[PDF] Characterizing Ethanol-Induced Glutamate Release in the Nucleus

1 Characterizing Ethanol-Induced Glutamate Release in the Nucleus Accumbens Stéphane Planche Integrated Program in Neuroscience McGill University, Montreal August 2017 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Stéphane Planche 2017

2 Table of contents ABSTRACT ........................................................................................................................................... 3RÉSUMÉ ............................................................................................................................................... 4ACKNOWLEDGMENTS .................................................................................................................... 5INTRODUCTION ................................................................................................................................ 6GLUTAMATE ........................................................................................................................................ 7GLUTAMATE IN SUBSTANCE USE ....................................................................................................... 7ETHANOL ............................................................................................................................................. 8ETHANOL & GLUTAMATE ................................................................................................................... 9ETHANOL & GABA ............................................................................................................................ 12RATIONALE ...................................................................................................................................... 13METHODS .......................................................................................................................................... 14SUBJECTS .......................................................................................................................................... 14CANNULATION .................................................................................................................................. 14MICRODIALYSIS PROBES ................................................................................................................... 15MICRODIALYSIS ................................................................................................................................ 16ANALYSIS OF LYSATE ....................................................................................................................... 17HISTOLOGY ....................................................................................................................................... 18STATISTICAL ANALYSIS .................................................................................................................... 18RESULTS ............................................................................................................................................ 21HISTOLOGY ....................................................................................................................................... 21ETHANOL-INDUCED GLUTAMATE RESPONSE .................................................................................... 21ETHANOL-INDUCED BEHAVIOURAL EFFECTS .................................................................................... 22CALCIUM CONTROL ........................................................................................................................... 22ETHANOL-INDUCED GABA RESPONSE ............................................................................................... 22DISCUSSION ...................................................................................................................................... 24SUMMARY & CONCLUSIONS ...................................................................................................... 29BIBLIOGRAPHY ............................................................................................................................... 30FIGURES ............................................................................................................................................. 42

3 Abstract Glutamate is the main excitatory neurotransmitter in the central nervous system. Studies in laboratory animals indi cate that it plays an import ant role in experience-dependent neuroplasticity and reward-related learning including t he acquisition of drug-seeking and taking behaviors, such as alcohol use. Despite this, clear evidence that ethanol promotes glutamate release is missing. To address this, the present investigation sought to characterize the effects of ethanol on glutam ate release in rodents in vivo using microdial ysis. The experiments tested the following two questions. First, does ethanol provoke glutamate release in the nucleus accumbens? Second, is this effect calcium dependent? The results suggest that (i) low-dose ethanol administration leads to increases in extracellular glutamate in the nucleus accumbens, and (ii) this glutamate response is sensitive to calcium depletion, suggesting a neuronal origin of the effect.

4 Résumé Le glutamate est le principal neurotransmetteur excitateur dans le système nerveux central. Des études chez l'animal démontrent qu'il joue un rôle important dans la neuroplasticité et l'apprentissage par la récompense, essentielles lors de la transition entre une consommation régulière et inoffensive et l'acquisition d'une conduite pharmacophile qui peut s'effectuer avec certaines substances telles que l'alcool . Malgré cela, les preuves d'une ac tion glutamatergique de l'éthanol, principal ingrédient actif dans les boissons alcooliques, ne sont pas encore venues étayer cette théorie. Afin d'adresser cette lacune, l'expérience présente chercha à caractériser, par microdialyse, les effets de l'éthanol sur la libération de glutamate in vivo . Nous cherchions en un premier temps à déterm iner si l'éthanol provoque une libération de glutamate dans le noyau accumbens et en un deuxième temps si cette libération dépendrait du calcium. Nos résultats indiquent que (i) l'administration d'éthanol à faible dose produit des augm entations dans les niveaux de glutamate extracell ulaire dans le noyau accumbens, et que (ii) cette réponse glutamatergique est sensible à une déplétion calcique, suggérant une origine neuronale de cet effet.

5 Acknowledgments I thank Drs. Marco Leyton, Alain Gratton and Pedro Rosa-Neto for their supervision as well as their feedback on earlier versions of this project. I would especially like to extend my gratitude to Dr. Marco Leyton for his mentorship as well as his editorial help that greatly improved this manuscript. I would also like to thank Luc Moquin for his assistance with the technical aspects of the microdialysis procedure as well as his valuable advice and training. The author wrot e the manusc ript in its ent irety; no contributions were made other than editorial help from Dr. Marco Leyton.

6 Introduction Since the mid 1970s, much of the neuropharmacological literature surrounding substance use has revolved around the neurotransmitter dopamine, particularly the ascending mesolimbic projections from the ventral tegmental area to the nucleus accumbens (Di Chiara & Imperato, 1988; Robinson a nd Berridge, 2001; Berridge, 2007). The latter, located in the ve ntral striatum, is a component of the basal ganglia, a group of subcortical nuclei interconnected with broad swaths of the limbic cortex, as well as the thalamus and brainstem, and involved in an array of processes integrating affective, contextual and motivational information. The nucleus accumbens is known to play an essential role in processing motivational salience and positive reinforcement, functions that have been largely attributed to dopaminergic activity (Pierce & Kumaresan, 2006; Yager et al, 2015). In the past two decades, however, a second neurotransmitter has been identified as playing critical roles in these behavioural effects, glutamate (Uys & Lalum iere, 2009). This might refle ct interactions between the two transmitters (Tzschentke & Schmidt, 2003), and supporting evidence included early work pointing towards effects of glutamate antagonists on the dopaminergic system and vice-versa (Moghaddam & Bolinao, 1994b; Reid et al, 1997), as well as later studies demonstrating co-release of the neurotransmitters from the same mesolimbic cells (Stuber et al, 2010). This noted, there is now accumulating evidence that glutamate also has dopamine-independent influences on processes related to substanc e use and other re ward seeking be haviours (Chiamulera et al, 2001; Britt et al, 2012; for review, see D'Souza, 2015), though the specific details remain poorly understood.

7 Glutamate Glutamate is the most abundant neurotransmitter in the vertebrate central nervous system, and is a major medi ator of neura l signaling enabling both fast ionotropi c and sl ower metabotropic neurotransmission (Watkins, 2000; Niciu et al, 2012). The former, in which glutamate acts as the brain's principal excitatory neurotransmitter, involves ligand-gated ion channels such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, while the latter, i n which gluta mate acts as a modulator of neuronal and glial activity, involves seven transmembrane domain metabotropic receptors (mGluRs) (Meldrum, 2000). These receptors are usually classified into three families, Groups I (mGluR1 & mGluR5), II (m GluR2 & mGluR3) and III (mGluR4, mGluR6, mGluR7 & mGluR8), and are known to contribute to a variety of neural functions such as neuronal excitability, neurotoxicity and synaptic plasticity (Conn, 2003). Group II and III receptors are Gi/Go-coupled and are located both pre- and post-synaptically, while Group I receptors are Gq-coupled and primarily post-synaptic (Pin et al., 2003). Glutamate in Substance Use In addition to mesolimbic dopaminergic projections, there is also evidence of glutamatergic afferents from the ventral tegmental area to the nucleus accumbens (Hnasko et al, 2012). These afferents intersect with a larger array of glutamatergic input to the nucleus accumbens from both cortical and subcortical regions including the prefrontal cortex, the hippocampus, the amygdala and the thalamus (Meredith et al, 1993; Gass & Olive, 2008; Britt et al, 2012). Moreover, there is c lear evidence for the expression of glutamatergic rece ptors, both ionotropic and metabotropic, in the nucleus accumbens (Albin et al, 1992; Tallaksen-Greene et al, 1998).

9 neurobiological effects are equally varied, ranging from anxiolysis, sociability, emotional lability and motor incoordination at lower doses to stupor, ataxia and anterograde amnesia at higher doses. Sufficiently high concentrations can lead to loss of consciousness and even death. Among the various neurobiological effects attributed to ethanol, particular emphasis has been placed on its role as a positive allosteric modulator of GABAA receptors and a negative allosteric modulator of NMDA glutamate receptors (Lovinger et al, 1989; Malenka et al, 2009). Ethanol & Glutamate A first line of evidence implicating glutamate in ethanol use points towards an effect of glutamate receptor antagonism on drug self-administration. Indeed, studies have shown that, as with other substances, the administration of NMDA antagonists decreases ethanol self-administration (Rassnick et al, 1992; Shelton & Balster, 1997). However, this phenomenon is not restricted to NMDA receptors, as antagoni sts of other glutamate rec eptors, both ionotropic (e.g., AM PA) and metabotropic (e.g., mG luR5), ca n attenuate ethanol self-administration (Stephens & Brown, 1999; Schroeder et al, 2005) There is also evidence that ethanol administration can induce glutamate release in multiple brain regions, such as the anterior cingulate, the hippocampus, the ventral tegmental area, the prefrontal cortex and the caudate-putamen (Zuo et al, 2007; Shimizu et al, 1998; Xiao et al, 2009; Fliegel et al, 2013). The most commonly studied site is the nucleus accumbens, but the results have been strikingly inconsistent. In an attempt to explain this variability, a recent review by Sarah Fliegel, Rainer Spanagel and their colleagues (2013) pooled most of these studies and identified evidence of a dose-dependent glutamatergic response to ethanol that could broadly be described as biphasic. Their model proposes that, within the rat nucleus

10 accumbens, lower doses (i.e., 2 g/kg or le ss) le ad to increa sed extracellular glutamate concentrations (e.g., Selim & Bradberry, 1996) whereas higher doses (i.e., over 2 g/kg) lead to decrea sed extracellular glutamate concentrations (e.g., Dahchour et al, 1994). Ethanol doses above 3 g/kg tend to lead to high rates of mortality (Moghaddam & Bolinao, 1994a). The above model might well account for many of the seeming discrepancies in the literature. However, there remains considerable variability in the studies included in the review, with some, for exa mple, reporting no significa nt e ffect of ethanol on ext racellular glutamate concentrations (Kashkin & De Witte, 2004) and others reporting results contrary to the model (Dahchour et al, 1996; Yan et al, 1998). There are also studies excluded from the review (as they did not meet the meta-analysis' inclusion criteria) that do not readily align with the model (Carboni et al, 1993; Moghaddam & Bolinao, 1994a). Moreover, differences in accumbal release of glutama te following ethanol administrat ion have also been reported between strains of rats selected for high vs. low behavioural sensitivity to ethanol (Selim & Bradberry, 1996; Piepponen et al, 2002; Quertemont et al, 2002), with high- vs. low-alcohol sensitive strains even displaying opposite glutamatergic responses in one case (Dahchour et al, 2000). In the present study, the strai n of rats was selec te d based on evidence that, compared to other strains, Lewis rats more rapidly acquire the characteristic behavioural and neurochemical aspects of drug-seeking behaviour (Cadoni, 2016). For example, experiments have shown that orally self-administered ethanol is a stronger positive reinforce in Lewis rats than in Fischer rats, the former displaying significantly higher response rates, quantities of ethanol consumed, blood ethanol levels and behavioural activation than the latter (Suzuki et al, 1988). Moreover, in a single study, Lewis rats also showed a larger and more consistent ethanol-induced glutamate response in the nucleus accumbens (Selim & Bradberry, 1996).

11 Beyond this inconsistency with respect to the effect of ethanol on extracellular levels of glutamate in the nucleus accumbe ns, there is also a dearth of information about the transmitter's source (Timmerman & Westerink, 1997). For exampl e, whi le neurons are traditionally thought of as the seat of ne uropharmacological a ctivity, other actors in the nervous system, such as glia, cannot a priori be excluded. Indeed, while research about glia's glutamatergic actions have historically focused on its role in preventing the accumulation of excitotoxic levels of the transmitter by transporter uptake and subsequent catabolic recycling, there is also evidence that they are involved in the tonic release of glutamate via the cystine-glutamate exchanger (Danbolt, 2001; Le Meur et al, 2007). This non-neuronal glutamatergic activity has been proposed to influence baseline extracellular glutamate levels (Baker et al, 2002; Herman & Jahr, 2007) and substance use related behaviours (Scofield & Kalivas, 2014; Lewitus et al, 2016). Gi ven these observati ons, the questi on arises whether acute drug administration-provoked glutamate release might also reflect extrasynaptic activity generated by glial cells. A key difference between the two glial vs. neuronal sources of glutamate is the calcium dependence of neurona l exocytosis. Indee d, this process, involving the docking of the presynaptic vesicle on the cell membrane and subsequent release of neurotransmitter into the synapse depends on calcium (Barclay et al, 2005). As such, calcium depletion is one method that can be used to determine whether changes in neurotransmitter levels reflect astrocytic or exocytotic activity. Consequently, if the glutamatergic response to ethanol is reduced or even eliminated following perfusion with Ca2+-free aCSF, the effect can be attributed, at least in part, to neuronal exocytosis.

12 Ethanol & GABA Gamma-Aminobutyric acid (GABA) is the prima ry inhibitory neurot ransmitter of the mammalian central nervous system, and, like glutamate, exerts its effects through ionotropic as well as metabotropic receptor types. As mentioned previously, the GABAergic system and its ionotropic GABAA receptors in particular are also important neuropharmacological targets of ethanol (Davies, 2003). An early study also seemed to point towards a direct effect of ethanol on extracellula r GA BA concentrations (Gordon, 1967), but subsequent research failed to replicate the findings and ultimately displayed significant variability (for review, see Kelm et al, 2011). More recent studies using microdialysis to observe extracellular GABA release in vivo show that et hanol can produce dec reases in extracell ular GABA concentrations in a few select area s but fai ls to elicit a GABAergic response in most investigated brain regions (Kelm et al, 2011), including the nucleus accumbens (Heidbreder & De Witte, 1993; De Witte et al, 1994; Smith et al, 2004). This noted, other studies using intracellular recordings have raised the possibility that ethanol may indeed elicit GABAergic responses, though in a more complex and indirect fashion involving other neurobiological actors such as serotonin and opioid receptors (Xiao & Ye, 2008; Theile et al, 2009). Crucially though, as with glutamate, factors such as strain and dose might be important. For example, strain studies have shown that ethanol-induced modulation of extracellular GABA levels in the nucleus accumbens was restricted to alcohol-dependent (Dahchour & De Witte, 2000) as well as alcohol-tolerant (Piepponen et al, 2002) rat lines. It would also be of interest to invest igate the dose-dependency of both glutamate rgic and GABA ergic responses to ethanol administration as well as the effects of prior use histories.

13 Rationale Specific Aims 1. Ethanol challenge: To de termine whether a glutamate rgic respons e to low-dose ethanol exists, concentrations of extracellular accumbens glutamate will be measured with microdialysis following the intraperitoneal administration of saline and 0.5g/kg ethanol. 2. Calcium control: To determine the calcium dependency of the hypothesized ethanol-induced glutamatergic response, concentrations of extracellular accumbens glutamate will be measured with microdialysis following the intraperitoneal administration of 0.5g/kg ethanol during a perfusion with Ca2+-free aCSF. Hypotheses Based on the lite rature summarized above, we hypothesize that low-dose ethanol administration will induce glutamat e release in the nucleus accumbens and that calcium depletion will abolish this effect.

14 Methods Subjects Male Lewis rats (obtained from Charles River, St.-Constant, Canada) were received in our animal facility on postnatal day (PND) 60 and housed under controlled conditions of light (12:12h light/dark cycle), temperature (24 -26 °C) and humidity (70 - 80%), with food and water available ad libitum. All procedures were approved by the Animal Care Committee at McGill University in accordance with the guidelines of the Canadian Council on Animal Care (CCAC). Since previous studies have found evidence of glutamate effects with four to nine rats per group; we planned to test eight rats in each condition. Cannulation At PND 67, after being given a week to habituate to the animal facility, animals were brought to the operating room to have the microdialysis guide cannula surgically inserted. They were placed in an anesthesia chamber and administered 2% sodium isoflurane at 5 L/min alongside a 2 L/min oxygen flow until anesthesia, after which they were weighed, shaved and placed on a stereotaxic apparatus. Anesthesia was maintained in the stereotax by a constant flow of isoflurane at a rate of 3-4 L/min. Artificial tears were applied to prevent ocular dryness. The target area was disinfected using iodine and an incision was made. The scalp was affixed using forceps and the underlying tissue was removed and the skull cleaned using peroxide. Saline (2 mL/kg) and carprofen (1 mL/kg) we re then i njected subcut aneously to prevent excessive dehydration and provide post-operative analgesia, respectively. The target location for the left nucleus accum bens was then calcula ted using the following coordinates: anteroposterior (AP): +1.20 mm; mediolateral (ML): -1.40 mm (Paxinos & Watson, 1998). Apertures for the cannula and three a nchor screw s were drille d into the skull using a handheld drill (Dremel ®), following which the screws were threaded into the cranium. The

15 22-gauge stainless steel guide cannula (Plastics One) was then inserted 6.50 mm along the dorsoventral (DV) axis so that the cannula tip be placed on the edge of the left nucleus accumbens core [Figure 1]. The cannula was then secured to the screws using acrylic dental cement to be anchored to the skull. A stainless steel obturator extending 2.0 mm beyond the end of the cannula was inserted to prevent infection and cerebrospinal fluid leakage. The incision was then sutured shut using ethilon suture kits (Ethicon ™) and a topical antibiotic ointment (Polysporin ®) was applied to the wound. The animals were allowed one week for recovery, housed one per cage, before testing; carprofen in edible gel form was used for three days to address postoperative analgesia. Microdialysis probes I-shaped microdialysis probes were assembled, constructed from side-by-side fused silica inlet-outlet lines [internal diameter (ID): 50 μm] encased in polyethylene tubing (ID: 0.58-0.38 mm). A length of regenerated, hollow cellulose membrane (Spectrum, molecular weight cutoff: 13 kDa, OD: 216 μm; ID: 200 μm) was secured to the end of a 26-gauge stainless steel cannula using cyanoacrylate adhesive and was sealed with epoxy. A stainless steel collar fitted to the probes provided a secure, threaded connection to the animals' indwelling guide cannula. The probes extend 2 mm beyond the end of the cannulae with 0.2 mm of inactive membrane at the top, where the probe is glued to the stainless steel cannula and 0.5 mm of inactive membrane at the bottom, where the probe is sealed with epoxy resin. This leaves an active membrane spanning 1.3 mm between -6.70 and -8.00 mm relative to the skull surface [Figure 1]. Probes were calibrated in artificial cerebrospinal fluid (26 mM NaHCO3, 0.5 mM NaH2PO4, 1.3 mM MgCl2, 2.3 mM CaCl2, 3.0 mM KCl, 0.126 mM NaCl, 0.2 mM L-ascorbic acid) containing 100 ng/ml aspartate, glutamate, and GABA. In vitro probe recovery ranges

16 from 14% to 19% at a flow rate of 2 μL/min., and the dialysate was collected from the fused silica outlet line (dead volume: 0.79 μL). Microdialysis The procedure was performed in awake, free ly moving animals. Following a 20-minute habituation period, the microdialysis probe was inserted into the animal's indwelling guide cannula and affixed to a stainless steel spring tethered to a liquid swivel (CMA). A syringe filled with sterile artificial c erebrospinal fluid at room tempera ture was secured to a computer-controlled microinfusion pump (CMA), used to deliver the perfusate to the probe. Perfusion of aCSF at a flow rate of 1μL/min then began. Dialysate accumulates in collection tips at 20-min intervals and each 20μL sample was then transferred to a vial preloaded with 1μL of 0.25 M perchloric acid (HClO4) to prevent degradation and immediately stored at 4°C for subsequent chromatographic peak analysis. Ethanol challenge The study parameters, particularly time to glutamate concentration stabilization, were based on the existing literature and pilot testing. As reported by the majority of previous studies (Carboni et al, 1993; Dahchour et al, 1994; Selim & Bradberry, 1996; Kashkin & De Witte, 2004; see Table 1 for summary), we found that the experimental setup requires around 120 minutes following probe insertion and aCSF perfusion for glutamate levels to stabilize. Once glutamate levels had stabiliz ed, three samples w ere collected to determine baseline extracellular glutamate concentrations, as in all previous studies [Table 1]. Each animal then received an intrape ritoneal injection of 0.5 g/kg saline and five fractions were collected , followed by an intraperitoneal injection of 0.5 g/kg 20% ethanol. Thi s dose is roughly equivalent to a blood alcohol level (BAL) of 60 mg/dL, in the range of "a low intoxicating

17 BAL" according to Spanagel (2009). Although for statistical purposes the acute effect of ethanol was circumscribe d to glutamate levels within the first two hours (six fra ctions) following its administration, we collected samples for three hours and forty minutes after injection to ensure that delayed effects would be captured [Figure 2]. Calcium control The calcium dependency of the ethanol-induced glutamate response was tested two weeks after using Ca2+-free aCSF, wherein CaCl2 was replaced with an equimolar concentration of MgCl2 (final concentration: 3.6 mM). The paradigm was similar to the ethanol challenge microdialysis described above. However, after collecting four baseline samples, the perfusate was changed from aCSF to Ca2+-free aCSF using a liquid switch (CMA), following which four fractions were collected. After the animal received an intraperitoneal injection of 0.5 g/kg 20% ethanol, eleven fractions were collected, as above [Figure 3]. Analysis of Lysate Glutamate levels were determined using a high-performance liquid chromatography (HPLC) precolumn derivatizati on with fluorescence detection (FD). The chromatographic system consists of a pump and an autosampler (ultimate 3000 RS) coupled to a Waters Xterra MS C18 3.0 mm x 50mm 5μm analytical column. The mobile phase was prepared as needed and consists of 3.5% acetonitrile, 20% methanol, and 100 mM sodium phosphate dibasic adjusted to pH 6.7 with 85% phosphoric acid. Flow rate was set at 0.5 mL/min. Working standards (100 ng/mL) and derivatization reagents were prepared fresh daily from stock solutions and loaded with samples into a refrigerated (10°C) Thermo scientific autosampler (ultimate 3000 RS). Before injection onto the analytical column, each fraction was sequentially mixed with 20 μL of o-phthaldehyde (0.0143 M) diluted with 0.1 M sodium tetraborate and 20 μL of 3-

18 mercaptopropionic acid (0.071 M) diluted with H2O and allowed to react for 10 minutes. After each injec tion, the inject ion loop was flushed with 20% met hanol to prevent contamination of subsequent samples. Under the se conditions, the retention time for glutamate was 2.4 minutes with a total run time of 30 minutes/sample. Fluorescence detection was achieved usi ng a fluoresce nce detector (ultimate 3000 RS) wit h excitation 323 and emission 455. Chromatographic peak analysis was accomplished by identification of unknown peaks in a sample matched according to retention times from known standards using Chromeleon 7 software. Histology After microdialysis, animals were euthanized and decorticated. Their brains were flash-frozen in a bea ker of 2-methylbutane and stored in a -80°C freezer. Using a cryostat (Leica Biosystems ©), the brains were sliced in 30 μm-thick coronal samples and stored in a -10°C freezer. The samples were then treated with cresyl violet (Nissl staining), yielding stained slices [Figure 4] that were coded for probe placement using a microscope (Leica Biosystems ©). Probe placement coding was tested for inter-rater reliability by two technicians. Statistical Analysis The effects of ethanol administration were tested using a one-way repeated measures analysis of variance (rmANOVA) with time as the within-group factor containing 17 levels (pooled baseline and each subsequent fraction; five saline samples and eleven ethanol samples). In light of the embedded factor arising from the repeated measures model, subsequent analyses delineated the three conditions (baseline, saline and ethanol). Average values were extracted for each condition (baseline: B1-3; saline: S1-5; ethanol: E1-6) and compared using a one-way rmANOVA with condition as the within-group fac tor containing three levels. Both

19 ANOVAs contained tests for violations of sphericity; Huynh-Feldt corrections were applied when necessa ry. Bonferroni-corrected paired t-tests were then used to compare the three experimental conditions. In cases where the ANOVA and follow-up tests were found to point in the direction of our hypothesis, that is, indicate a significant effect of ethanol on extra-cellular glutamate concentrations, six Bonferroni-corrected paired t-tests were then applied to determine if any individual ethanol fracti on was significantly different from the saline condition. We also calculated the average of the individual peak responses as well as the peak percent of baseline, defined as the percent value of the highest divergence from baseline of average extracellular glutamate concentrations, and the time at which it occurs. Experimental effects of calcium depletion were tested in a similar fashion, using a one-way rmANOVA with time as the within-group factor containing 16 levels corresponding to a pooled baseline (four samples) and each subsequent fra ction (four Ca2+-free baselines samples and eleven Ca2+-free ethanol samples) and a one-way rmANOVA with condition as the within-group factor containing the three conditions (baseline: B1-4, Ca2+-free baseline: S1-4 and Ca2+-free ethanol: E1-6). In cases where the ANOVA results were significant after having applied Huynh-Feldt correction for event ual violations in spherici ty, fol low-up Bonferroni-corrected paired t-tests were used to direct ly compa re the three experimental conditions, and six Bonferroni-corrected paired t-tests were applied to determine if any individual Ca2+-free ethanol fraction was significantly different from the Ca2+-free baseline condition.

20 Unless stated otherwise, the statistical analyses were performed using the raw data expressed in µg/mL; the graphic representations of the data were expressed as the mean (+/-SEM) percentage change in glutamate levels relative to the pooled average of the three baseline samples. Analyses were performed using SPSS software (version 23, IBM®). Differences were considered statistically significant at p < 0.05.

21 Results Histology Figure 5 depicts the probe placement of rats displaying clear tissue lesions consistent with cannula and probe inserti on. Of the ni ne rats that had succes sful cannulation and microdialysis (on a total cohort of fourteen rats), histological analysis indicated that two had misplaced cannulae, both more dorsally placed. Indeed, the active membrane was most often between -5 and -7 mm relative to the skull surface, rather than -6.7 and -8 mm as targeted. Of the seven rat s included in the final analyses, five displayed clear probe placement. The remaining two rats displayed lesions suggestive of correct probe placement but did not yield unequivocal coding. Ethanol-induced glutamate response Figure 6 depicts the percent change from baseline (B1-3) of ext racellular glutamate concentrations in the nucleus accumbens dialysate, at baseline, following intraperitoneal injection of 0.5 g/kg saline, and following intraperitoneal injection of 0.5 g/kg ethanol in a sample of seven rats. Repeated measures analyses of variance revealed main effects of ti me (Huynh-Feldt F(4.891,29.346) = 2.901; p = .035) and condition (Huynh-Feldt F(1.240,7.439) = 10.621; p = .010) on e xtracellular concentrations of glutamate within the nucleus ac cumbens. Holm-Bonferroni-corrected paired t-tests indicated that these effect s reflected ethanol-induced increases in glutamate levels, as compared to baseline (t(6) = 3.372; p = .045) and saline control (t(6) = 3.322; p = .048) conditions. As expected, no differences were seen between the baseline and saline conditions (p > .50). Subsequent paired t-tests contrasting individual

22 ethanol fractions to the average s aline response revealed that one fraction, E3, the third ethanol sample collected between 40 minutes and a n hour after ethanol administ rati on, approached significance (t(6) = 2.430; p = .051, uncorrected). The average pe ak response was 246 ± 21.3% of baseline. The peak perce nt of baseli ne reached 190.5 ± 34.7% of baseline during E3. Ethanol-induced behavioural effects The behavioural effects of ethanol administration were not evaluated quantitatively in this study; our observations, however, tended to fall in line with those described in Moghaddam & Bolinao (1994) and earlier studies, with a general increase in behavioural activation such as locomotion and grooming following the injection of 0.5 g/kg of ethanol. This phenomenon was of greater magnitude and duration than the arousal induced by the earlier saline injection manipulation. Calcium control Figure 7 depicts the percent c hange from baseline (B1-4) of e xtrac ellular glutamate concentrations in the nucleus accumbens dialysate, at baseline, following calcium depletion, and following intraperitoneal injection of 0.5 g/kg ethanol in a sample of four rats. Neither rmANOVA yielded significant effects, indicating that ethanol failed to elicit a glutamate response when calcium levels were depleted. Ethanol-induced GABA response Figure 8 depicts the percent c hange from baseline (B1-3) of e xtrac ellular GABA concentrations in the nucleus accumbens dialysat e, at basel ine, following intraperit oneal

23 injection of 0.5 g/kg saline, and following intraperitoneal injection of 0.5 g/kg ethanol in a sample of seven rats. Neither rmANOVA yielded significant effects, indicating that ethanol failed to elicit a GABA response in the nucleus accumbens.

24 Discussion The present study lends support to our first hypothesis that administration of 0.5 g/kg ethanol to awa ke freely moving rats increases extra-cellular glutamate levels in the nucleus accumbens. These data align well with the increases predicted by a meta-analysis (Fliegel et al, 2013 [Fig. 3]). Extrapolating from their trend line constructed from peak percent increase following the administration of 1, 2 and 3 g/kg ethanol, one would expect a peak percent of baseline to be around 200% for 0.5 g/kg. The peak percent of baseline reported here was 190.5%. A plurality (43%) of animals displayed peaks in extracellular glutamate concentrations during the third ethanol sample (40 to 60 min), but individual variability in this response was noted and peak responses were present in each of the first five fractions. As a consequence, the average peak response, irrespective of sample, was larger than the group average peak (246 ± 21.3% vs. 190.5 ± 34.7%). All peaks in extra-cellular glutamate concentrations were found within the first five ethanol samples, other than a single animal whose extracellular glutamate concentration at E11 was minimally higher than its peak during the first five ethanol samples. This corroborates our choice to delimit the ethanol condition to the first two hours after drug administration. This also provides an explanation so as to why no significant increases in extra-cellular glutamate were observed in any individual ethanol fraction. Finally, this raises the possibility that the predictions made in the meta-analysis based on average values rather than raw data might be underestimating the average peak response by failing to take into account individual variability in the timecourse of the glutamatergic response to ethanol.

25 The data presented above also support our second hypothesis that calcium depletion abolishes the glutamatergic effect of ethanol in the nucleus accumbens. This novel finding suggests that the ethanol-induced glutamate response observed here and elsewhere is dependent on the calcium-dependent synaptic release of the neurotransmitter, pointing towards the neuronal source of the glutamatergic effect of ethanol. To further bolster our novel finding regarding the source of the ethanol-induced glutamate response, it would be informative to compare our results with the effects of tetrodotoxin (TTX) and (S)-4-carboxyphenylglycine (CPG) (Lupinsky et al, 2010). TTX is a sodi um channel blocker that inhibits the firing of action potentials (Narahashi et al, 1964). A TTX control could plausibl y shed further l ight on whether this neuronal a ctivity is impulse dependent - that is, reliant on action potentials. CPG, on the other hand, is a glial cystine-glutamate exchanger blocker that prevents glial glutamate release. While the abolition of a glutamatergic peak following calcium depletion does suggest that this phenomenon relies at least in part on calcium-dependent exocytotic glutamate release at the level of the synapses, a CPG control would allow us to determine more conclusively what role - if any - glial activity plays in ethanol-induced glutamate release, given that the two are not necessarily mutually exclusive. A limitation of these pilot experiments involves the paradigm. Since it was designed with the longer-term objective of testing multiple doses, the internal saline control was selected to best control for inter-individual differences while minimizing the number of animal s used. However, this internal control generated a methodol ogical confound, in that et hanol administration always followed a first s aline injection. As a c onsequence, the effect of repeated stress exposure or intraperitoneal injection as well as the diurnal passage of time

26 could account for some of the variability in extracellular glutamate concentrations observed. One way to address this issue would have been to counterbalance the order of ethanol and saline administration or to test a group of rats being administered saline a second time rather than receiving an ethanol injection. A similar limitation, concerning only the second experiment, is that the animals used for the calcium depletion had also been tested in the first experiment. It is possible therefore, that the absence of a neurochemical effect of ethanol on the second test day reflected a tolerance-like effect rather than an ability of the calcium depletion to prevent the ethanol response. This too should be tested in future experiments. This noted, experiments in behavioural tolerance to alcohol do provide some perspective. Such tolerance has only been demonstrated following longer exposures and at higher doses, and there exists no evidence substantiating t he development of tolerance to ethanol a fter a single administration or with t he low doses administered here (Kalant et al, 1971). While there exists electrophysiological evidence that chronic ethanol exposure has an effect on synaptic strength at the level of dopaminergic neurons in the mesolimbic system (Stuber et al, 2008; Bernier et al, 2011), indications of such an effect following single acute ethanol exposure remain sparse. One electrophysiological study found that acute ethanol exposure led to the potentiation of glutamatergic synapses on midbrain dopamine neurons when observed 24 hours later (Saal et al, 2003), while a later study reported opposite findings (Wanat et al, 2011). Studies investigating the effect of other substances, namely cocaine, a mphetamine, morphine and nicotine, on m esol imbic glutamatergic neurotransmission have shown that these produce long-term potentiation, though this effect did not last longer than a week (Ungless et al, 2001; Saal et al, 2003), while others investigating the effect of ethanol on mesolimbic GABAergic transmission have found that a single ethanol exposure can induce changes in GABAergic activity also lasting up to a

27 week (Melis et al, 2002; Wanat et al, 2011). It remains to be seen however whether acute ethanol exposure would provoke changes in glutamatergic neurotransmission lasting the two weeks separating the first and second experiments in the present study. The mechanis m by which ethanol promotes glutamate remains poorly understood. One plausible mechanism is through its effects as a negative modulator of NMDA receptors. Both the non-competitive NMDA receptor antagonist dizocilpine (MK-801) and the competitive antagonist (2R)-amino-5-phosphonovaleric acid (AP5) increase extracellular glutamate levels in the striatum (Bustos et al, 1992; Liu & Moghadda m, 1995. Ke tamine, anot her non-competitive NMDA receptor antagonist, seems to produce the same dose-dependent effect on extracellular glutamate levels as has been proposed with ethanol, w ith low (or 'subanesthetic') doses producing increases in glutamate levels and higher ('anesthetic') doses producing decreases (Moghaddam et al, 1997). The increase in extracellular glutamate levels seen at subanesthetic doses has been attributed to AMPA receptor activation (Maeng et al, 2008; Duman et al, 2012). It has be en proposed, by m eans of linking NMDA rece ptor antagonism to AMPA receptor activat ion and consequent glutamate efflux, tha t NMDA receptors located at synaptic si tes might be principally involved in tonic i nhibition of glutamate neurotransmission via excitatory projections to inhibitory GABAergic interneurons. Thus , antagonism of these rec eptors would block spontaneous GABAergic neurotransmission and therefore ultimately disinhibit glutamatergic neurotrans mission (Homayoun & Moghaddam, 2007). A similar mechanism could be imagined following the administration of low doses of ethanol. The behavioural significance of ethanol-induced glutamate release within the nucleus accumbens will require additional study. As mentioned previously, the behavioural effects of

28 low-dose ethanol administration include anxiolysis, sociability and emotional lability. The latter two are more abstract concepts difficult to study in laboratory rodents. The anxiolytic effects of pharmacological substances has been a more fruitful area of study, and there is evidence that a number of NMDA receptor ligands, mainly antagonists, decrease anxiety-like behaviours (Plaznik et al, 1994; Wierońska & Pilc, 2013). There is also some evidence for an anxiolytic effect of glutamate at targets farther downstream, particularly group I metabotropic glutamate receptors (Palucha & Pilc, 2007). These observations rais e the possibility that anxiolytic and other disinhibiting and euphoric properties of low-dose ethanol administration are related to NMDA antagonism-induced glutamate release. As noted earlier, there exist extensive glutamatergic projections to the nucleus accumbens, some of which synapse onto mesolimbic dopaminergic system afferents and are essential for synaptic plasticity. Moreover, the expression of certain metabotropic glutamate receptors in the nucleus accumbens has been implicated in the rewarding effects of compulsively self- administered substances. The se two factors constitute a putat ive mechanism by whi ch ethanol-induced glutamate release could influence the development of the motivational salience and positive reinforcement essential to the development and persistence of drug-seeking behaviours.

29 Summary & Conclusions This investigation sought to characterize the effects of ethanol on glutamate release in the nucleus accumbens. The experiments provide new evidence that ethanol administration, at a low dose of 0.5 g/kg, produces increases in extracellular glutamate levels in the nucleus accumbens, as predicted by a recent review. The experiments described here also suggest that this glutamatergic response to ethanol is sensitive to calcium depletion, pointing to a neuronal origin of the effect that demands further investigation. Finally, we have also shown that, at the low dose tested here, ethanol does not affect nucleus accumbens extracellular GABA concentrations. Together, these neurobiological effects might make important contributions to the behavioural profile elicited by low-dose ethanol ingestion.

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42 Figures Table 1: Study parameters of previous publications. STR: striatum; NA: nucleus accumbens; AP: anteroposterior; ML: mediolateral; VD: ventrodorsal; i.p.: intraperitoneal Figure 1: Schematized cross-section of a rat brain indicating the target area for cannula implantation (grey with solid border) and probe placement including both active (grey with dotted border) and inactive (black) membrane (overlay on Paxinos & Watson, 1998). AcbC: nucleus accumbens core; AcbSh: nucleus accumbens shell; CPu: caudate-putamen; aca: anterior part of the anterior commissure

43 Figure 2: Microdialysis procedure design - ethanol challenge Figure 3: Microdialysis procedure design - calcium control Figure 4: Microphotograph of 30 μm-thick coronal slice stained with cresyl violet

44 Figure 5: Schematized cross-section of a rat brain indicating target (red line), correctly placed (black line) and misplaced (grey lines) probes (overlay on Paxinos & Watson, 1998). AcbC: nucleus accumbens core; AcbSh: nucleus accumbens shell; CPu: caudate-putamen; aca: anterior part of the anterior commissure.

45 Figure 6: Percent change from baseline (B1-3) of extracellular glutamate concentrations in the nucleus accumbens quotesdbs_dbs35.pdfusesText_40