Timing-dependent reduction in ethanol sedation and drinking preference by NMDA receptor co-agonist d -serine

Alcohol 46 (2012) 389e400

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Timing-dependent reduction in ethanol sedation and drinking preference by NMDA receptor co-agonist D-serine Amber Lockridge a, Gabriel Romero a, Justin Harrington a, Brett Newland a, Zi Gong a, Andrew Cameron b, Li-Lian Yuan a, * a b

Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Portland Alcohol Research Center, Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR 97239, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2011 Received in revised form 31 October 2011 Accepted 9 November 2011

NMDA receptors become a major contributor to acute ethanol intoxication effects at high concentrations as ethanol binds to a unique site on the receptor and inhibits glutamatergic activity in multiple brain areas. Although a convincing body of literature exists on the ability of NMDA receptor antagonists to mimic and worsen cellular and behavioral ethanol effects, receptor agonists have been less well-studied. In addition to a primary agonist site for glutamate, the NMDA receptor contains a separate co-agonist site that responds to endogenous amino acids glycine and D-serine. D-serine is both selective for this co-agonist site and potent in boosting NMDA dependent activity even after systemic administration. In this study, we hypothesized that exogenous D-serine might ameliorate some acute ethanol behaviors by opposing NMDA receptor inhibition. We injected adult male C57 mice with a high concentration of D-serine at various time windows relative to ethanol administration and monitored sedation, motor coordination and voluntary ethanol drinking. D-serine (2.7 g/kg, ip) prolonged latency to a loss of righting reflex (LoRR) and shortened LoRR duration when given 15 min before ethanol (3 g/kg) but not when it was injected with or shortly after ethanol. Blood samples taken at sedative recovery and at fixed time intervals revealed no effect of D-serine on ethanol concentration but an ethanol-induced decrease in L-serine and glycine content was prevented by acute D-serine pre-administration. D-serine had no effect on ethanol-induced (2 g/kg) rotarod deficits in young adult animals but independently and interactively degraded motor performance in a subset of older mice. Finally, a week-long series of daily ip injections resulted in a 50% decrease in free choice ethanol preference for D-serine treated animals compared to saline-injected controls in a two-bottle choice experiment. Ó 2012 Elsevier Inc. All rights reserved.

Keywords: NMDA receptor D-serine Ethanol Intoxication Tolerance Preference

Introduction The negative side effects of acute ethanol intoxication can have severe implications for an individual’s health and place a heavy economic burden on health care services through accidents and reckless behavior. Consuming high concentrations of ethanol can disrupt motor function and induce sedative effects in humans resulting in slurred speech, poor judgment, staggering gait, weak reflexes, and loss of memory and consciousness although the neurological processes driving these impairments are not fully understood. Ethanol acts on many receptor targets in the brain, including GABAA and 5HT1, but substantial evidence suggests that the down-regulation of glutamatergic transmission through NMDA * Corresponding author. Department of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. Tel.: þ1 612 625 8613; fax: þ1 612 626 5009. E-mail address: [email protected] (L.-L. Yuan). 0741-8329/$ e see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.alcohol.2011.11.004

receptor (NMDAR) inhibition plays a dominant role in mediating high-dose intoxicant effects (Grant & Colombo, 1993; Krystal, Petrakis, Mason, Trevisan, & D’Souza, 2003). Ethanol binds to a unique hydrophobic pocket of the NMDAR NR1 subunit and has been shown to dose-dependently suppress receptor function in vitro between 5 and 100 mM concentrations (22 mM w 0.10% blood alcohol level) (Krystal et al., 2003; Ronald, Mirshahi, & Woodward, 2001; Weitlauf & Woodward, 2008). NMDAR antagonists, such as ketamine and MK-801, mimic subjective intoxication in humans and prolong ethanol sedation in rodents (Krystal et al., 2003; Palachick et al., 2008). These antagonist studies collectively suggest that NMDAR inhibition becomes the primary contributor to perceived ethanol effect at or above intragastric doses of 2 g/kg in rodents or 4 standard alcoholic drinks (Grant & Colombo, 1993; Krystal et al., 2003). In response to the exogenous inhibition, NMDARs are upregulated in sensitivity and function leading to cellular and behavioral ethanol tolerance even during the course of a single exposure. These temporary NMDAR

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changes compound over time and are accompanied by genetic and receptor trafficking adjustments that more permanently elevate glutamate transmission, contributing to withdrawal symptoms and longer-term alcohol drinking patterns (Nagy, 2008). The NMDA receptor is a calcium permeable membrane channel comprised of two NR1 subunits and two NR2 subunits (or in some cases NR3). NMDARs are activated by a variety of coincident events including local depolarization, glutamate binding to the NR2 primary ligand site and co-agonist binding to a secondary NR1 site resulting in increased excitability of the post-synaptic cell. The coagonist binding site is responsive to the endogenous agonists glycine and D-serine as well as to a variety of other exogenous or manufactured modulators (Jansen & Dannhardt, 2003). While direct pharmacologic manipulation of glutamate or the NMDAR itself can induce serious aversive side effects, the relatively mild consequences of co-agonist modulation have increasingly made these factors a target of psychiatric research for diseases and conditions associated with glutamatergic dysfunction (Jansen & Dannhardt, 2003). A large body of research exists on the ability of NMDAR antagonists to mimic or worsen the acute negative effects of ethanol but less is known about the potential impacts of co-agonist site activators. Past acute ethanol based studies have primarily relied on glycine and the partial agonist D-cycloserine (Blum, Wallace, & Friedman, 1974; Cebers, Cebere, Zharkovsky, & Liljequist, 1996; Khanna, Morato, & Kalant, 2002; Rabe & Tabakoff, 1990; Trevisan et al., 2008; Williams, Ferko, Barbieri, & DiGregorio, 1995). However, the results of these studies are inconsistent with each other and data interpretation is confounded by the presence of alternate target receptors, intestinal absorption interference, poor binding affinity and/or partial antagonism at high doses (Krystal et al., 2011; Molander, Lido, Lof, Ericson, & Soderpalm, 2007). By contrast, D-serine has a higher brain penetration, excellent binding affinity and strong selectively as an NMDAR co-agonist (Wolosker, Dumin, Balan, & Foltyn, 2008). Furthermore, D-serine has been showing impressive success in clinical trials as a treatment for conditions marked by NMDAR hypofunction such as schizophrenia (Heresco-Levy et al., 2009; Labrie & Roder, 2010). A small number of studies have looked at the interaction of D-serine and acute ethanol application on the behavioral and cellular level (Blum et al., 1974; Blum, Wallace, & Geller, 1972; Rabe & Tabakoff, 1990; Williams et al., 1995). As with the other co-agonist modulation experiments, the results of these studies were contradictory and subject to numerous confounding or mitigating factors (see discussion for more detail). In addition, differences in administration method and timing between these papers preclude any definitive summary of D-serine’s potential to modulate acute ethanol intoxication. In this study, we investigated whether the time window of intraperitoneal (ip) D-serine administration relative to ethanol might change its effect on sedation in mice using the loss of righting reflex (LoRR) test. We further examined D-serine’s impact on blood ethanol concentration and amino acids (L-serine, glycine), motor coordination, rapid sedative tolerance and voluntary ethanol preference.

Drugs D-serine (Sigma Aldrich, St. Louis, MO, USA) was prepared daily by dissolving varying concentrations into 0.9% saline proportioned for an average injection volume of 0.3 mL for a 25-g mouse. Ethanol injections were prepared by mixing 200-proof ethanol (pharmco-aaper) in saline at 20% v/v immediately before administration. Ethanol bottles were stored at 4  C, and a new bottle was opened each week to minimize the impact of water absorption. All injections were intraperitoneal (ip) made with a sterile 1 mL 27 1/2 gauge tuberculin syringe. Blood samples were processed for gas chromatography with ZnSO4 and Ba(OH)2 (Sigma Aldrich, St. Louis, MO, USA) prepared in distilled water and for capillary electrophoresis in standard bicarbonate ringer according to the following concentrations (mM): 111.0 NaCl, 32.0 NaHCO3, 3.0 KCl, 1.0 MgSO4, 0.5 NaH2PO4, 2.0 CaCl2, 12.0 Dextrose (Sigma Aldrich, St. Louis, MO, USA).

Loss of righting reflex (LoRR) Procedures were adapted from Palachick et al. (2008) and Radcliffe, Floyd, and Lee (2006). Immediately after receiving an ip injection of ethanol (3 g/kg), the subject was gently flipped over onto its back in a plastic coated v-shaped trough. Subject was turned over again every 3 s until it did not right itself for a 30 s duration. Latency to LoRR was recorded at the start of the 30-s period. Mice were then monitored and turned over following any self-righting event until the subject could turn over twice within 30 s. Duration was calculated as the time between reflex loss and recovery. The v-shaped trough was situated inside a clear plastic cage with a loose-fitting lid and a reptile pad heater affixed to one side, which reduced noise and maintained an ambient internal temperature of 26e27  C. For time window testing, subjects received a saline or D-serine injection (2.7 g/kg) 15 min before, at the same time or 15 min after ethanol. For concentration testing, Dserine was administered prior to ethanol at doses of 0.6, 1.8 and 2.7 g/kg (Kanahara et al., 2008). During rapid tolerance testing, animals were tested a second time with a single injection of ethanol 24 h after acute pre-administration treatment. Mice that did not show LoRR after 3 min or recover after 90 min were removed from the data pool on the assumption of an improper injection. Blood collection Blood samples were taken at LoRR recovery or at fixed time intervals (baseline, 20 and 40 min post-ethanol injection). Several drops of blood were collected into a sterile microcentrifuge tube from the facial vein by making a submandibular puncture with an 18 1/2 gauge needle tip. Two 20 ml volumes were then pipetted immediately into either ZnSO4 or Ringer solution for subsequent assays. Samples were stored on ice until further processing every 4 collections; saline and D-serine treated animals were tested in parallel. Blood ethanol concentration (BEC)

Methods Subjects Adult male C57BL/6J mice (2e3 months old unless otherwise stated) were housed in groups of 3e4 on a 12:12 light cycle with ad libitum access to food and water. The use of animals for these studies was approved by the University of Minnesota Institutional Animal Care and Use Committee.

BEC was assessed by gas chromatography (GC) in a manner similar to previously published methods (Gallaher, Jones, Belknap, & Crabbe, 1996). During collection, 20 ml of blood was transferred into a tube with 50 ml of cold ZnSO4 solution. Subsequent processing started with the addition of 50 ml of 0.3 N Ba(OH)2 and 300 ml deionized water to each tube. Samples were then vortexed for 10 s and spun down for 5 min at 12,000 RPM in a 4  C centrifuge. 350 ml of supernatant was drawn off into a GC vial and tightly capped. These samples were kept frozen at 80  C until BEC

A. Lockridge et al. / Alcohol 46 (2012) 389e400

assay. Samples were processed through an Agilent 6890N gas chromatograph with attached 7683 autosampler and a J&W scientific 30 m  0.53 mm column. Detector output was quantified by computer using Chemstation software. Results were calculated relative to standards that bracketed the unknown sample concentrations. Amino acid concentrations Capillary electrophoresis (CE) was used to measure L-serine, and glycine levels in a procedure adapted from previously published work (Sullivan & Miller, 2010). During collection, 20 ml of blood was transferred into a tube with 185 ml of cold mammalian bicarbonate ringer solution and stored at 20  C until they could be fully processed. Once thawed, samples were then spun down in a centrifuge. The supernatant was removed and spun a second time through a cellulose acetate membrane (Corning Costar) to remove remaining particles or cells. Amino acids in the supernatant were fluorescently labeled with 0.7 mg/mL 4-fluro-7-nitrobenz-2 oxa1,3-diazole (NBD-F; Molecular Probes, Eugene, OR) at 60  C for 15 min. Labeled amino acids were analyzed with laser-induced fluorescence (LIF) detection on a commercial CE machine (Beckman-Coulter MDQ, Fullerton, CA) in a (2-Hydroxypropyl)-b-cyclodextrin buffer at 15 kV (70 mA). Fluorescently labeled amino acids were detected by a photomultiplier tube and plotted as fluorescence vs. time. 32 Karat software (Beckman-Coulter, version 7.0) was used for plotting, peak integration, and analysis. Mass of desired amino acids were determined by comparing unknown samples to gradients of known standards.

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bottles for two additional days. The position of the bottles was alternated every day relative to the cage walls to avoid any place preference bias. Volume levels were recorded at noon to determine daily consumption and subsequently tubes were refilled with the appropriate solution. Mice were also weighed each day and this value was used to calculate individual ethanol concentrations over a 24-h period. Starting on day 2, mice received an ip injection of either D-serine (2.7 g/kg) or saline at approximately 5:00 pm, 30 min before the start of dark phase. Injections continued once a day to the end of the experiment for a total of 6 treatments.

D-serine

Data analysis Recovery from LoRR, rotarod performance, animal weights and drinking tube volumes were all assessed and recorded by an experimenter blind to treatment condition. These data were preliminarily analyzed and graphed in Excel. Formal statistical analysis was conducted in MATLAB using a multiway (n-way) analysis of variance (ANOVA) that included age and weight as minimum variance factors as well as experimentally-dependent factors such as drug treatment and time window. When significant factors were identified, we employed the multiple comparison test function on the ANOVA output to identify dissimilar groups under each factor condition. In some cases, where indicated in the text, Student’s t-test (two-tailed, unequal variance) was then used to obtain p-values for specific pairs. Significance threshold was set at p < 0.05. All reported uncertainties and graph error bars refer to standard error or the mean, calculated in Excel with the formula: standard deviation/square root (n).

Accelerating rotarod

Results

Each rotarod trial consisted of a single mouse placed on the rotating rod for 30 s at a constant speed of 4 rpm followed by an acceleration of 0.12 rpm/s up to a maximum of 120 s (15 rpm). At the beginning of the day, each mouse was given up to 15 consecutive attempts to reach 120 s (pre-training) with all mice being evaluated to this threshold before the start of formal testing. Any younger animal that did not meet the criterion was eliminated from further testing. For experiments on older animals, mice received similar pre-training but none were eliminated resulting in 4 mice across all conditions that did not meet the 120-s threshold (3 were within a 10 s margin). During formal testing, subjects were given two back-to-back trials on the rotarod for each time point but only the longest latency to fall was recorded. For co-injection conditions, each mouse was tested for time 0 immediately before receiving a single injection (ethanol 2 g/kg, D-serine 2.7 g/kg, or both) and then every 15 min up to 90 min after injection. For the pre-injection time window, mice received the first injection (saline or D-serine) 11 min prior to time 0 testing and the ethanol injection immediately after (approximately 15 min since first injection) with the subsequent behavioral evaluations occurring at 15 min intervals relative to the ethanol injection. Adapted from (Taslim, Al-Rejaie, & Saeed Dar, 2008).

Injection of D-serine prior to ethanol ameliorates acute sedation

Short-term drinking Mice were individually housed for 2 days prior to the start of experiments. Each cage was then outfitted with two drinking bottles made of graduated serological pipettes attached to ball bearing sipper tubes and plugged with silicone stoppers. Joints were sealed with parafilm to reduce evaporation and the bottles were secured to the wires of a normal cage insert using binder clips. Both bottles were filled with water for three days, then one each with water or 8% ethanol for two days and finally 8% ethanol in both

We first set out to determine the effects of a high concentration of systemic acute D-serine (ip 2.7 g/kg) on mouse sedation in the LoRR test. Subjects received a pre-injection (15 min), co-injection, or post-injection (þ15 min) of D-serine relative to ethanol injection (ip 3.0 g/kg) and were monitored for latency to lose their righting reflex and the duration of LoRR until reflex recovery (Fig. 1A). Saline controls were combined when time window of application was found to be an insignificant factor (p ¼ 0.21 latency; p ¼ 0.28 duration). Saline and ethanol-injected animals (n ¼ 23) demonstrated LoRR within 74.8  2.1 s and recovered after 41.0  2.7 min. By contrast, subjects receiving pre-injected D-serine (n ¼ 14) showed a prolonged latency to LoRR at 110.8  6.3 s (F(3,54) ¼ 17.9, p < 0.0000001) and a dramatically shorter duration average of 24.0  2.3 min (F(3,54) ¼ 10.1, p < 0.0001) (Fig. 1B). The application of D-serine did not impact sedation behavior values in the co-ethanol and post-ethanol injection groups (Fig. 1B). One mouse in the post-ethanol group woke up briefly after the D-serine injection but spontaneously re-entered a LoRR sedation period 2 min later. Duration was recorded from the ethanol injection to the second recovery time. Given the robustness of D-serine’s impact on ethanol sedation at the 2.7 g/kg dose, we decided to further probe the concentration threshold at which modulatory effects begin to occur. Using the pre-administration time window only, we tested dosages of 0, 0.6 and 1.8 g/kg. 1.8 g/kg of D-serine (n ¼ 9) elevated LoRR latency from 77.5  2.6 s (saline, n ¼ 15) to 107.3  4.5 s (F(3,46) ¼ 13.78, p < 0.00001) but the lowest dose (0.6 g/kg, n ¼ 9) was no different from controls (Fig. 1C). The concentration curve of duration revealed that only the maximum quantity of D-serine (2.7 g/kg, n ¼ 14) significantly diminished the period of time between initial reflex loss and recovery (F(3,46) ¼ 6.42, p < 0.01) (Fig. 1D).

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Fig. 1. The effect of D-serine on acute sedative response to ethanol. (A) Experimental schematic demonstrating the three time windows for administering D-serine: 15 min prior to ethanol injection (Pre-EtOH), with ethanol (Co-EtOH), or 15 min after ethanol (Post-EtOH) (3 g/kg, ip). (B) D-serine (2.7 g/kg, ip) prolonged latency to loss of righting reflex (LoRR) and shortened LoRR duration when administered Pre-EtOH. (C) 1.8 g/kg of pre-injected D-serine was also able to delay latency. (D) Only the maximum dose of D-serine had a significant impact on duration. (E) Scatter plot of blood ethanol concentration (BEC) values determined at recovery following D-serine or saline pre-EtOH injections. One saline outlier was removed from subsequent analysis as it was 2 standard deviations outside of the group mean. (F) Slight elevation in D-serine BEC was not statistically dissimilar from saline BEC (p ¼ 0.065). N numbers given in parentheses of legends (B) or x-axis labels (C, D, F). **p < 0.01, ***p < 0.001 represent significance vs. saline.

These behavior results highlight the importance of time window and concentration on D-serine’s modulatory interaction with ethanol. The data do not elucidate whether the observed significant effects arise from a metabolic interference with ethanol or a synaptic opposition. To evaluate these hypotheses, we collected facial vein blood at the time of LoRR recovery to measure blood ethanol concentration (BEC). We took samples from a subset of D-serine (2.7 g/kg, n ¼ 8) and saline (n ¼ 8) pre-injected animals within 1 min of their demonstrated reflex recovery time and processed these for ethanol concentration using gas chromatography. One saline outlier was identified (2.97 mg/mL BEC, 27.4 min LoRR duration) and removed from cumulative analysis (Fig. 1E). LoRR latency and duration values for this subset were compared to the entire set, as presented in Fig. 1B, and found to be no different among saline controls (p ¼ 0.39 latency; p ¼ 0.70 duration) or Dserine treated animals (p ¼ 0.61 latency; p ¼ 0.49 duration). Among the BEC data itself, saline controls averaged 2.49  0.05 mg/mL while D-serine values were slightly higher at 2.61  0.06 mg/mL but fell just below the significance threshold in statistical analysis (F(1,14) ¼ 4.1, p ¼ 0.065) (Fig. 1F).

D-serine

prevents ethanol-induced reductions of blood glycine and

L -serine

Above BEC data was suggestive of D-serine effects on ethanol concentration at LoRR recovery but not statistically definitive. We therefore designed a further experiment to evaluate the potential of D-serine to interfere with ethanol metabolism. We took facial vein blood samples at baseline, prior to any treatment, and at 20 and 40 min post-ethanol injection (3 g/kg) for animals pre-treated with either saline (n ¼ 9) or D-serine injection (2.7 g/kg, n ¼ 9). We assayed the post-ethanol blood samples for BEC as previously described and used capillary electrophoresis to evaluate all three time points for concentrations of D-serine, L-serine and glycine. Sedative behavior was not monitored because of disturbances from a high degree of handling during sample collection. Three D-serine treated animals were removed from the data set prior to analysis based on improper injection in two cases and a damaged sample vial in the other. BEC in the saline-treated animals started at 2.90  0.11 mg/mL at 20 min and 2.82  0.06 mg/mL at 40 min, although this change over

A. Lockridge et al. / Alcohol 46 (2012) 389e400

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Fig. 2. D-serine pre-treatment on blood levels of ethanol and amino acids. (A) Scatter plot of individual blood ethanol concentrations (BEC) at 20 and 40 min after ethanol injection (3 g/kg, ip) for saline and D-serine (2.7 g/kg, ip) pre-treated groups. (B) Summary of BEC data showing no significant impact of treatment on BEC at 20 or 40 min post-ethanol but a decline in BEC over time was only significant in D-serine animals. (C) Saline and ethanol injections did not change D-serine blood concentrations from baseline (0). (D) D-serine pretreatment increased D-serine blood concentrations by 1000-fold to similar levels at both 20 and 40 min following ethanol injection. (E) In saline animals, ethanol significantly reduced L-serine concentration relative to baseline at both 20 and 40 min but no change was seen in D-serine treated animals. (F) Similarly, D-serine prevented the ethanol-induced loss of glycine at 20 min post-ethanol injection. N numbers are given in panel A and B legends. *p < 0.05 represents significance vs. baseline value, unless otherwise indicated.

time was not statistically significant (p ¼ 0.56) (Fig. 2B). BEC in D-serine animals did undergo a significant decrease from 2.86  0.04 mg/mL to 2.72  0.06 mg/mL (F(1,16) ¼ 6.85, p < 0.05, 20 vs. 40 min) but there was no significant drug treatment effect for either raw concentration values (p ¼ 0.33) (Fig. 2B) or for slope between the two time points (p ¼ 0.54) (data not shown). Baseline D-serine levels started at 19.45  5.14 and 24.75  4.61 nmol/mL for saline and D-serine treatment groups, respectively. Subsequent saline and ethanol treatment had no effect on endogenous D-serine at any time point (p ¼ 0.42) (Fig. 2C). Exogenous D-serine injection caused a 700% increase in serum D-serine concentration to 12.84  1.15 at 20 min and 12.11  1.57 mmol/mL at 40 min (F(1,53) ¼ 63.1, p < 0.000000001) (Fig. 2D). Acute ethanol did significantly reduce endogenous L-serine from 70.49  5.92 nmol/mL at baseline to 47.78  3.80 and 47.82  4.33 nmol/mL by 20 and 40 min in saline-treated animals

(F(2,26) ¼ 5.13, p < 0.05) (Fig. 2E). Glycine was also diminished at 20 min in the saline group from 182.22  27.84 to 118.74  12.04 nmol/mL (F(2,26) ¼ 5.99, p < 0.05, baseline vs. 20 min) (Fig. 2F). By contrast, D-serine treated animals showed no time-dependent loss in serum concentration of either L-serine (p ¼ 0.81) or glycine (p ¼ 0.78) (Fig. 2E and F). These data show that while Dserine did not impact ethanol metabolism directly, acute D-serine did prevent the impact of ethanol on L-serine and glycine metabolism. D-serine acute effects do not alter rapid sedation tolerance on the second day

Recovery from LoRR is in part due to a natural reduction in brain ethanol concentration over time but also to internal adjustments in the sensitivity and performance of NMDA receptors representing

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acute tolerance (Fadda & Rossetti, 1998; Nagy, 2008). This cellular remodeling continues after recovery and produces a characteristic rapid tolerance phenomena of reduced ethanol effect 24 h later (Radcliffe et al., 2006). In order to evaluate whether acute D-serine pre-administration might be hastening the acquisition of ethanol tolerance, we repeated the pre-injection experiment of the first section and then tested these same animals 24 h later on ethanol response alone (Fig. 3A). Because our interest was in the behavior difference between day 1 and day 2, we included some previously tested animals that had been allowed a month of rest (naïve n ¼ 6, saline experienced n ¼ 4, D-serine experienced n ¼ 10). The variously experienced mice were randomly assigned to either saline or D-serine day 1 injection conditions but we found no impact of prior experience on latency to LoRR (p ¼ 0.77) or total duration (p ¼ 0.79) values. Similarly day 1 saline values in this experimental group were no different from the saline data previously gathered (p ¼ 0.16 latency; p ¼ 0.12 duration) or between the two sets of D-serine data (p ¼ 0.51 latency; p ¼ 0.41 duration). Animals treated with saline on day 1 (n ¼ 10) showed no cumulative difference in LoRR latency between the first and the second day (p ¼ 0.45). D-serine treated animals (n ¼ 10) demonstrated the characteristic boost in acute latency (p < 0.001) but day

A

Saline or D-serine (2.7 g/kg) 15 min Latency

2 ethanol-only latency was no different than in the controls (p ¼ 0.77) (Fig. 3B and C). This resulted in an apparent gain in rapid tolerance of 27  4% (F(1,19) ¼ 20.65, p < 0.001 day 1 vs. day 2 D-serine group) (Fig. 3C). Although whether this tolerance was in fact due to cellular remodeling or to the absence of the acute presence of D-serine cannot be determined from this experiment. The development of rapid tolerance in the LoRR duration data showed just the opposite trends relative to treatment. Initial saline reflex loss periods were longer than D-serine loss intervals (p < 0.01) but whereas day 2 saline values dropped by 26  7% in a significant display of tolerance (F(1,19) ¼ 7.74, p < 0.05), day had no impact on duration values for acutely-treated D-serine animals (p ¼ 0.99) (Fig. 3D and E). With regards to ethanol-induced LoRR duration, therefore, day 2 values were the same for all treatment groups (p ¼ 0.17) suggesting that the acquisition of rapid tolerance was neither retarded nor enhanced by day 1 D-serine administration (Fig. 3E). Systemic D-serine does not change ethanol motor coordination deficits In addition to sedation, slightly lower concentrations of ethanol produce motor deficits that can be observed by monitoring rodent

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Fig. 3. The impact of acute D-serine on rapid tolerance to ethanol sedation. (A) Experimental schematic showing day 1 treatment conditions with saline or D-serine (2.7 g/kg) pre-injection and day 2 treatment condition with ethanol-only (3 g/kg). (B) Scatter plot showing the change in latency to LoRR behavior for each individual over the two-day period. (C) Bar graph summarizing the lack of change in saline group latency in contrast to the reduction in day 2 latency for mice treated with D-serine on day 1. (D) Scatter plot showing the change in LoRR duration for each individual mouse. (E) Summary bar graph demonstrating the acquisition of rapid tolerance on day 2 in the saline group but no change in the D-serine animals between first and second day reflex loss periods. N numbers are given in the figure legends. *p < 0.05 represent significance vs. day 1 values.

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Systemic D-serine diminishes voluntary ethanol drinking preference There is some concern in the field of ethanol research that reducing the aversive consequences of heavy intoxication could promote increased drinking (Fadda & Rossetti, 1998; Krystal et al., 2011). To test whether high concentration D-serine has an impact on short-term voluntary drinking we devised a week-long test in which animals received daily injections of D-serine (2.7 g/kg, n ¼ 10) or saline (0.9%, n ¼ 9) in conjunction with a two-bottle drinking design. Animals received water in both bottles before the first injection (water, untreated), after the first two injections (water,

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behavior over time on an accelerating rotarod. After pre-training all mice on an accelerating rotarod to a 120 s performance threshold, we injected each with either ethanol (2.0 g/kg), D-serine (2.7 g/kg) or a mixture of both and monitored their latency to fall off at 15 min intervals up to 1.5 h. Because the nature of the experiment runs over an extended time course, we started with a co-injection time window. 15 min after the ethanol-only injection (n ¼ 7), mouse latency had dropped from 106  9.12 s (time 0) to 7.3  3.0 s but improved steadily from there to a 94  15% recovery to baseline at 90 min (Fig. 4A). The ethanol/D-serine co-injected group (n ¼ 7) was nearly identical to ethanol controls with an 18.1  8.4 s deficit at 15 min (p ¼ 0.26 by Student t-test) and a 92  12% final recovery (p ¼ 0.89 by Student t-test) to the baseline value of 104  9.23 s (Fig. 4A). D-serine alone (n ¼ 7) showed no change in motor performance over time (p ¼ 0.69) (Fig. 4A). From this data set, D-serine appeared to have a neutral effect on both baseline and ethanol-induced rotarod behavior. In a pilot run of these studies, we included a few older animals and found that their performance greatly differed from the younger test subjects. Therefore, we kept these mice from the previous experiment (n ¼ 6 per condition) and tested them again 2e3 months later under the same conditions (Fig. 4B). All animals performed significantly worse in this second test, regardless of injection type (F(1,251) ¼ 33.07; p < 0.0000001). Ethanol-only animals had an average 15 min deficit of 1.7  0.8 s and ultimately recovered to only 62  5% of baseline. More interesting is the fact that D-serine displayed an aversive effect on rotarod behavior in this experiment that worsened over time. Although maximum deficit at 15 min was no different in the ethanol/D-serine group (p ¼ 0.24 by Student ttest), recovery at 90 min was significantly stunted by comparison at 43  35% (F(2,17) ¼ 8.11; p < 0.05). D-serine alone induced progressively worsening motor performance scores with a 90-min latency only 50  23% of the average time 0 value. The difference in the three groups was most apparent in an analysis of recovery slope (slope of latency points between 15 and 90 min) which was dissimilar between every condition (F(2,17) ¼ 38.78, p ¼ 0). Ethanol slope at 1.0  0.2 moved upward indicating some degree of recovery while D-serine sank downwards along a 0.6  0.3 slope and the combination group stagnated levelly at the maximum deficit level with a 0.3  0.3 slope. The LoRR experiments demonstrated a profound effect of time window on D-serine’s ability to modulate ethanol behavior. It seemed possible, therefore, that the lack of D-serine effect on rotarod performance in younger animals might be due to the use of co-injection. To test this hypothesis, we repeated the rotarod tests in naïve young adult animals but administered either saline (n ¼ 5) or D-serine (n ¼ 7) 15 min before the ethanol injection (Fig. 4C). We found that treatment had no effect on either latency (p ¼ 0.21) or recovery slope (p ¼ 0.20) among this set. Furthermore, there was no difference between the pre-injection and co-injection latency data for ethanol (p ¼ 0.86) or ethanol/D-serine (p ¼ 0.18) groups.

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treated), then two days of water vs. 8% ethanol (free choice) and two days of ethanol-only (forced ethanol) (Fig. 5A). Baseline total volume consumption during the untreated water phase was 3.8  0.1 mL/day. Overall volume was somewhat reduced during the forced ethanol phase to 3.5  0.1 mL/day (F(3,109) ¼ 3.82, p < 0.05) but there was no difference between D-serine and

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Fig. 5. The impact of D-serine on voluntary water and ethanol consumption. (A) Experimental schematic indicating the different phases of water (treated and untreated), free choice (water vs. 8% ethanol), and forced ethanol (8% ethanol-only) offered over a week within a two-bottle choice paradigm. Arrows indicate the timing of saline or D-serine (2.7 g/kg) injections relative to volume recording times. (B) Treatment group had no significant impact on total volume consumption regardless of experimental phase. (C) Daily ethanol volume and concentration was reduced in D-serine treated animals during free choice but similar to saline controls during the subsequent forced ethanol phase. (D) A scatter plot of ethanol preference (ethanol volume/total volume*100%) for all saline and D-serine treated individuals over both days of free choice. (E) Bar graph summarizing the reduction in free choice ethanol preference among the D-serine treatment group. N numbers are given in the figure legend. *p < 0.05, ***p < 0.001 represent significance vs. saline controls.

saline-treated groups in any phase (p ¼ 0.16) (Fig. 5B). By contrast, treatment and phase had a significant interactive effect on ethanol volume consumption (F(1,37) ¼ 5.72, p < 0.05). During free choice, saline-injected mice consumed on average 2.4  0.2 mL/day over the two-day period while D-serine-injected mice only drank 1.4  0.2 mL/day in the same time (F(1,37) ¼ 5.72, p < 0.05) (Fig. 5C). This pattern of reduced ethanol intake by D-serine treated mice was also reflected when drinking was adjusted by weight to reflect a daily concentration discrepancy of 5.9  0.4 g/kg/day among the saline group and 3.5  0.7 g/kg/day for D-serine animals (F(1,37) ¼ 4.81, p < 0.05) (Fig. 5C). Both ethanol consumption and daily concentration were higher during the forced ethanol phase but treatment had no impact on outcome (p ¼ 0.38 volume; p ¼ 0.33 concentration) (Fig. 5C). Ethanol volume divided into total volume consumption produced an estimate of ethanol preference for each mouse on each day of the free choice phase of the experiment (Fig. 5D). Neither day (p ¼ 0.86) nor weight (p ¼ 0.45) significantly impacted these values but treatment revealed a dramatic group segregation (F(1,37) ¼ 18.99, p < 0.001). While a control analysis for preference of one random tube rendered values around 50% in the other phases, free choice groups showed saline

animals with a 67  4% preference for the ethanol tube while the group avoided ethanol with a 33  5% preference (Fig. 5E).

D-serine

Discussion Our primary finding in this investigation was the ability of systemic pre-administered D-serine to reduce acute ethanol sedation in the LoRR test and to reduce voluntary ethanol drinking preference in a two-bottle free choice paradigm. D-serine also prevented ethanol-suppression of L-serine and glycine blood serum concentrations. D-serine did not appear to impact ethanol-induced motor coordination deficits in young adult animals but was independently and interactively detrimental to rotarod performance in older mice. NMDAR co-agonist modulation of ethanol sedation Only a couple of studies have attempted to examine the effects of D-serine on ethanol-induced sedation. In the first, an ip injection of DL-isoserine was observed to prolong LoRR duration when coadministered with ethanol or after a 5-min delay (Blum et al.,

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1972). There are concerns, however, over the reliability of these results as the sedative behavior of the saline controls was inconsistent between experiments and with other studies relying on a similar mouse strain (Chan, 1978; Ferko, 1992; Giknis & Damjanov, 1983; Williams et al., 1995). A more recent study also found that post-recovery D-serine (ICV) enhanced the central depressant effect of ethanol, triggering a second LoRR lasting several minutes (Williams et al., 1995). Nonetheless, the secondary depression was prevented by the addition of strychnine, suggesting that this effect was not fueled by action at the strychnine-insensitive NMDAR coagonist binding site (Williams et al., 1995). A greater number of studies have examined the interaction of ethanol sedation and other NMDAR co-agonists, particularly glycine and D-cycloserine. Relying on various drug delivery timing and conditions such as oral, intraperitoneal, and intracerebroventricular (ICV) administration, these studies have found co-agonist treatment to improve (Breglia, Ward, & Jarowski, 1973; Chan, 1978), worsen (Blum et al., 1972; Chan, 1978; Karcz-Kubicha & Liljequist, 1995; Williams et al., 1995) or have no effect (Breglia et al., 1973; Trevisan et al., 2008) on LoRR behavior. There have also been a very small number of studies examining acute co-agonist site antagonism which generally found an enhancement of sedative-like behavior (Pietrzak & Czarnecka, 2004). The results of these studies do not lend themselves to a unified understanding of the impact of NMDAR co-agonist site modulation on ethanol sedative behavior. Further experimentation is also somewhat hindered by the interpretational difficulties of using D-cycloserine and glycine, which until recently were the best available options for in vivo testing. D-cycloserine is only a partial agonist and can inhibit NMDAR responses 40e50% at fairly low cellular concentrations of 10e100 mM (Karcz-Kubicha et al., 1997; Watson, Bolanowski, Baganoff, Deppeler, & Lanthorn, 1990). Oral glycine appears to run some metabolic interference with ethanol (Breglia et al., 1973) and glycine furthermore acts as a primary ligand at its own inhibitory CNS receptors which may themselves be involved in regulating ethanol sedation (Basu et al., 2009; Williams et al., 1995). Despite some difficulties in interpretation, a consistently emerging observation from these studies was the importance of drug administration time window. It is interesting to note that all of the studies reporting reduced co-agonist depressant effects did so for pre-ethanol treatment conditions only. In our experiments, D-serine was able to reduce initial ethanol sensitivity at the two highest concentrations and to hasten acute tolerance at the maximum dose in sedation tests. The saline control values we obtained were consistent with other reports using the same mouse strain and ethanol concentration (Palachick et al., 2008). The ameliorative effects of D-serine were only observed when the drug was administered 15 min before ethanol (or up to 60 min prior in our preliminary tests, data not shown). D-serine administered with ethanol or 15 min afterward had no effect on LoRR behaviors. The potentially stimulative effects of extra handling in the post-ethanol treatment group were effectively ruled out by the similarity between saline controls. In addition to acute tolerance we also assessed rapid tolerance to ethanol sedation. We found that LoRR behavior was very similar among all animals on day two. Latency to reflex loss did not undergo tolerance in the control group so the loss of prolonged latency in the D-serine group likely represents the necessity of an acute high concentration of drug to occur. Significant tolerance was seen for LoRR duration in saline-treated mice but not in the D-serine animals, suggesting that this acute treatment neither promoted nor interfered with the total accumulation of cellular ethanol rapid tolerance mechanisms. These results do not necessarily reject the notion that D-serine altered the initial pace of tolerance. It is also possible that low day one duration values in D-serine mice resulted in a floor effect.

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Timing-dependent mechanisms There are two immediately obvious hypotheses to explain our acute LoRR results that take time window into account: metabolic interference and synaptic opposition. The metabolic explanation argues that the presence of D-serine may slow the absorption of ethanol into the blood stream, suppressing BEC and both prolonging initial latency to LoRR and reducing the degree of cellular tolerance required to achieve recovery. If the initial absorption period is critical for influencing acute sensitivity and controlling total ethanol accumulation, it is reasonable that D-serine would need to be present prior to ethanol for the effect to occur. It is unclear, however, why this mechanism would fail to improve rotarod deficits in our mice. A second metabolic argument is the potential ability of D-serine to hasten the degradation of ethanol but this would not explain the prolonged latency and or why other time windows of application did not have at least some impact on recovery time. We found D-serine to have no significant effect on BEC values at 20 or 40 min after injection, contrary to expectations in a metabolically manipulated system. This result is also in a good agreement with a previous study (Blum et al., 1972). The synaptic opposition hypothesis posits that after crossing into the CNS, D-serine binds to post-synaptic NMDARs and weakens the action of ethanol at these same receptors. Bound D-serine could physically reduce ethanol binding by partially blocking ethanol’s NR1 binding site or by inducing a conformational change that reduces the ethanol binding affinity of that receptor. This theory particularly explains why D-serine would need to reach to the receptor before ethanol to be effective. Alternately, D-serine’s agonism of the receptor could functionally oppose ethanol’s antagonism thereby reducing the drive toward cellular homeostasis via increased NMDAR activity. D-serine might require an administrative lead time because it is retarded at the blood brain barrier while ethanol passes into the CNS unhindered. Under these circumstances, we would expect BEC values to be influenced only by elapsed time and D-serine mice should demonstrate higher values given their early recovery. A significant decline in average BEC was found for D-serine treated mice but despite a promising trend, no significant effect of treatment on BEC was found in either experiment. We note, however, that individual scatter was high, particularly in the saline control animals. All together, the data are more supportive of a synaptic mechanism of D-serine modulation but the potential contribution of metabolic interference cannot be definitively ruled out. Neuromodulatory actions of NMDAR co-agonist modulators and ethanol The viability of a synaptic location of effect is only plausible given the assumption that D-serine reaches NMDARs in behaviorally relevant brain locations at sufficient concentration and has the capability of antagonizing ethanol’s cellular actions on that system. The high concentrations of D-serine we found in blood samples after D-serine injection suggests an effective absorption of drug by the time behavioral endpoints were reached. As to location, chronic ethanol use upregulates NMDARs most heavily in the hippocampus, cortex, and striatum (Gonzales & Brown, 1995; Gulya, Grant, Valverius, Hoffman, & Tabakoff, 1991), the same areas where endogenous D-serine is concentrated (Schell, Molliver, & Snyder, 1995) and where the greatest elevations in CNS D-serine occur subsequent to an ip injection (Hashimoto & Chiba, 2004). At the cellular level, D-serine reduced ethanol inhibition of NMDAstimulated Caþþ uptake by 65% in cultured rat cerebellar granule cells (Rabe & Tabakoff, 1990) and somewhat reduced ethanol inhibition of NMDA-stimulated norepinephrine release in rat

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hippocampal slices (Woodward, 1994). Furthermore, a previous study demonstrated that D-serine is capable of reversing behavioral deficits caused by another NMDAR antagonist, MK-801, at the same doses we found to be effective in this study (Kanahara et al., 2008). It is also possible that D-serine has an indirect synaptic impact via secondary effects on other systems. Ethanol reduced blood serum concentrations of L-serine and glycine, in agreement with previously published studies (Eriksson & Carlsson, 1980; Milakofsky, Miller, & Vogel, 1989). D-serine may have prevented these drops by stoichiometrically retarding the enzymatic conversion of L-serine and by driving up the catabolic byproduct serine, a glycinergic precursor. The nature of a molecular interaction between NMDAR coagonist modulators and ethanol has been investigated on multiple occasions but remains unresolved. The most recent model posits that ethanol has a unique intracellular binding site on the third transmembrane segment (TM3) of the NR1 subunit (Krystal et al., 2003; Ronald et al., 2001). The co-agonist site has also been localized to TM3 but extracellularly (Krystal et al., 2003), which reduces the likelihood of direct physical interference between ligands. TM3 phenylalanine residue 639 seems to be of particular importance as alterations to this amino acid can change receptor sensitivity to both ethanol and co-agonist modulators (Ronald et al., 2001). The electrophysiological effects of ethanol binding are best mimicked by reducing open channel frequency and duration and it has been suggested that Phe639 assists in transducing the effects of TM3 ligand binding to the channel opening machinery (Ronald et al., 2001). The story is more complicated, however, as identification of the remaining receptor subunits plays a heavy role in determining the outcome of NR1 binding. NR2A and NR2B containing receptors are more sensitive to ethanol and some co-agonist binding than NR2C or 2D (Masood, Wu, Brauneis, & Weight, 1994; Mirshahi & Woodward, 1995). The phosphorylation state of the NR2 subunit has been shown to contribute to both receptor modulatory efficacy and ethanol-induced behavioral responses (Miyakawa et al., 1997; Ronald et al., 2001; Yaka, Tang, Camarini, Janak, & Ron, 2003). Possibly due to the distribution of receptors with different subunit compositions, there are also brain regional differences in sensitivity. For example, glycine reverses NMDARmediated ethanol action in the striatum and cerebellum but has no impact on ethanol-induced norepinephrine releases in the cortex and hippocampus (Gonzales & Brown, 1995). It should also be noted, we have described D-serine as a highly selective drug and thus a better predictor of the influence of NMDAR co-agonism to ethanol behavior compared to other coagonists. Nevertheless, D-serine has been shown to bind to NR3-containing NMDARs, which have a very different electrophysiological and pharmacological profile from NR2-containining receptors (Smothers & Woodward, 2007). NR3-NMDARs appear to be mildly ethanol sensitive (20% current inhibition) but their role in the neurobiological effects of ethanol is currently unknown (Smothers & Woodward, 2007).

glycine have shown some acute positive effects on alleviating motor impairment (Blum et al., 1974; Breglia et al., 1973; Khanna et al., 2002; Ogawa, 2004). NMDAR co-agonist site inhibitors have conversely been seen to exacerbate ethanol-induced motor deficits (Pietrzak & Czarnecka, 2004; Vanover, 1999) as well as producing independent ataxic effects (Bienkowski, Koros, Kostowski, & Danysz, 1999). On the other hand, improved rotarod coordination was noted during voluntary and forced ethanol consumption in Grin1D481N mice, a transgenic model with an 80% reduction in NMDAR glycine binding (Kiefer et al., 2003). One possibility for the discrepancy in efficacy between sedation and ethanol ataxia is the heavy involvement of the cerebellum in regulating motor behavior. This brain region showed the smallest change in D-serine concentration following ip injection (Hashimoto & Chiba, 2004) and no change in NMDAR co-agonist dependent fMRI activity in rats (Panizzutti et al., 2005). The lack of functional activity could be due to full occupancy of NMDARs in the cerebellum rendering additional D-serine irrelevant. On the other hand, the adult cerebellum contains a very high concentration of the catabolic enzyme D-amino acid oxidase (DAAO) (Konno, Hamase, Maruyama, & Zaitsu, 2010), which keeps endogenous D-serine low in the region and may degrade exogenous molecules more quickly than they can act at the synapse. It would be interesting to test acute ethanol effects in the currently available strain of DAAO knockout mice, which show elevated levels of cerebellar D-serine (Hashimoto et al., 1993) and reduced NMDAR-modulated ataxia (Hashimoto, Yoshikawa, Niwa, & Konno, 2005). The rotarod experiments also revealed an unanticipated age effect in which D-serine appeared to have a detrimental impact on motor performance in older animals only. Some degradation of baseline and ethanol-induced motor coordination was expected given the age and weight of the test subjects (Draski, Bice, & Deitrich, 2001). The ethanol-only treated animals did have a prolonged period of high deficit but ultimately showed some recovery by the end of the test. By contrast, D-serine had both an independent and progressively negative impact on rotarod performance and also suppressed recovery in ethanol-treated animals. The relevance of age as the primary factor is supported by the neutral D-serine response previously exhibited by the same animals several months earlier. It is unlikely that prior exposure to D-serine had any long-term effect on these results given the lack of acute treatment impact in the rapid tolerance experiment. There is no ready explanation in the extant literature for this senescence-dependent impairment. One possibility, learning plays a role in rotarod performance and D-serine has a nootropic effect in younger animals (Karasawa, Hashimoto, & Chaki, 2008) but the number of NMDAR binding sites is diminished with age (Nagata, Uehara, Kitamura, Nomura, & Horiike, 1998) as well as the efficacy of D-serine as a promoter of hippocampal plasticity (Mothet et al., 2006; Oliet & Mothet, 2009). It should be noted, however, that the co-agonists glycine and D-cycloserine can damage memory processes in alcoholic subjects with elevated NMDAR concentrations (Krystal et al., 2011).

The impact of D-serine on ethanol motor deficits in young adult and older mice

D-serine

It is somewhat surprising that D-serine had no effect on ethanolinduced motor coordination deficits or recovery in young adult animals. In other studies, oral serine prior to ethanol increased rotarod performance but also depressed BEC (Blum et al., 1974). D-serine (ICV) reduced ataxia in a mouse model of spinocerebellar atrophy based on cerebellar hypoglutamatergia (Tanabe, Nakano, Honda, & Ono, 2009) and antagonized motor deficits induced by multiple NMDAR antagonists (Contreras, 1990; Tanii, Nishikawa, Hashimoto, & Takahashi, 1994). Additionally, D-cycloserine and

modulation of voluntary ethanol drinking

Perhaps the most exciting and intriguing result in this study was that daily ip D-serine reduced short-term ethanol preference in mice given a free choice between drinking 8% ethanol and water. Rodents typically do not spontaneously consume ethanol to intoxicating concentrations so it is unlikely that the acute sedative or tolerant effects we observed were responsible for D-serine’s inhibition of ethanol choice drinking. Similarly, the absence of change in total volume consumption argues against reductions in thirst or alterations in taste perception as contributing factors. Subjective aversion is also unlikely given that ethanol preference was stable

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with time and a recent publication that could not induce conditioned place aversion in rats with D-serine (Yang et al., 2010). There are a variety of neurological mechanisms that might have contributed to D-serine’s suppression of ethanol preference. Co-agonist site modulators have been shown to influence drinking patterns previously. The partial NMDAR antagonists ACPC and HA-966 both diminished voluntary ethanol consumption when administered ip (McMillen, Joyner, Parmar, Tyer, & Williams, 2004; Stromberg, Volpicelli, O’Brien, & Mackler, 1999) or for ACPC when injected directly into the nucleus accumbens, a brain region heavily involved in reward (Stromberg et al., 1999). The full antagonist L-701,324 suppressed rodent withdrawal/relapse-drinking (Alen et al., 2009; Vengeliene, Bachteler, Danysz, & Spanagel, 2005). On the other hand, glycine injections into the nucleus accumbens also reduced ethanol preferences (Molander, Lof, Stomberg, Ericson, & Soderpalm, 2005) as did systemic administration of Org 25935, an inhibitor of glycine transporter 1 (GlyT1) (Molander et al., 2005, 2007). Some of these effects were sensitive to strychnine, however, suggesting the involvement of glycine receptor activation (Molander et al., 2005). Whether through NMDARs or through glycine receptors, the contribution of elevated glycine to reduced ethanol drinking seems particularly probable given our own data showing D-serine’s prevention of ethanol-induced glycine plasma suppression. Glutamate is not the only system that impacts drinking behavior. GABAA and 5HT1 activity may arguably have a larger influence on sub-intoxication ethanol effects given their dominant role in mimicking the discriminative stimulus quality of ethanol consumption at lower concentrations (Grant & Colombo, 1993; Krystal et al., 2003). In the very short-term, delayed and diminished alcohol sedation has immediate individual and societal benefits as a drowsy intoxicated person is more likely to fall into unsafe situations. In spite of this, there is a larger question as to what the impact of faster acute tolerance might be on chronic patterns of alcohol abuse. Some experts argue that it is the negative physical side effects of intoxication that drive the cessation of drinking and therefore reduced sedation will only encourage more drinking and long-term dependence (Fadda & Rossetti, 1998; Krystal et al., 2011). This argument presupposes that drinkers imbibe ethanol to get drunk and will not stop until forced to do so. While this may be true of some drinkers, a plausible alternate theory is that people drink alcohol to achieve a certain subjectively pleasurable state and will generally halt consumption when this is achieved (Boyce-Rustay & Cunningham, 2004). Our findings more strongly support the latter hypothesis as reduced acute sedation was paired with a smaller volume of ethanol consumption. However, it is not necessarily true that mice modulate their consumption of ethanol for the same reasons that humans do. For this reason, clinical studies on the impact of D-serine on alcohol use will be imperative to clarify the resolution of these competing theories. Further implications The potential impact of D-serine on short and long-term ethanol abuse takes on an added urgency considering that this drug is currently being used in high-end clinical trials. D-serine is showing considerable success as a treatment option against psychiatric disorders with suspected NMDAR hypofunction such as posttraumatic stress disorder, depression and schizophrenia (HerescoLevy et al., 2009; Labrie & Roder, 2010). These conditions, however, already demonstrate an increased comorbidity of substance abuse, including alcoholism (Coyle, 2006; Krystal et al., 2006; Palomo, Archer, Kostrzewa, & Beninger, 2007). Despite the heightened vulnerability and the push to bring D-serine to market as an approved pharmaceutical intervention, no studies have been

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undertaken to determine its potential side effects on the rates of drug and alcohol abuse in these sub-populations. Clinical evaluations are certainly necessary, but it may also be advantageous to pursue preclinical explorations in murine transgenic models. Serine racemase (SR) is the endogenous enzyme that converts neurologically inactive L-serine into D-serine. Mice with the SR gene removed or functionally inactivated demonstrate an 89e95% reduction in D-serine concentrations in the brain. These mice also display some characteristic behavior symptoms of schizophrenia such as increased startle response and social and spatial learning deficits that are reversible by D-serine treatment (Basu et al., 2009; Labrie et al., 2009). It would be interesting, therefore, to assess basic acute and chronic ethanol responses in these mice to see if the model mimics the kind of increased alcohol abuse observed in human schizophrenics and then to determine whether D-serine has any impact on these findings. Acknowledgments We would like to thank Drs John Crabbe and Robert Meisel for their valuable comments on the initial experimental design and subsequent manuscripts. We also thank Dr. Walter Low for sharing lab equipment, Drs. Robert Miller, Steve Sullivan, and Manuel Esguerra for their advice on capillary electrophoresis experiments.

References Alen, F., Santos, A., Moreno-Sanz, G., Gonzalez-Cuevas, G., Gine, E., Franco-Ruiz, L., et al. (2009). Cannabinoid-induced increase in relapse-like drinking is prevented by the blockade of the glycine-binding site of N-methyl-D-aspartate receptors. Neuroscience, 158, 465e473. Basu, A. C., Tsai, G. E., Ma, C. L., Ehmsen, J. T., Mustafa, A. K., Han, L., et al. (2009). Targeted disruption of serine racemase affects glutamatergic neurotransmission and behavior. Molecular Psychiatry, 14, 719e727. Bienkowski, P., Koros, E., Kostowski, W., & Danysz, W. (1999). Effects of N-methyl-Daspartate receptor antagonists on reinforced and nonreinforced responding for ethanol in rats. Alcohol, 18, 131e137. Blum, K., Wallace, J. E., & Friedman, R. N. (1974). Reduction of acute alcoholic intoxication by alpha amino acids: glycine and serine. Life Sciences, 14, 557e565. Blum, K., Wallace, J. E., & Geller, I. (1972). Synergy of ethanol and putative neurotransmitters: glycine and serine. Science, 176, 292e294. Boyce-Rustay, J. M., & Cunningham, C. L. (2004). The role of NMDA receptor binding sites in ethanol place conditioning. Behavioral Neuroscience, 118, 822e834. Breglia, R. J., Ward, C. O., & Jarowski, C. I. (1973). Effect of selected amino acids on ethanol toxicity in rats. Journal of Pharmaceutical Sciences, 62, 49e55. Cebers, G., Cebere, A., Zharkovsky, A., & Liljequist, S. (1996). Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells. Naunyn-Schmiedeberg’s Archives of Pharmacology, 354, 736e745. Chan, A. W. (1978). Interaction of glycine with ethanol. Biochemical Pharmacology, 27, 1913e1918. Contreras, P. C. (1990). D-serine antagonized phencyclidine- and MK-801-induced stereotyped behavior and ataxia. Neuropharmacology, 29, 291e293. Coyle, J. T. (2006). Substance use disorders and schizophrenia: a question of shared glutamatergic mechanisms. Neurotoxicity Research, 10, 221e233. Draski, L. J., Bice, P. J., & Deitrich, R. A. (2001). Developmental alterations of ethanol sensitivity in selectively bred high and low alcohol sensitive rats. Pharmacology, Biochemistry, and Behavior, 70, 387e396. Eriksson, T., & Carlsson, A. (1980). Ethanol-induced increase in brain concentrations of administered neutral amino acids. Naunyn-Schmiedeberg’s Archives of Pharmacology, 314, 47e50. Fadda, F., & Rossetti, Z. L. (1998). Chronic ethanol consumption: from neuroadaptation to neurodegeneration. Progress in Neurobiology, 56, 385e431. Ferko, A. P. (1992). NMDA enhances the central depressant properties of ethanol in mice. Pharmacology, Biochemistry, and Behavior, 43, 297e301. Gallaher, E. J., Jones, G. E., Belknap, J. K., & Crabbe, J. C. (1996). Identification of genetic markers for initial sensitivity and rapid tolerance to ethanol-induced ataxia using quantitative trait locus analysis in BXD recombinant inbred mice. The Journal of Pharmacology and Experimental Therapeutics, 277, 604e612. Giknis, M. L., & Damjanov, I. (1983). The transplacental effects of ethanol and metronidazole in Swiss Webster mice. Toxicology Letters, 19, 37e42. Gonzales, R. A., & Brown, L. M. (1995). Brain regional differences in glycine reversal of ethanol-induced inhibition of N-methyl-D-aspartate-stimulated neurotransmitter release. Life Sciences, 56, 571e577. Grant, K. A., & Colombo, G. (1993). Pharmacological analysis of the mixed discriminative stimulus effects of ethanol. Alcohol and Alcoholism, 2, 445e449.

400

A. Lockridge et al. / Alcohol 46 (2012) 389e400

Gulya, K., Grant, K. A., Valverius, P., Hoffman, P. L., & Tabakoff, B. (1991). Brain regional specificity and time-course of changes in the NMDA receptorionophore complex during ethanol withdrawal. Brain Research, 547, 129e134. Hashimoto, A., & Chiba, S. (2004). Effect of systemic administration of D-serine on the levels of D- and L-serine in several brain areas and periphery of rat. European Journal of Pharmacology, 495, 153e158. Hashimoto, A., Nishikawa, T., Konno, R., Niwa, A., Yasumura, Y., Oka, T., et al. (1993). Free D-serine, D-aspartate and D-alanine in central nervous system and serum in mutant mice lacking D-amino acid oxidase. Neuroscience Letters, 152, 33e36. Hashimoto, A., Yoshikawa, M., Niwa, A., & Konno, R. (2005). Mice lacking D-amino acid oxidase activity display marked attenuation of stereotypy and ataxia induced by MK-801. Brain Research, 1033, 210e215. Heresco-Levy, U., Vass, A., Bloch, B., Wolosker, H., Dumin, E., Balan, L., et al. (2009). Pilot controlled trial of D-serine for the treatment of post-traumatic stress disorder. International Journal of Neuropsychopharmacology, 12, 1275e1282. Jansen, M., & Dannhardt, G. (2003). Antagonists and agonists at the glycine site of the NMDA receptor for therapeutic interventions. European Journal of Medicinal Chemistry, 38, 661e670. Kanahara, N., Shimizu, E., Ohgake, S., Fujita, Y., Kohno, M., Hashimoto, T., et al. (2008). Glycine and D: -serine, but not D: -cycloserine, attenuate prepulse inhibition deficits induced by NMDA receptor antagonist MK-801. Psychopharmacology (Berl), 198, 363e374. Karasawa, J., Hashimoto, K., & Chaki, S. (2008). D-Serine and a glycine transporter inhibitor improve MK-801-induced cognitive deficits in a novel object recognition test in rats. Behavioural brain research, 186, 78e83. Karcz-Kubicha, M., Jessa, M., Nazar, M., Plaznik, A., Hartmann, S., Parsons, C. G., et al. (1997). Anxiolytic activity of glycine-B antagonists and partial agonistseno relation to intrinsic activity in the patch clamp. Neuropharmacology, 36, 1355e1367. Karcz-Kubicha, M., & Liljequist, S. (1995). Effects of post-ethanol administration of NMDA and non-NMDA receptor antagonists on the development of ethanol tolerance in C57B1 mice. Psychopharmacology, 120, 49e56. Khanna, J. M., Morato, G. S., & Kalant, H. (2002). Effect of NMDA antagonists, an NMDA agonist, and serotonin depletion on acute tolerance to ethanol. Pharmacology, Biochemistry, and Behavior, 72, 291e298. Kiefer, F., Jahn, H., Koester, A., Montkowski, A., Reinscheid, R. K., & Wiedemann, K. (2003). Involvement of NMDA receptors in alcohol-mediated behavior: mice with reduced affinity of the NMDA R1 glycine binding site display an attenuated sensitivity to ethanol. Biological Psychiatry, 53, 345e351. Konno, R., Hamase, K., Maruyama, R., & Zaitsu, K. (2010). Mutant mice and rats lacking D-amino acid oxidase. Chemistry & Biodiversity, 7, 1450e1458. Krystal, J. H., D’Souza, D. C., Gallinat, J., Driesen, N., Abi-Dargham, A., Petrakis, I., et al. (2006). The vulnerability to alcohol and substance abuse in individuals diagnosed with schizophrenia. Neurotoxicity Research, 10, 235e252. Krystal, J. H., Petrakis, I. L., Limoncelli, D., Nappi, S. K., Trevisan, L., Pittman, B., et al. (2011). Characterization of the interactive effects of glycine and D-cycloserine in men: further evidence for enhanced NMDA receptor function associated with human alcohol dependence. Neuropsychopharmacology, 36, 701e710. Krystal, J. H., Petrakis, I. L., Mason, G., Trevisan, L., & D’Souza, D. C. (2003). N-methylD-aspartate glutamate receptors and alcoholism: reward, dependence, treatment, and vulnerability. Pharmacology & Therapeutics, 99, 79e94. Labrie, V., Fukumura, R., Rastogi, A., Fick, L. J., Wang, W., Boutros, P. C., et al. (2009). Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model. Human Molecular Genetics, 18, 3227e3243. Labrie, V., & Roder, J. C. (2010). The involvement of the NMDA receptor D-serine/ glycine site in the pathophysiology and treatment of schizophrenia. Neuroscience & Biobehavioral Reviews, 34, 351e372. Masood, K., Wu, C., Brauneis, U., & Weight, F. F. (1994). Differential ethanol sensitivity of recombinant N-methyl-D-aspartate receptor subunits. Molecular Pharmacology, 45, 324e329. McMillen, B. A., Joyner, P. W., Parmar, C. A., Tyer, W. E., & Williams, H. L. (2004). Effects of NMDA glutamate receptor antagonist drugs on the volitional consumption of ethanol by a genetic drinking rat. Brain Research Bulletin, 64, 279e284. Milakofsky, L., Miller, J. M., & Vogel, W. H. (1989). Effect of ethanol on plasma amino acids and related compounds of stressed male rats. Pharmacology, Biochemistry, and Behavior, 32, 1071e1074. Mirshahi, T., & Woodward, J. J. (1995). Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2þ)-insensitive mutants. Neuropharmacology, 34, 347e355. Miyakawa, T., Yagi, T., Kitazawa, H., Yasuda, M., Kawai, N., Tsuboi, K., et al. (1997). Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function. Science, 278, 698e701. Molander, A., Lido, H. H., Lof, E., Ericson, M., & Soderpalm, B. (2007). The glycine reuptake inhibitor Org 25935 decreases ethanol intake and preference in male wistar rats. Alcohol and Alcoholism, 42, 11e18. Molander, A., Lof, E., Stomberg, R., Ericson, M., & Soderpalm, B. (2005). Involvement of accumbal glycine receptors in the regulation of voluntary ethanol intake in the rat. Alcoholism: Clinical and Experimental Research, 29, 38e45. Mothet, J. P., Rouaud, E., Sinet, P. M., Potier, B., Jouvenceau, A., Dutar, P., et al. (2006). A critical role for the glial-derived neuromodulator D-serine in the age-related deficits of cellular mechanisms of learning and memory. Aging Cell, 5, 267e274. Nagata, Y., Uehara, T., Kitamura, Y., Nomura, Y., & Horiike, K. (1998). D-serine content and D-[3H]serine binding in the brain regions of the senescence-accelerated mouse. Mechanisms of Ageing and Development, 104, 115e124. Nagy, J. (2008). Alcohol related changes in regulation of NMDA receptor functions. Current Neuropharmacology, 6, 39e54.

Ogawa, M. (2004). Pharmacological treatments of cerebellar ataxia. Cerebellum, 3, 107e111. Oliet, S. H., & Mothet, J. P. (2009). Regulation of N-methyl-D-aspartate receptors by astrocytic D-serine. Neuroscience, 158, 275e283. Palachick, B., Chen, Y. C., Enoch, A. J., Karlsson, R. M., Mishina, M., & Holmes, A. (2008). Role of major NMDA or AMPA receptor subunits in MK-801 potentiation of ethanol intoxication. Alcoholism: Clinical and Experimental Research, 32, 1479e1492. Palomo, T., Archer, T., Kostrzewa, R. M., & Beninger, R. J. (2007). Comorbidity of substance abuse with other psychiatric disorders. Neurotoxicity Research, 12, 17e27. Panizzutti, R., Rausch, M., Zurbrugg, S., Baumann, D., Beckmann, N., & Rudin, M. (2005). The pharmacological stimulation of NMDA receptors via co-agonist site: an fMRI study in the rat brain. Neuroscience Letters, 380, 111e115. Pietrzak, B., & Czarnecka, E. (2004). Influence of felbamate on selected central effects of ethanol in experimental animals. Acta Poloniae Pharmaceutica, 61, 209e213. Rabe, C. S., & Tabakoff, B. (1990). Glycine site-directed agonists reverse the actions of ethanol at the N-methyl-D-aspartate receptor. Molecular Pharmacology, 38, 753e757. Radcliffe, R. A., Floyd, K. L., & Lee, M. J. (2006). Rapid ethanol tolerance mediated by adaptations in acute tolerance in inbred mouse strains. Pharmacology, Biochemistry, and Behavior, 84, 524e534. Ronald, K. M., Mirshahi, T., & Woodward, J. J. (2001). Ethanol inhibition of N-methylD-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue. Journal of Biological Chemistry, 276, 44729e44735. Schell, M. J., Molliver, M. E., & Snyder, S. H. (1995). D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proceedings of the National Academy of Sciences of the United States of America, 92, 3948e3952. Smothers, C. T., & Woodward, J. J. (2007). Pharmacological characterization of glycine-activated currents in HEK 293 cells expressing N-methyl-D-aspartate NR1 and NR3 subunits. The Journal of Pharmacology and Experimental Therapeutics, 322, 739e748. Stromberg, M. F., Volpicelli, J. R., O’Brien, C. P., & Mackler, S. A. (1999). The NMDA receptor partial agonist, 1-aminocyclopropanecarboxylic acid (ACPC), reduces ethanol consumption in the rat. Pharmacology, Biochemistry, and Behavior, 64, 585e590. Sullivan, S. J., & Miller, R. F. (2010). AMPA receptor mediated D-serine release from retinal glial cells. Journal of Neurochemistry, 115, 1681e1689. Tanabe, M., Nakano, T., Honda, M., & Ono, H. (2009). Glycine transporter blockade ameliorates motor ataxia in a mouse model of spinocerebellar atrophy. Journal of Pharmacological Sciences, 109, 444e448. Tanii, Y., Nishikawa, T., Hashimoto, A., & Takahashi, K. (1994). Stereoselective antagonism by enantiomers of alanine and serine of phencyclidine-induced hyperactivity, stereotypy and ataxia in the rat. The Journal of Pharmacology and Experimental Therapeutics, 269, 1040e1048. Taslim, N., Al-Rejaie, S., & Saeed Dar, M. (2008). Attenuation of ethanol-induced ataxia by alpha(4)beta(2) nicotinic acetylcholine receptor subtype in mouse cerebellum: a functional interaction. Neuroscience, 157, 204e213. Trevisan, L., Petrakis, I. L., Pittman, B., Gueorguieva, R., D’Souza, D. C., Perry, E., et al. (2008). Absence of significant interactive effects of high-dose D-cycloserine and ethanol in healthy human subjects: preliminary insights into ethanol actions at the glycine B site of NMDA glutamate receptors. Alcoholism: Clinical and Experimental Research, 32, 36e42. Vanover, K. E. (1999). Interaction of ethanol with excitatory amino acid receptor antagonists in mice. European Journal of Pharmacology, 368, 137e142. Vengeliene, V., Bachteler, D., Danysz, W., & Spanagel, R. (2005). The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect. Neuropharmacology, 48, 822e829. Watson, G. B., Bolanowski, M. A., Baganoff, M. P., Deppeler, C. L., & Lanthorn, T. H. (1990). D-cycloserine acts as a partial agonist at the glycine modulatory site of the NMDA receptor expressed in Xenopus oocytes. Brain Research, 510, 158e160. Weitlauf, C., & Woodward, J. J. (2008). Ethanol selectively attenuates NMDARmediated synaptic transmission in the prefrontal cortex. Alcoholism: Clinical and Experimental Research, 32, 690e698. Williams, K. L., Ferko, A. P., Barbieri, E. J., & DiGregorio, G. J. (1995). Glycine enhances the central depressant properties of ethanol in mice. Pharmacology, Biochemistry, and Behavior, 50, 199e205. Wolosker, H., Dumin, E., Balan, L., & Foltyn, V. N. (2008). D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration. FEBS Journal, 275, 3514e3526. Woodward, J. J. (1994). A comparison of the effects of ethanol and the competitive glycine antagonist 7-chlorokynurenic acid on N-methyl-D-aspartic acid-induced neurotransmitter release from rat hippocampal slices. Journal of Neurochemistry, 62, 987e991. Yaka, R., Tang, K. C., Camarini, R., Janak, P. H., & Ron, D. (2003). Fyn kinase and NR2Bcontaining NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward. Alcoholism: Clinical and Experimental Research, 27, 1736e1742. Yang, F. Y., Lee, Y. S., Cherng, C. G., Cheng, L. Y., Chang, W. T., Chuang, J. Y., et al. (2010). D-cycloserine, sarcosine and D-serine diminish the expression of cocaine-induced conditioned place preference. Journal of Psychopharmacology.