-mediated synaptic responses in the neuroadaptive effects of CIE on hippocampal physiology and suggest that such changes may contribute to ethanol-induced changes in processes dependent on NMDA-receptor-mediated synaptic responses such as learning and memory, neural development, hyperexcitability and seizures, and neurotoxicity. D 2005 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Ligand-gated ion channels Keywords: Alcohol; Glutamate; Rat; Slice; Field potential; Electrophysiology; Immunoblot

1. Introduction Chronic alcohol (i.e., ethanol) consumption causes severe detrimental effects on central nervous system (CNS) function. Key targets of ethanol action are neurotransmitter receptor systems, in particular the GABAergic and glutamatergic systems. Of the glutamatergic receptors, ethanol has prominent effects on neuronal responses * Corresponding author. Fax: +1 858 784 7393. E-mail address: [email protected] (D.L. Gruol). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.041

mediated by the N-methyl-d-aspartate (NMDA) receptor subtype (for a review, see [15]). Acute ethanol exposure depresses NMDA-receptor-activated ionic currents [40] and NMDA-receptor-mediated Ca2+ influx in various brain regions [29]. In contrast, chronic exposure to ethanol results in a compensatory increase in NMDA receptor binding density [24,25] and elevated mRNA and protein expression of NMDA receptor subunits in the CNS [3,9,21,22,27,63]. As a result of increased NMDA receptor expression, altered NMDA-receptor-mediated responses contribute to the hyperexcitability and excito-

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toxicity associated with withdrawal from chronic ethanol [8,24,62,66]. In addition, NMDA receptors are involved in many fundamental CNS processes, such as neurotrophic activity [26] and synaptic plasticity [41], that may be strongly impacted by chronic ethanol exposure. Chronic alcohol consumption in humans typically follows an episodic, or intermittent, pattern characterized by regular bouts of intoxication interspersed with multiple periods of withdrawal from ethanol. In animal models, the CNS effects of chronic ethanol were found to be more severe when an intermittent pattern of ethanol administration and withdrawal is used. For example, chronic intermittent ethanol (CIE) exposure can lead to enhanced neurotoxicity [47], kindling-like increases in seizure susceptibility [38] and EEG spiking, [65], and a reduction of GABAergic inhibition in the hippocampus [37]. However, the effects of in vivo CIE exposure on NMDA receptor expression have not been directly investigated. The hippocampus is a brain region that is particularly sensitive to ethanol [56]. This region expresses high levels of NMDA receptors [23,43,51] and is critically involved in learning and memory formation [58], which are severely impacted by ethanol abuse [20,34]. The leading candidate for a cellular mechanism underlying learning and memory is long-term potentiation (LTP) [42]. Acute ethanol diminishes NMDA-receptor-dependent LTP in the hippocampus [4,44,59]. Long periods of chronic ethanol treatment (e.g., several months) produce a lasting impairment of NMDA-receptor-dependent LTP formation in the hippocampus [18], whereas we have shown that short-term CIE treatment (e.g., 2 weeks) produces a transient impairment of LTP induction that partially recovers after several days of withdrawal [52]. An effect of CIE exposure on LTP raises the possibility that CIE treatment alters NMDA-receptor-mediated synaptic transmission. In addition, enhanced NMDA-receptormediated synaptic transmission could potentially induce a recovery of LTP as was observed at later time points during withdrawal from CIE treatment in our previous studies. Therefore, to address this issue, in the current study, we investigated the effects of short-term CIE treatment on NMDA-receptor-mediated synaptic transmission in the CA1 of the hippocampus. In parallel studies, we also assessed the effects of CIE treatment on the expression of NMDA receptor subunits within the CA1 region. NMDA receptor expression and NMDAreceptor-mediated synaptic function were assessed after 2 weeks of CIE treatment. To determine the persistence of the effects of CIE treatment on NMDA receptor expression in relation to the effects on LTP formation, measurements were also made after 1 or 7 days of withdrawal from CIE. These time points were chosen based on our previous studies showing that NMDA-receptor-mediated LTP is transiently impaired following the CIE treatment period, and recovers by 5 –7 days of withdrawal from CIE.

2. Materials and methods 2.1. Chronic ethanol treatment Seventy-three naive male Wistar rats (42 days old; 140– 160 g; Charles River) were used in the study. The rats were housed 2– 3 per cage with a 6 AM to 6 PM light cycle and with free access to food and water. The animals were divided into 4 treatment groups: chronic intermittent ethanol (CIE) treatment only, 1 day of ethanol withdrawal, 7 days of ethanol withdrawal, and age-matched controls. Animals in the CIE-treated, 1-day withdrawn, and 7-day withdrawn groups received CIE treatment using the vapor inhalation chamber method [54]. This method of ethanol administration offered several advantages for our studies including the ability to control the timing of ethanol exposure and blood alcohol levels (BALs). The animals were exposed intermittently to ethanol on a 14-h on/10-h off cycle for a period of 12 – 14 days. Animals in the ethanol withdrawal groups were then withdrawn from the CIE treatment for a period of 1 day or 7 days. 1- and 7-day withdrawn animals were maintained in identical chambers as the CIE-treated animals, but were not exposed to ethanol vapor during the withdrawal period. Age-matched control animals were maintained in identical chambers for the same duration as the CIE-treated and withdrawn animals, but were not exposed to ethanol vapor at any time. The animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care. 2.2. Blood alcohol level, body weight, and brain weight BALs were determined twice per week during the 2week CIE treatment period. When necessary, adjustments in the ethanol vapor concentration were made after the first BAL measurement to achieve a target BAL of 150– 200 mg/ dl. BALs were determined by drawing tail blood (0.5 ml) into heparinized Eppendorf tubes. Tail blood was also collected from control animals. The samples were centrifuged, the plasma extracted by trichloroacetic acid, and assayed for ethanol by the nicotinamide adenine dinucleotide – alcohol dehydrogenase (NAD – ADH) enzyme spectrophotometric method (Sigma). The mean BAL of each rat was determined by averaging the measurements of all blood samples drawn during the CIE treatment period. Results for individual rats were then compiled to determine the mean BAL in each ethanol treatment group. All treatment groups had a similar mean BAL measured during the CIE treatment period (Table 1). The mean BAL of all animals receiving the CIE treatment was 181.3 T 4.5 mg/dl (mean T SEM; n = 43) over the 2 weeks of ethanol exposure. Following the CIE treatment period, the mean body weight of CIE-treated animals and 1-day withdrawn animals were significantly

T.E. Nelson et al. / Brain Research 1048 (2005) 69 – 79 Table 1 Summary of mean blood alcohol levels and mean body and brain weights BAL (mg/dl T SEM) Control – (55 days) CIE 184.1 T 7.6 (n=17) 1-day WD 191.5 T 7.2 (n=11) Control – (62 days) 7-day WD 170.8 T 7.6 (n=15)

Body wt. (g T SEM)

Brain wt. (g T SEM)

271 T 5 (n=16)

1.83 T 0.03 (n=16)

238 T 5 (n=17)* 1.80 T 0.03 (n=17) 252 T 5 (n=11)* 1.91 T 0.07 (n=11) 305 T 5 (n=14)* 1.94 T 0.04 (n=13) 296 T 7 (n=16)

1.94 T 0.03 (n=15)

* Significant from Control (55 days).

reduced compared to age-matched control animals (55 days) (Table 1). Thus, CIE treatment significantly reduced the amount of weight gained during the 2-week treatment period. Control rats gained approximately 120 g during this period, compared to about 90 – 100 g for the CIE-treated rats. This difference presumably reflects a reduced dietary intake in the CIE-treated rats (we did not directly measure dietary intake). Body weights of control rats were significantly greater at 62 days of age than at 55 days of age, but the body weights of 7-day withdrawn rats were not significantly different from age-matched control rats (62 days), indicating some recovery of normal weight gain following the CIE treatment. To determine if the treatment paradigm also affected brain growth, brain weight was estimated for each animal by doubling the weight of the unused half of the brain. Brain weights were similar between the CIE-treated, 1-day withdrawn, and agematched control rats (55 days) as well as between the 7day withdrawn and age-matched control rats (62 days) (Table 1). The lack of effect of ethanol on brain weight indicates that CIE treatment did not significantly impact brain growth. 2.3. Preparation of hippocampal slices Experiments using slices from control and CIE/withdrawn animals were performed on consecutive days. On experiment days, animals in the CIE treatment group were maintained in the ethanol vapor chamber until preparation of the hippocampal slices, which were prepared in the morning shortly after the ethanol exposure period ended. Slices were prepared from control animals at similar time of day. The animals were weighed, anesthetized with halothane, and decapitated. Brains were rapidly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF). Hippocampal slices (400 Am) were prepared using a McIlwain tissue chopper (Mickle Laboratory Engineering Co. Ltd., Surrey, UK). Slices were held (60 min) in a gas – fluid interface chamber maintained at approximately 33 -C and a perfusion rate of 0.55 ml/min until use. Slices from control and 1- or 7-day withdrawn animals were maintained in normal (ethanol-free) ACSF, whereas slices from CIE-treated animals were prepared and stored in ACSF containing 150 mg/dl (33 mM)

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ethanol until they were transferred to the recording chamber. The slices from the CIE-treated animals were stored in ACSF to ensure that physiological changes that may result from ethanol withdrawal did not occur prior to recording and therefore go undetected. Ethanol was not present during the recording to prevent acute actions of ethanol. The composition of the normal ACSF was (in mM): 130.0 NaCl, 3.5 KCl, 1.25 NaH 2PO 4, 24.0 NaHCO3, 2.0 CaCl2, 2.0 MgSO4, and 10.0 glucose (all chemicals from Sigma). During the slicing procedure, the following substitutions were made in the ACSF in order to maintain slice viability: 0.20 CaCl2 and 5.0 MgSO4. All solutions were gassed continuously with 95% O2/5% CO2 (pH 7.2– 7.4). 2.4. Isolated NMDA-receptor-mediated field potential recordings Hippocampal slices were transferred to a second gas – fluid interface chamber for recording (2 ml/min perfusion rate, 33 -C) and allowed to stabilize for 20– 30 min prior to data collection. All recordings were obtained in ethanol-free ACSF. Synaptic responses were elicited by electrical stimulation (50 As duration; Grass S48 Stimulator, Quincy, MA) of the Schaffer collateral–commissural afferent pathway using a concentric bipolar stimulating electrode (Rhodes Medical Instruments Inc., Woodland Hills, CA) placed at the border of the CA2 and CA1 subregions. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum (dendritic region of pyramidal neurons) of area CA1 with microelectrodes (1– 3 MV) filled with 3 M NaCl. Only slices that had a maximal fEPSP amplitude >2 mV while recording in normal ACSF (prior to isolation of the NMDA-receptormediated field potentials) were used in this study. In order to isolate NMDA-receptor-mediated field potentials, slices from both control and CIE-treated animals were recorded in ACSF containing 1 AM CGP-55845A (Novartis), 100 AM picrotoxin (Sigma), and 10 AM NBQX (Tocris), and the Mg2+ concentration was lowered to 1 mM. In addition, a cut in the Schaffer collateral pathway was made between the CA3 and CA2 subregions of the slices to reduce potential hyperexcitability generated in area CA3 induced by the GABA receptor antagonists. The signals were amplified with an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). The data were acquired using the pClamp software program (Axon Instruments) and analyzed with the AxoGraph software program (Axon Instruments). To determine the response parameters for each slice, an input/output (I/O) protocol was performed. The slices were stimulated at a range of input voltages (typically between 8 and 24 V) starting at the threshold voltage required to elicit a presynaptic fiber volley (PSV) measured in the dendritic region of the CA1. The stimulus strength was increased in steps of 2 V (stimulation rate of 1 pulse per 30 s) until the maximum fEPSP magnitude was reached. The maximal

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fEPSP was an approximation because the enhanced excitability resulting from the pharmacological isolation of NMDA-receptor-activated synaptic responses at high stimulus intensities prevented an exact determination. Presynaptic function was assessed in each slice using a standard paired-pulse stimulation protocol to elicit paired-pulse facilitation (PPF). A paired-pulse interval of 100 or 200 ms was used, which was long enough to prevent summation of the conditioning and test responses. PPF was examined in each slice after adjusting the stimulus intensity such that the fEPSP amplitude was equal to approximately 50% of the maximal fEPSP amplitude. Measurements were made of the initial slope of the fEPSP (defined as the line of best fit covering the initial 2 ms of each postsynaptic response) in all protocols. For paired-pulse data, the relative amount of facilitation for each slice was expressed as the ratio of the second response (slope) with respect to the first response. Compiled data were expressed as the mean T SEM. Statistical analyses were done using ANOVA (factorial) and the Fisher’s Protected Least Significant Difference (PLSD) post hoc test. Statistical significance was set at the P < 0.05 level. 2.5. Immunoblotting In a separate series of experiments, hippocampal slices (600 – 800 Am) were prepared from CIE-treated, 1-day withdrawn, 7-day withdrawn, and age-matched control animals as described above. The CA1 region was isolated and the collected tissue was placed on ice and homogenized with a Potter– Elvehjem tissue grinder (Wheaton Science Products, Millville, NJ) in 10 mM Tris –HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% NP-40, and a Protease Inhibitor Cocktail Tablet (Boehringer Mannheim). After incubation on ice for 1 h, the homogenates were spun for 30 min in an Eppendorf microfuge at 15,000 rpm. The supernatants were saved and the protein concentrations were determined against a series of BSA standards using a Protein Assay Kit (BioRad, Hercules, CA). Samples (10 Ag of protein) for each treatment (in duplicate) were diluted with 2 Laemmli sample buffer, heated at 100 -C for 3 min, and centrifuged to remove the insoluble material. The samples were separated by SDS-PAGE using 8 –16% Tris –glycine gels (Novex, Carlsbad, Ca) and transferred overnight onto Immobilon-P membranes (Millipore, Bedford, MA). Uniform transfer was confirmed by Ponceau S staining. The membranes were blocked for 30 min at room temperature in 3% non-fat milk, PBS, and 0.1% Tween 20 and incubated for 2 –3 h at room temperature with the respective primary antibody. The NR1 subunit (120 kDa) was immunolabeled using a rabbit affinity-purified polyclonal antibody (Chemicon, Temecula, CA; diluted 1:100). The NR2A and NR2B subunits (180 kDa) were immunolabeled using rabbit affinity-purified polyclonal antibodies (Chemicon; diluted 1:1000). After washing 5 times for 5 min with PBS and Tween 20 (0.1%), the membranes were blocked as above and

incubated for 60 min with anti-rabbit secondary IgG coupled to horseradish peroxidase (Amersham, Piscataway, NJ), washed as above, and visualized using the ECL system (Amersham). To determine the relative level of neuronal tissue in each sample, the membranes were stripped with 2mercaptoethanol at 56 -C for 30 min, washed in PBS/Tween20 (0.1%), and reprobed using an antibody against neuronspecific enolase (Chemicon; diluted 1:20000). Protein signals were digitized and quantified from photographic film using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) and were calibrated with photographic step tablet no. 2 (Kodak).

3. Results 3.1. Treatment groups Rats receiving CIE treatment belonged to one of three different treatment groups: (1) rats receiving CIE treatment only (CIE-treated); (2) rats that were withdrawn from CIE treatment for 1 day (1-day withdrawn); and (3) rats that were withdrawn from CIE treatment for 7 days (7-day withdrawn). Compiled data from each group of CIE-treated and withdrawn rats were compared to data from age-matched control rats at either 55 days of age (control group for CIEtreated and 1-day withdrawn rats) or 62 days of age (control group for 7-day withdrawn rats). 3.2. Effects of CIE treatment on NMDA-receptor-mediated synaptic responses Field excitatory postsynaptic potentials (fEPSPs) were elicited by brief electrical stimulation of the Schaffer collateral –commissural afferent pathway and recorded in the dendritic region (stratum radiatum) of hippocampal CA1 pyramidal neurons in ethanol-free ACSF. In order to measure synaptic activity elicited solely by activation of NMDA receptors, slices were recorded in the presence of 5 AM NBQX, an antagonist of the AMPA receptor subtype, as well as 100 AM picrotoxin and 1 AM CGP55845A, antagonists of GABAA and GABAB receptor subtypes, respectively. In addition, extracellular Mg2+ was reduced from 2 mM to 1 mM to relieve the blockade of the NMDA receptor ion channel pore. Under these conditions, NMDAreceptor-mediated fEPSPs recorded in stratum radiatum were characterized by prolonged negative-deflecting potentials preceded by a rapid negative deflection, representing the presynaptic fiber volley (Figs. 1A and B). At higher stimulus intensities, multiple sequential positive deflections, representing reflections of population spikes generated in the somatic region, were superimposed on the peak and recovery phase of the dendritic fEPSP as a result of increased excitability of pyramidal neurons under conditions of pharmacologic blockade of inhibitory synaptic input

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Fig. 1. NMDA-receptor-mediated synaptic function is unaltered shortly after CIE treatment but is enhanced following 7 days of withdrawal from CIE treatment. (A – B) Representative traces of NMDA-receptor-mediated fEPSPs recorded in the CA1 of hippocampal slices taken from CIE-treated, 1-day withdrawn (WD), and age-matched control rats (A) or from 7-day WD and age-matched control rats (B). Sample recordings are at stimulus intensities that produced threshold, 1/2 maximal, and maximal fEPSPs. All recordings were obtained in the absence of ethanol. Inset, NMDA-receptor-mediated fEPSPs were completely blocked by the addition of APV (10 AM). Scale bars in panel B apply to all traces in this figure. (C – D) Representative traces of the initial slopes (first 3 ms) of NMDAreceptor-mediated fEPSPs recorded in slices taken from CIE-treated, 1-day WD, and age-matched control rats (C) or from 7-day WD and age-matched control rats (D). Horizontal bars in panels A and B indicate the time periods shown in panels C and D. (E) NMDA-receptor-mediated fEPSPs were similar to agematched controls in slices from CIE-treated or 1-day withdrawn rats. (F) NMDA-receptor-mediated fEPSPs were significantly larger in slices from 7-day withdrawn rats than in age-matched control slices. *P < 0.05, ANOVA.

within area CA1. All synaptic responses were completely blocked by the addition of 10 AM APV, a specific antagonist of the NMDA receptor, confirming that the fEPSPs were mediated solely by the activation of this receptor subtype (Fig. 1A, inset). There were no differences in the activation threshold of NMDA-receptor-activated fEPSPs between the treatment groups. Immediately following CIE treatment and after 1 day of withdrawal from CIE treatment, NMDA-receptor-mediated synaptic responses were similar in general appearance (i.e., the waveforms were as described above) to responses recorded in slices from age-matched control rats (Fig. 1A). Measurement of the initial slopes of NMDA-receptormediated fEPSPs (Fig. 1C) obtained from input/output protocols revealed no statistically significant difference between slices prepared from CIE-treated animals or animals withdrawn from CIE treatment for 1 day and slices from age-matched control animals (Fig. 1E). However, after

a longer period of withdrawal (7 days) from CIE treatment, NMDA-receptor-mediated fEPSPs were significantly larger than fEPSPs recorded in age-matched control slices, although the general appearance of the responses was similar (Figs. 1B and D). Mean fEPSP slopes were significantly increased across a range of stimulus intensities (Fig. 1F). Thus, NMDA-receptor-mediated synaptic function was altered during the 7-day period of withdrawal from CIE treatment. 3.3. Effects of CIE treatment on paired-pulse facilitation To determine if presynaptic mechanisms contribute to the changes in NMDA-receptor-mediated synaptic responses following CIE treatment, a standard paired-pulse stimulation protocol was used to monitor presynaptic facilitation in each slice. Alterations in paired-pulse facilitation (PPF) primarily reflect changes in presynaptic function that affect neuro-

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transmitter release. Changes in PPF are inversely related to transmitter release such that an enhancement of PPF is indicative of a decreased probability of transmitter release in response to the initial stimulus pulse, whereas a decrease in PPF is indicative of an increased probability of transmitter release [1,10,12,14,17,49]. Following the 2-week CIE treatment period, measurement of the PPF using interpulse intervals of 100– 200 ms revealed no statistically significant difference between slices prepared from CIE-treated animals and slices from age-matched control animals (Figs. 2A and C). However, slices taken from rats that were withdrawn from the CIE treatment for 1 day exhibited a significant reduction in PPF relative to control slices using a 100-ms (Figs. 2A and C), but not a 200-ms (not shown), interpulse interval, indicating a higher probability of glutamate release at Schaffer collateral – CA1 pyramidal neuron synapses. This increase in presynaptic function is consistent with results from our previous study of CA1 synaptic transmission in animals exposed to the same treatment paradigm [52] and could account for the modest, though non-significant, increase in mean fEPSP slope observed in the 1-day withdrawn treatment group (Fig. 1E). In contrast, no difference in PPF (100 or 200 ms interpulse intervals) was observed between slices taken from rats following 7 days of withdrawal from CIE treatment and slices from age-matched control animals (Figs. 2B and D). Thus, the enhancement of fEPSP slopes measured in the input/output curves from 7-day withdrawn slices was not a result of increased transmitter release from presynaptic terminals.

3.4. Effect of NMDA receptor subunit expression The lack of correlation between the CIE/withdrawalinduced changes in the NMDA-receptor-mediated fEPSPs and the CIE/withdrawal-induced changes in PPF suggests that the changes in NMDA-receptor-mediated fEPSPs primarily involve postsynaptic mechanisms. One contributing factor could be a change in NMDA receptor subunit expression. To address this possibility, we examined the effects of CIE treatment and withdrawal on the relative level of expression of various NMDA receptor subunits in hippocampal tissue obtained from area CA1 of CIE-treated, 1-day withdrawn, 7-day withdrawn, and age-matched control rats. The primary subunits of the NMDA receptor expressed in the hippocampus are the NR1 (required for functional receptors), NR2A, and NR2B subunits [5,6,23,43,51]. Antibodies specific to each of these receptor subunits were utilized in a standard immunoblotting procedure, as well as an antibody to neuron-specific enolase to determine the relative neuronal content in each sample. NMDA receptor subunit expression (NMDA R1, 2A, and 2B) in area CA1 of CIE-treated and 1-day withdrawn animals showed modest increases relative to age-matched control slices (Fig. 3A) but the differences were not significant (Fig. 3B). In contrast, expression of the NR2A and NR2B subunits of the NMDA receptor was markedly increased relative to age-matched (62 days of age) controls following 7 days of withdrawal from CIE treatment (Figs. 3C and D). However, NR1 expression was only slightly enhanced (non-significant) in slices from 7-day withdrawn

Fig. 2. Changes in presynaptic function do not contribute to the altered NMDA-receptor-mediated synaptic function following withdrawal from CIE treatment. (A – B) Representative recordings using a 100-ms paired-pulse protocol to monitor changes in presynaptic facilitation in slices taken from CIE-treated, 1-day withdrawn (WD), and age-matched control rats (A) or from 7-day WD and age-matched control rats (B). Traces having first responses of similar magnitude are shown so that the relative facilitation of the second responses is more clearly illustrated. (C) PPF was significantly reduced in slices from 1-day withdrawn rats relative to age-matched control slices but not in slices from rats immediately removed from CIE treatment. (D) PPF was similar in slices from 7-day withdrawn and age-matched control rats. Numbers in parentheses indicate the number of slices in each treatment group. *P < 0.05, ANOVA.

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Fig. 3. NMDA receptor subunit expression is enhanced during withdrawal from CIE treatment. (A) Representative immunoblots of NMDA receptor subunit (run on separate gels) and neuron-specific enolase expression in CA1 tissue taken from CIE-treated, 1-day withdrawn, and age-matched control rats. (B) NMDA receptor subunit expression in hippocampal CA1 was not significantly altered shortly after cessation of CIE treatment. Measurements made of the CIEtreated and 1-day withdrawn lanes were normalized to the age-matched control lane of each blot. The dashed line represents the normalized control value (=1). (C) NMDA receptor subunit and neuron-specific enolase expression in CA1 tissue taken from 7-day withdrawn and age-matched control rats. (D) NR2A and NR2B subunit expression was significantly enhanced following 7 days of withdrawal from CIE treatment when compared to age-matched controls. Measurements made of the 7-day withdrawn lanes were normalized to the age-matched control lane of each blot. *P < 0.05, ANOVA.

rats compared with slices from age-matched controls (Figs. 3C and D). Levels of neuron-specific enolase were consistent across the treatment groups, indicating that the changes in NMDA receptor subunit expression were not a result of differences in the quantity of neuronal tissue collected from each treatment group (Figs. 3A –D). These results suggest that an increase in NMDA receptor subunit expression contributes to the enhancement of NMDAreceptor-mediated synaptic responses observed in the hippocampal CA1 region following 7 days of withdrawal from the CIE treatment.

4. Discussion In the current study, we found that a relatively short period of CIE treatment (2 weeks) resulted in a significant enhancement of NMDA-receptor-mediated synaptic responses in area CA1 of the hippocampus. Interestingly, this enhancement was not observed immediately following the cessation of the CIE treatment period or after 1 day of withdrawal from CIE. Rather, the augmentation of NMDAreceptor-mediated synaptic activity occurred between 1 and 7 days after termination of the CIE treatment period. In addition, the enhancement of NMDA-receptor-mediated

fEPSPs observed at 7 days of withdrawal from CIE treatment was paralleled by an increase in expression of NMDA receptor subunits in the CA1 at this time point. Together, these data highlight a unique time course of increased NMDA receptor expression and function following CIE treatment in which the upregulation does not reach a significant level until several days after cessation of ethanol exposure. A number of studies have shown that chronic ethanol treatment in vivo by various routes of administration increases the binding or expression of NMDA receptors in the hippocampus and other brain regions [3,9,11,13,21, 22,24,25,27,36,55,57,60,63]. In these studies, which were carried out in a variety of animal species and strains, NMDA receptor expression was elevated at the time of chronic ethanol withdrawal or when measured shortly thereafter. Moreover, the increased NMDA receptor expression returned to control levels within several days after the chronic alcohol treatment had ended [21,57]. In contrast, our results showed a delay (between 1 and 7 days after cessation of CIE treatment) before significant elevations in NMDA receptor expression and function were observed. These differences are likely to relate to the patterns and routes of administration and BALs achieved. The previous studies utilized treatment paradigms that involved either inclusion

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of ethanol in the drinking water for long periods (e.g., several weeks to months) or forced ethanol administration (e.g., by gastric gavage or injections) for short periods (e.g., several days). With each of these routes of administration, BALs remained relatively constant throughout the treatment period, whereas with the CIE treatment paradigm used in our studies, BALS are varied in a controlled manner on a daily basis. In addition, studies using ethanol-drinking paradigms did not achieve BALs as high as were achieved with the ethanol vapor inhalation method. In vitro studies using chronic ethanol treatment of cultured neurons have also shown upregulated binding or expression of NMDA receptors [7,9,22,30 – 32]. Interestingly, when cultured cortical neurons received CIE treatment for 5 days, NMDA receptor binding and function were enhanced immediately after the CIE treatment [31], an effect similar to that observed with continuous ethanol exposure of cultured cortical neurons [30,32]. However, whereas NMDA receptor expression and function returned to basal levels within 2 – 3 days following continuous in vitro ethanol exposure, enhanced NMDA receptor function persisted in CIE-treated cultures following 1 week of withdrawal. Thus, the mechanisms regulating chronic ethanol-induced NMDA receptor expression differ between continuous and intermittent ethanol exposure paradigms. Although the in vitro and in vivo CIE treatment paradigms used in the culture studies and our current study are not identical, similarity in the persistence of the NMDA receptor expression and function following CIE treatment in vitro and the delay in expression observed in our studies using CIE treatment in vivo suggests that these effects may be mechanistically related. With the CIE paradigm used in our studies, animals were subjected to daily periods of ethanol exposure and withdrawal. With this pattern of ethanol exposure, NMDA receptor function is likely to be suppressed by the acute effects of ethanol during the exposure phase, whereas during the withdrawal phase, NMDA receptor function is no longer acutely suppressed by ethanol and NMDA-receptor-mediated synaptic activity appears to be normal (as shown by comparing the slices from CIE-treated and age-matched control animals). Interestingly, hippocampal expression of mRNA for the NR2A and NR2B subunits was shown to peak 9 h following continuous chronic ethanol treatment and return to normal within 48 h [21], demonstrating that transient or reversible alterations of NMDA receptor expression can occur in response to chronic ethanol administration. Thus, the pattern of ethanol consumption and withdrawal may be a critical determining factor in the time course of NMDA receptor upregulation and enhancement of NMDA-receptor-mediated synaptic transmission. The CIE-treated rats in our study exhibited a statistically significant reduction in weight gain compared to agematched controls. However, the body weights remained within a range appropriate for Wistar rats at the ages used in this study and the alterations in weight gain did not coincide

with increases in NMDA receptor subunit expression, or with enhanced NMDA receptor-activated synaptic transmission. Furthermore, brain weights were not adversely affected by CIE treatment. Therefore, although we cannot completely exclude possible deficits in nutrition as a contributing factor to the changes in NMDA receptor, it is unlikely that this is the primary cause of the changes observed in our study. Several studies have demonstrated chronic ethanolinduced alteration of NR1 subunit expression. For example, Trevisan et al. [63] described increased NR1 subunit immunoreactivity in the hippocampus following chronic ethanol ingestion. Devaud and Morrow [13] reported increased hippocampal NR1 subunit expression in ethanoldependent rats, although NR1 mRNA levels were not changed in an earlier report by the same group [46]. Snell et al. [60] observed a lack of correspondence between both NR1 and NR2A protein (upregulated) and mRNA expression (no change) in chronic ethanol-treated mice. A lack of chronic ethanol-induced changes in NR1 mRNA expression in the hippocampus, despite increases in NR2A and NR2B mRNA expression, has also been reported by others [11,21], indicating that changes in NR1 subunit expression were not dependent on transcriptional changes in NR1 mRNA levels. In our studies, expression of NR1 protein in area CA1 was not significantly altered by CIE treatment and withdrawal whereas expression of NR2A and NR2B subunits was enhanced. These studies indicate that the expression of NMDA receptor subunits is differentially affected by chronic ethanol exposure. Differential subunit expression could result in altered subunit composition of NMDA receptors and, thus, modified receptor function (e.g., channel gating, Mg2+ sensitivity). Although we cannot directly determine from the present results whether the altered expression of NMDA receptor subunits underlies the enhanced synaptic responses observed in our studies, our results from immunoblotting and electrophysiological experiments are consistent with such a mechanism. Our results did not determine whether the expression of NMDA receptor subunits represents functional receptors or the precise regional or cellular localization of the receptors. However, recent data have shown that in vitro chronic ethanol exposure results in activity-dependent upregulation of NMDA receptor subunits in synaptic, but not extrasynaptic, sites in hippocampal neurons [7]. In this study, which bears many methodological differences from our own study, both NR1 and NR2B subunits were localized to synaptic targets by chronic ethanol. In our study, although total NR1 subunit expression levels did not change, it is possible that functional NR1 expression was enhanced, perhaps by NR1 translocation to synaptic sites, or by combination with newly synthesized NR2A/NR2B subunits at synaptic sites. Withdrawal from chronic ethanol is often associated with neuronal hyperexcitability [45] and seizure activity [24] attributable to increased NMDA-receptor-mediated synaptic activity. Consistent with our previous study in which

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behavioral signs of ethanol withdrawal were assessed after cessation of CIE treatment [52], we did not observe any indications of hyperexcitability (e.g., seizures) in the rats during any phase of the CIE treatment or withdrawal period. In the current study (before wash-in of non-NMDA and GABA receptor antagonists) and in our previous studies [48,52], we also did not observe indications of hyperexcitability in field potential recordings of hippocampal slices from CIE-treated/withdrawn rats. However, both the behavioral and electrophysiological withdrawal signs may be below the level of detection by our methods. Mild forms of hyperexcitability/seizures (e.g., Racine’s stages 1– 2) can be difficult to detect behaviorally [50]. A modest increase in excitability may contribute to subsequent upregulation of NMDA receptor expression at later stages of withdrawal observed in our studies. Although there is disagreement among studies that have investigated changes in NMDA receptor expression induced by seizure activity [2,28,39], there are a number of examples of increased NMDA receptor expression in various experimental models of epileptiform activity [16,33,35,61,64]. Thus, the delayed NMDA receptor upregulation occurring at 7 days of withdrawal could be a secondary effect of CIE treatment, stimulated by small increases in excitability during the withdrawal period rather than by a direct effect of chronic ethanol on NMDA receptor expression. Following 1 day of withdrawal from CIE treatment, we observed a significant reduction in PPF, indicating an enhancement of glutamate release from presynaptic terminals at the Schaffer collateral – CA1 pyramidal neuron synapse. This finding is in agreement with results from our previous studies using the same CIE treatment paradigm [52] and corresponds to the small enhancement of the NMDAreceptor-mediated fEPSPs observed at 1 day of withdrawal in the current study. As would be predicted for a change in presynaptic function, these changes were not specific to NMDA-receptor-mediated postsynaptic responses because we previously observed similar changes (i.e., decreased PPF concomitant with increased synaptic response) in AMPAreceptor-mediated fEPSPs after 1 day of CIE withdrawal [52]. Thus, changes in synaptic responses resulting from short-term (1 day) withdrawal from CIE treatment are associated with changes in presynaptic function. In contrast, the changes in NMDA-receptor-mediated synaptic responses resulting from long-term (7 days) withdrawal were most likely postsynaptic changes, related to increased NMDA receptor subunit expression. The changes were specific to NMDA receptors because we found no change in AMPAreceptor-mediated fEPSPs after a similar period (5 days) of ethanol withdrawal in our previous study. In addition, PPF was not altered after 7 days of CIE withdrawal, indicating that the enhancement of NMDA-receptor-mediated fEPSPs observed at this stage was not a result of an increase in the probability of transmitter release from presynaptic terminals. The selective enhancement of NMDA-receptor-mediated fEPSPs, but not AMPA-receptor-mediated fEPSPs, also

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makes it unlikely that other presynaptic changes that do not affect PPF ratio (e.g., the number of functional synapses or release sites) are involved. In a previous study, we found that CIE treatment resulted in a complete suppression of NMDA-receptor-dependent LTP induction in area CA1 of the hippocampus when measured at the cessation of the treatment period. However, the ability to induce LTP recovered by 5 days of withdrawal from CIE treatment [52]. In the present study, NMDA receptor expression was unaltered by CIE treatment at the end of the treatment period and at 1 day of withdrawal. Our results showing that the NMDA-receptor-mediated fEPSP and receptor expression is unaltered by CIE treatment at these time points indicates that the impairment of LTP cannot be directly attributed to changes in NMDA receptor expression or function at 1 day of withdrawal. However, the increased expression of NMDA receptor subunits and the increase in NMDA-receptor-mediated synaptic responses observed at later stages of withdrawal could contribute to the recovery of LTP induction in hippocampal CA1 reported in our earlier study [52]. Additional investigation of the mechanisms underlying the impairment of LTP by CIE treatment indicated that the MAPK signaling pathway may be involved [53]. Basal and HFS-induced levels of phosphorlyated (activated) MAPK were reduced immediately following CIE treatment and after 1 day of withdrawal from CIE. MAPK activation is critical for LTP induction in the CA1 region and is induced by NMDA receptor activation [19]. After 5 days of withdrawal, recovery of LTP induction was accompanied by a recovery of MAPK signaling and a greater sensitivity to the MEK inhibitor PD098059, which blocks MAPK activation. This enhanced sensitivity to blockade suggests that the MAPK pathway may undergo neuroadaptive changes during withdrawal from CIE treatment. The coincident enhancement of NMDA receptor expression and activity could contribute to the recovery of MAPK signaling and LTP induction observed during this time period.

Acknowledgments The authors wish to thank Ms. Vi Nguyen for assistance in the analysis of raw electrophysiological data, Dr. Anne Prieto for guidance with the Western blotting techniques, Mr. Maury Cole and Ms. Tess Kimber for assistance with the ethanol vapor treatments and determination of blood alcohol levels, and Ms. Floriska Chizer for secretarial support. This work was supported by AA06420. References [1] M. Andreasen, J.J. Hablitz, Paired-pulse facilitation in the dentate gyrus: a patch-clamp study in rat hippocampus in vitro, J. Neurophysiol. 72 (1994) 326 – 336. [2] J.R. Atack, S.M. Cook, P.H. Hutson, S.E. File, Kindling induced by

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