Jun Wang

Maria Fe Lanfranco

Stuart L Gibb

Quinn V Yowell

Sebastien Carnicella

Dorit Ron
email: dron@gallo.ucsf.edu

addressed. Dorit Ron

Wang

Long-Lasting Adaptations of the NR2B-containing NMDA Receptors in the Dorsomedial Striatum Play a Crucial Role in Alcohol Consumption and Relapse

Introduction

Increasing evidence in recent years suggests that the dorsal striatum plays an important role in the development of compulsive drug seeking and taking [START_REF] Gerdeman | It could be habit forming: drugs of abuse and striatal synaptic plasticity[END_REF][START_REF] Everitt | Neural systems of reinforcement for drug addiction: from actions to habits to compulsion[END_REF][START_REF] Hyman | Neural mechanisms of addiction: the role of rewardrelated learning and memory[END_REF]. The dorsal striatum can be divided into two parts: the DMS and the DLS, which differ in connectivity, receptor distribution, synaptic plasticity and behavioral functions [START_REF] Joel | The connections of the dopaminergic system with the striatum in rats and primates: an analysis with respect to the functional and compartmental organization of the striatum[END_REF][START_REF] Gerdeman | It could be habit forming: drugs of abuse and striatal synaptic plasticity[END_REF][START_REF] Voorn | Putting a spin on the dorsal-ventral divide of the striatum[END_REF][START_REF] Yin | The role of the basal ganglia in habit formation[END_REF][START_REF] Belin | Parallel and interactive learning processes within the basal ganglia: relevance for the understanding of addiction[END_REF]. In rodents, the DMS receives inputs primarily from several cortical areas, the medial part of the substantia nigra pars compacta (SNc), the ventral tegmental area and the basolateral amygdala, whereas the DLS receives inputs from the sensorimotor cortex and the lateral part of the SNc [START_REF] Mcgeorge | The organization of the projection from the cerebral cortex to the striatum in the rat[END_REF][START_REF] Joel | The connections of the dopaminergic system with the striatum in rats and primates: an analysis with respect to the functional and compartmental organization of the striatum[END_REF][START_REF] Reep | The associative striatum: organization of cortical projections to the dorsocentral striatum in rats[END_REF][START_REF] Voorn | Putting a spin on the dorsal-ventral divide of the striatum[END_REF]. Moreover, long-term potentiation (LTP) is preferentially expressed in the DMS as opposed to the DLS, in which long-term depression (LTD) is more commonly found [START_REF] Partridge | Regional and postnatal heterogeneity of activitydependent long-term changes in synaptic efficacy in the dorsal striatum[END_REF]. Interestingly, greater levels of CB1 cannabinoid receptors, which have been shown to promote LTD [START_REF] Gerdeman | Postsynaptic endocannabinoid release is critical to long-term depression in the striatum[END_REF], are found in the DLS as compared to the DMS [START_REF] Hohmann | Localization of cannabinoid CB(1) receptor mRNA in neuronal subpopulations of rat striatum: a double-label in situ hybridization study[END_REF].

In addition, rodent and human data suggest that the DMS is important for the formation and processing of action-outcome (A-O) associations that mediate goal-directed behaviors, whereas the DLS has been implicated in the development of habit, stimulus-response (S-R), and learning [START_REF] O'doherty | Dissociable roles of ventral and dorsal striatum in instrumental conditioning[END_REF][START_REF] Tricomi | Modulation of caudate activity by action contingency[END_REF][START_REF] Yin | The role of the basal ganglia in habit formation[END_REF][START_REF] Balleine | The role of the dorsal striatum in reward and decision-making[END_REF][START_REF] Tanaka | Calculating consequences: brain systems that encode the causal effects of actions[END_REF][START_REF] Balleine | The integrative function of the basal ganglia in instrumental conditioning[END_REF][START_REF] Tricomi | A specific role for posterior dorsolateral striatum in human habit learning[END_REF]. Interestingly, exposure to amphetamine can enhance both goal-directed and habit formation behaviors in rats [START_REF] Nelson | Amphetamine exposure enhances habit formation[END_REF][START_REF] Nordquist | Augmented reinforcer value and accelerated habit formation after repeated amphetamine treatment[END_REF]. Furthermore, operant response under a procedure that favors the development of habit formation becomes more rapidly habitual in rats trained to orally Wang et al. 5 self-administer cocaine or alcohol (ethanol) compared to a non-drug reinforcer such as sucrose [START_REF] Dickinson | Alcohol seeking by rats: action or habit?[END_REF][START_REF] Miles | Oral cocaine seeking by rats: action or habit?[END_REF]. Together, these studies suggest that the DMS and DLS may be involved in different aspects that lead to the development of addiction.

Previously, we found that acute ex vivo exposure of striatal slices to, and withdrawal from, ethanol results in LTF of NR2B-NMDAR activity in the dorsal but not the ventral striatum [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. Here, we set out to test whether ethanol acts preferentially within a specific subregion of the dorsal striatum to alter the activity of the NMDARs, and determine whether ethanolmediated LTF of NR2B-NMDAR plays a role in mechanisms underlying increased motivation to consume ethanol.

Materials and Methods

Reagents

The monoclonal anti-NR1 and anti-NR2B antibodies, and polyclonal anti-[pY1472]NR2B antibodies were obtained from Cell Signaling Technology (Beverly, MA). The polyclonal anti-NR2A, anti-Fyn, anti-NR2B, anti-Actin, anti-GAPDH and the secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The polyclonal anti-[pY418/420]Src/Fyn (for measuring active Fyn) and polyclonal anti-[pY529/531]Src/Fyn (for measuring inactive Fyn) antibodies, NuPAGE 4-12% Bis-Tris gradient gels, and protein G agarose were obtained from Invitrogen (Carlsbad, CA). Bicinchoninic acid (BCA) TM protein assay kit was obtained from Pierce (Rockford, IL). Nitrocellulose membrane was purchased from Millipore (Billerica, MA). 4--Amino--5--(4--chlorophenyl)--7--(t--butyl)pyrazolo[3,4--D]pyrimidine (PP2) was purchased from Calbiochem (La Jolla, CA). 2,3--Dioxo--6--nitro--1,2,3,4-tetrahydrobenzo [f]quinoxaline--7--sulfonamide disodium salt (NBQX), (αR,βS)--α--(4-hydroxyphenyl)--β--methyl--4--(phenylmethyl)--1--piperidinepropanol hydrochloride (Ro 25-6981) and ifenprodil were obtained from Tocris (Ellisville, MO). Sodium orthovanadate, picrotoxin, and phosphatase inhibitor cocktails 1 and 2 were obtained from Sigma (St. Louis, MO). Complete™ mini, EDTA-free protease inhibitor cocktail was purchased from Roche (Indianapolis, IN). Enhanced Chemiluminescence (ECL) TM plus was purchased from Amersham Biosciences (Pittsburg, PA).

Animals

Male Sprague-Dawley rats (3-4 weeks old) and male Long-Evans rats (300 g) were obtained from Harlan (Indianapolis, IN). Animals were housed under a light:dark cycle of 12 hrs, with lights on at 7:00 a.m. and food and water available ad libitum. All animal procedures in this report were approved by the Gallo Center Institutional Animal Care and Use Committee and were conducted in agreement with the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996.

In vivo ethanol administration

Sprague-Dawley rats were injected intraperitoneally (i.p.) with ethanol (20% v/v, 2 g/kg) or saline once a day for 7 or 14 days. Sixteen or 40 hrs after the last injection, striatal slices were prepared for electrophysiological recordings, or striatal tissues were dissected for biochemical measurements.

Blood ethanol concentration (BEC) measurements

Trunk blood was collected in heparinized capillary tubes 15 min after systemic administration of 2 g/kg of ethanol. Serum was extracted with 3.4% trichloroacetic acid followed by a 5-min centrifugation at 420 g (2000 rpm) and assayed for ethanol content using the NAD-NADH enzyme spectrophotometric method [START_REF] Weiss | Oral alcohol self-administration stimulates dopamine release in the rat nucleus accumbens: genetic and motivational determinants[END_REF]Zapata et al., 2006). BECs were determined by using a standard calibration curve. BEC measurements were performed on 3

Sprague-Dawley rats (3-4 weeks old), and the BEC average value was 34 ± 3 mM.

Electrophysiology

Slice preparation. The preparation of striatal slices and whole-cell recordings of striatal neurons were done as previously described [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. Specifically, Sprague-Dawley rats were anesthetized with isoflurane. Coronal sections of the striatum (150 µm) were cut with a vibratome The method for preparation of slices from adult Long-Evans rats was the same as above except for the following: (1) Rats were anesthetized and intracardially perfused with ice-cold cutting solution as described in [START_REF] Wang | Photolysis of postsynaptic caged Ca2+ can potentiate and depress mossy fiber synaptic responses in rat hippocampal CA3 pyramidal neurons[END_REF]; (2) Slices were incubated in a cutting and the external solutions (1:1 in volume) for 45 min before being transferred into the external solution.

Whole-cell voltage-clamp recordings. NMDARs-mediated Excitatory Postsynaptic Currents (NMDAR-EPSCs) were recorded at -70 mV and in the presence of 50 µM Mg 2+ , 10 µM NBQX, and 100 µM picrotoxin and 200 µM sodium orthovanadate in the bath solution [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. To measure NMDA-induced currents, NMDA (10 µM) was bath applied for 30 sec and holding currents were measured every 5 sec. To generate input-output curves for NMDAR-EPSCs, currents responding to increasing intensities of stimulation (3.6 to 7.2 V with an increment of 0.9 V) were recorded, as previously described [START_REF] Palop | Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease[END_REF]. Slices were superfused at 3-4 ml/min, and cells were visualized using infrared differential interference contrast video microscopy (BX50WI, Olympus, Japan). that were placed in the DMS or the DLS and used to stimulate excitatory afferents at 0.05 Hz.

Neurons were voltage-clamped at -70 mV. Data were filtered at 2 kHz, digitized at 5-10 kHz, and collected on-line using pClamp 9 (Molecular Devices, Union City, CA).

DMS and DLS tissue collection and Homogenization

DMS and DLS tissues were collected as described in [START_REF] Jeanblanc | Endogenous BDNF in the dorsolateral striatum gates alcohol drinking[END_REF]. Specifically, after rats were anesthetized by isoflurane and decapitated, brains were removed and one section per animal was cut between +1.70 mm and +0.5 mm anterior to bregma. Following removal of the cortex and the ventral striatum (-6.00 mm below the brain surface), the dorsal striatum was divided into the DLS and the DMS at +2.5 mm from the middle line. DMS and DLS were dissected on a cool (4 o C) tray and immediately homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer (in mM: 50 Tris-Cl, 5 EDTA, 120 NaCl, and 1 % NP-40, 0.1% deoxycholate, 0.5% SDS, protease and phosphatase inhibitors). Samples were homogenized using a sonic dismembrator. Protein content of samples was determined using the BCA TM protein assay kit.

Synaptosomal Membrane preparation

Synaptosomal membranes were prepared as described in [START_REF] Carvelli | PI 3kinase regulation of dopamine uptake[END_REF][START_REF] Daws | Cocaine increases dopamine uptake and cell surface expression of dopamine transporters[END_REF].

Briefly, immediately after being collected, DMS tissue was homogenized in a glass homogenizer containing 200 µl of ice-cold Krebs-sucrose buffer (in mM: 125 NaCl, 1.2 KCl, 1.2 MgSO 4 , 1.2

CaCl 2 , 22 Na 2 CO 3 , 1.2 NaH 2 PO 4 , 10 glucose, and 320 mM sucrose, pH 7.4) in the presence of protease and phosphatase inhibitors. The homogenate was centrifuged at 1,000 g for 10 min at 4°C to pellet heavy membranes and debris (P1). The supernatant (S1) was collected and was centrifuged at 16,000 g at 4°C for 20 min to pellet the synaptosomal membrane fraction (P2). P2

was re-suspended in 100 µl RIPA buffer. Total protein concentration was determined using the BCA TM protein assay kit.

Immunoprecipitation

DMS tissues (n = 3 rats per treatment) were homogenized in 1x immunoprecipitation (IP) buffer (in mM: 150 NaCl, 10 Tris-HCl, 1 EDTA, 1 EGTA, 1% Triton X-100, pH 7.4) in the presence of protease and phosphatase inhibitor cocktails. DMS homogenates were pre-cleared by incubation with protein G agarose for 1 hr at 4°C. Samples were then centrifuged and protein quantity was determined using the BCA TM protein assay. IPs were performed by combining 5 µg of the appropriate antibody with approximately 1 mg protein diluted in 1 × IP buffer to a total volume of 1 ml. Following overnight incubation at 4°C, protein G agarose was added and the mixture was incubated at 4°C for 4 hrs. The protein G was washed extensively with 1 x IP buffer and pellets were re-suspended in 25 µl of 2x Laemmli buffer and incubated at 95°C for 10 min. The resulting supernatant was separated by SDS-PAGE using 10% BisTris pre-cast gels for 2 hrs at 130V.

Western blot analysis

Samples were separated on NuPAGE 4-12% Bis-Tris gradient gels at 130V for 2-3 hrs unless stated otherwise. After overnight transfer at 4 o C onto nitrocellulose membranes, blots were blocked in 5% milk/TBST before probing with specific antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Blots were visualized using ECL plus and a Storm® 860 imagining system (Molecular Dynamics Inc, Sunnyvale, CA). Results were quantified with the Scion Image-Analysis Software (Scion Corporation, Frederick, MD).

Intermittent access 2-bottle choice procedure

The procedure is similar to the one described in [START_REF] Carnicella | Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor[END_REF]. Briefly, Long-Evans rats were given 24-hr concurrent access to 1 bottle of 20% ethanol in water and 1 bottle of water starting at 11:00 a.m. on Monday, Wednesday and Friday, with 24 or 48 hrs ethanol-deprivation periods in-between the ethanol-drinking sessions. The placement (left or right) of each solution was alternated between each session to control for side preference. The water and ethanol bottles were weighed after 24 hrs of access. Experiments started after a stable baseline of consumption was achieved (average of 5-6 g/kg/24 hrs, Supplemental Table 1). The control group had access to 2 bottles of water throughout the duration of the experiment.

Operant ethanol self-administration after history of high voluntary ethanol consumption

Long-Evans rats were first trained to voluntary consume high levels of ethanol using the intermittent-access 20% ethanol (v/v) 2-bottle choice drinking procedure as described above.

After achieving a stable baseline of consumption, rats were trained to self-administer a 20% ethanol solution in an operant self-administration chamber. The chambers contain two levers: an active lever, for which presses result in delivery of 0.1 ml of the ethanol solution, and an inactive lever, for which responses are counted but no programmed events occur. After 2-3 nights in the chamber to allow acquisition of lever-press response for ethanol under a fixed ratio 1 (FR1), operant sessions were conducted 5 days per week, with the schedule requirement increased to FR3 and the length of session shortened from 60 to 30 min over the first 2 weeks. Because the level of presses on the inactive lever was extremely low after acquisition of the selfadministration procedure (< 10 presses), and the activity on this lever was not affected by any of the experimental treatments, this measure was excluded from the figures and the analysis for better clarity. After 1 month of training, surgery was conducted to implant cannulae.

Operant self-administration test in extinction and reinstatement of ethanol-seeking

The following behavioral manipulations began after acquisition of a stable operant responding for ethanol as described above.

Test in extinction. Rats experienced a single 30 min session of extinction per week in which responses on the ethanol lever were counted but no ethanol was delivered.

Reinstatement of ethanol-seeking. Rats underwent a minimum of 9 extinction sessions until the average of active lever presses across 3 consecutive extinction sessions was lower than 10. The following session, reinstatement of ethanol-seeking, was induced by the non-contingent delivery of 0.2 ml of 20% ethanol into the reward port immediately at the beginning of the session, followed by an additional 0.1 ml of 20% ethanol after the completion of the first 3 presses on the ethanol lever. During the remainder of the 30-min session, subsequent responses on the active lever were counted but no ethanol was delivered. This oral ethanol priming is not sufficient to produce a pharmacological effect (intake < 0.1 g/kg), however enables it access to the exteroceptive properties of ethanol (i.e., taste and odor) and has been shown to reinstate leverpressing on the ethanol lever [START_REF] Carnicella | GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse[END_REF][START_REF] Chaudhri | Reinstated ethanol-seeking in rats is modulated by environmental context and requires the nucleus accumbens core[END_REF]. The experiment was conducted by using a counterbalanced within-subjects design (resulting in 3 tests per subject) and as such, reinstatement tests were followed by 1 week of reacquisition of ethanol selfadministration and then further extinction sessions before the next reinstatement test [START_REF] Carnicella | GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse[END_REF]. This procedure of reacquisition between 2 extinction and reinstatement procedures prevents any decrease in the efficacy of the prime due to repeated exposure, as the level of reinstatement for the vehicle condition was similar during the 3 tests (data not shown).

Operant sucrose self-administration

Long-Evans rats were initially trained under FR1 using 8% sucrose (w/v) during 2 overnight sessions. The FR schedule was then progressively increased to FR3, and the sucrose concentration was progressively decreased to 2.5%, while the length of the session was shortened to 30 min. These parameters were chosen in order to have a lever-press activity similar to that observed in operant ethanol self-administration.

Surgery and microinjection of PP2 and ifenprodil

Long-Evans rats were anesthetized continuously with isoflurane (Baxter Health Care Corporation, Deerfield, IL). Bilateral guide cannulae (C235G, 26 ga, Plastics One, Roanoke, VA)

were implanted into the DMS (1.2 mm anterior to bregma, 1.5 mm mediolateral, 4.2 mm ventral to bregma) or into the DLS (1.2 mm anterior to bregma, 3.4 mm mediolateral, 4.2 mm ventral to bregma), according to [START_REF] Paxinos | The Rat Brain in stereotaxic coordinates, four Edition[END_REF], and were based on [START_REF] Corbit | Inactivation of the lateral but not medial dorsal striatum eliminates the excitatory impact of Pavlovian stimuli on instrumental responding[END_REF].

Drug or vehicle was administered via an injection cannulae extending 0.5 mm beyond the guide cannula tip. The volume infused with a Hamilton 25 µl syringe (# 1702) was 1 µl/side at a rate of 0.5 µl/min, with the injector kept in position for an additional 2 min. All subjects received each Wang et al. 14 drug in a counter-balanced fashion, with at least 1 week between 2 infusions. The doses used were 0.3 and 1.5 ng/µl for PP2, and 0.5 and 2.5 µg/µl for ifenprodil. PP2 and ifenprodil were initially dissolved in DMSO and brought to a final concentration of 1% DMSO with PBS. Doses and time of infusion of PP2 and ifenprodil were based on [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF].

Histology

Locations of cannulae were verified in 60-μm coronal sections of paraformaldehyde-fixed tissue stained with thionin. Only data from subjects with cannulae located in the region of interest were included in the analysis (Supplemental Figure 1).

Statistical analysis

All data are expressed as mean ± SEM unless stated otherwise.

Statistical analyses for electrophysiological data were performed using two-way ANOVA for 2D, 6B, and 6D, the ethanol treatment was analyzed as a between-subject factor and the stimulation intensity as a within-subject factor, which consists of 5 different stimulation intensities (from 3.6 to 7.2 V with an increment of 0.9 V).

For the biochemical analyses, the ratio of the intensity of the immunoreactive band of interest to GAPDH was determined for each sample to normalize for the amount of protein loaded, and the resultant data were analyzed using unpaired, two-tailed t-test. When data did not conform to a normal distribution, the analyses were conducted using the Mann-Whitney Rank Sum Test.

Behavioral data were analyzed using a one-way ANOVA with repeated measures (one-way RM-ANOVA), followed by SNK test when indicated. As the latency data to the third press (i.e., first reward) did not conform to a normal distribution, the analyses were conducted using the Friedman repeated-measures ANOVA on ranks.

Results

Acute ethanol exposure and withdrawal upregulates the NMDAR activity in the DMS

We first determined whether acute exposure of striatal slices to, and withdrawal from, ethanol differentially alters the activity of NMDAR in the DMS and the DLS. After a stable recording of NMDAR-mediated excitatory post-synaptic currents (EPSCs) for 10 min, ethanol (40 mM) was bath applied for 15 min, then washed out (withdrawal) for another 30 min, and the EPSCs were continuously measured. As shown in Figure 1A, the NMDAR-EPSCs were reduced in the presence of ethanol [P < 0.001 vs. baseline in the DMS and DLS], and the amplitude of NMDAR-mediated EPSCs was greatly enhanced immediately after ethanol withdrawal in both subregions. However, the EPSCs remained elevated in the DMS (P < 0.001 vs. baseline), but not in the DLS (P = 0.48 vs. baseline) for 30 min after ethanol withdrawal (Figure 1A). The analysis of NMDAR-EPSCs by two-way RM-ANOVA further reveals a significant interaction between the brain region and the ethanol treatment (F 1,20 = 4.47, P < 0.05, see Methods). Post hoc comparison by SNK tests reveals that NMDAR-EPSC amplitudes are different between the DMS and the DLS 30 min after ethanol washout (post-EtOH, P < 0.01), but not during ethanol application (EtOH, P = 0.29). This result therefore suggests that exposure of striatal slices to, and withdrawal from ethanol induces LTF of NMDAR activity in the DMS, but not in the DLS.

Next, we tested whether the differential effects of ethanol exposure on the activity of the NMDAR in the subregions of the dorsal striatum are due to different protein levels of the NR2A and/or the NR2B subunits, the major regulatory NR2 subunits in the dorsal striatum [START_REF] Monyer | Heteromeric NMDA receptors: molecular and functional distinction of subtypes[END_REF][START_REF] Standaert | Organization of N-methyl-Daspartate glutamate receptor gene expression in the basal ganglia of the rat[END_REF]. As shown in Figure 1B, we did not observe any differences between the protein levels of NR2A or NR2B in the DMS and the DLS (t 16 = 0.05, P = 0.96 for NR2A and t 16 = 0.39, P = 0.71 for NR2B). Furthermore, the degree of inhibition of NMDAR-mediated EPSCs in the presence of an NR2B-NMDAR-specific antagonist, Ro 25-6981,was similar in the DMS and DLS of naïve rats (data not shown), suggesting that the level of synaptic responses are similar in the two subregions.

Repeated ethanol administration increases NMDA-induced currents in the DMS

Next, we tested whether in vivo exposure to, and withdrawal from, ethanol result in changes in the activity of NMDARs in the DMS. To correlate the results obtained ex vivo described above with an in vivo procedure, we chose to systemically administer a dose of 2 g/kg, which results in a peak blood ethanol concentration (BEC) of 34 ± 3 mM (see Methods) that is similar to the concentration of ethanol that induces LTF of NMDARs in striatal slices (Figure 1A). Rats were given a single systemic injection of ethanol or saline and NMDA-induced currents were measured in DMS neurons 16 hrs later. A single systemic injection of ethanol did not result in a detectable alteration of NMDAR activity in the DMS (data not shown). We therefore hypothesized that prolonged exposure to, and withdrawal from ethanol, lead to a gradual long-lasting increase in the activity of the channel in the DMS (diagram, Figure 10). To model this possibility, rats received a daily systemic administration of ethanol (2 g/kg, i.p.) or saline for 7 days, and the activity of the channel was measured 16 hrs after the 7 th injection. We found that daily repeated administration of ethanol produced a greater NMDA-elicited change in holding currents in DMS neurons compared to saline-treated rats (Figure 2A) (t 26 = -3.02, P < 0.01). Since the NMDA current was elicited by bath-application of NMDA, which activates both synaptic and extrasynaptic NMDARs [START_REF] Carpenter-Hyland | Chronic ethanol induces synaptic but not extrasynaptic targeting of NMDA receptors[END_REF], the differences in the NMDA-induced currents between saline and ethanol treatments may result from changes in synaptic and/or extrasynaptic NMDARs. To address this question, we measured the input-output relation for NMDAR-EPSCs in DMS neurons from rats that received saline or ethanol treatments.

As shown in Figure 2B, EPSCs are higher in DMS neurons from ethanol-than from saline-treated animals [main effect of stimulation intensity: F 4, 146 = 30.54, P < 0.001; main effect of ethanol treatment: F 1, 146 = 5.47, P < 0.05; interaction between both factors: F 4, 146 = 3.44, P = 0.01],

suggesting that repeated ethanol administration induces an increase in the activity of synaptic NMDARs in the DMS. Interestingly, the frequency of miniature EPSCs (mEPSCs) was not altered 16 hrs after the last administration of ethanol (data not shown), suggesting that the changes in NMDAR activity in response to ethanol exposure and withdrawal are postsynaptic.

Seven daily treatments of rats with ethanol did not produce a change in the activity of the NMDAR in the DMS 40 hrs after the last administration (data not shown). We therefore tested whether there is a correlation between the duration of the repeated ethanol exposure and withdrawal, and the extent of enhancement of NMDAR activity. Rats were treated daily with 2 g/kg of ethanol or saline for 14 days and the activity of the channel was determined 40 hrs after the last injection. We observed a significantly higher amplitude of NMDA-induced currents in the DMS of ethanol-treated animal as compared to saline treated ones (Figure 2C) (t 49 = -3.72, P < 0.001), and a higher amplitude of NMDAR-EPSCs (Figure 2D) [main effect of stimulation intensity: F 4, 92 = 69.06, P < 0.001; main effect of ethanol treatment: F 1, 92 = 10.22, P < 0.01; interaction between both factors: F 4, 92 = 8.89, P < 0.001]. Together, our results suggest that repeated exposure of rats to ethanol results in a progressive and long-lasting upregulation of the activity of NMDAR in the DMS.

Repeated administration of ethanol leads to a long-lasting increase in the contribution of NR2B subunits to the activity of the channel in the DMS

Next, we tested for the contribution of the NR2B subunit to the long-lasting upregulation of the activity of the channel after daily exposure to ethanol. Rats were treated with ethanol (2 g/kg, i.p., once daily for 7 days) or saline, slices were prepared 16 hrs after the last injection, and the activity of the channel in the DMS was determined in presence of Ro 25-6981. As shown in Figure 3, a greater inhibition of the NR2B-containing NMDAR-mediated synaptic transmission was observed in the DMS of ethanol-treated rats as opposed to the saline-treated ones (t 30 = -2.63, P < 0.05), suggesting that repeated in vivo exposure to, and withdrawal from ethanol results in a long-lasting increase in the contribution of NR2B subunits to the overall activity of the channel.

Repeated ethanol administration leads to increased phosphorylation in the DMS

A potential mechanism to account for the prolonged upregulation of NR2B-NMDAR activity in response to ethanol exposure and withdrawal is an increase in the forward trafficking of the NR2B-containing NMDARs to the membrane [START_REF] Qiang | Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons[END_REF]. To address this possibility, we isolated synaptosomes from the DMS of rats 16 hrs after the 7 th injection of saline or ethanol and measured the level of synaptosomal NR2B subunit in the DMS. As shown in Fig.

4A residue Y1472 has been linked to the forward trafficking of the NR2B-NMDARs from intracellular compartments to the synaptic membrane [START_REF] Dunah | Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane[END_REF][START_REF] Hallett | Dopamine D1 activation potentiates striatal NMDA receptors by tyrosine phosphorylation-dependent subunit trafficking[END_REF]. Tyrosine phosphorylation of Y1472 was also shown to stabilize the subunit at the membrane by preventing endocytosis of the receptor [START_REF] Prybylowski | The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2[END_REF][START_REF] Hayashi | Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking[END_REF]. In addition, we previously showed that acute ex vivo exposure of hippocampal or striatal slices to ethanol leads to increased tyrosine phosphorylation of the NR2B subunit in total homogenates [START_REF] Yaka | Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol[END_REF][START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF], and that acute systemic administration of ethanol leads to an increase in the phosphorylation state of the NR2B subunit in the dorsal striatum [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. We therefore tested whether the NR2B subunit in the synaptosomal fraction was tyrosine phosphorylated 16 hrs after the 7 th administration of ethanol or saline. As shown in Figure 4A upper panel panel, right bar graph, repeated ethanol exposure resulted in a striking long-lasting increase in the phosphorylation level of synaptosomal NR2B subunits (t 10 = -4.05, P < 0.01).

Together, these results suggest that repeated in vivo exposure to ethanol causes a longterm increase in the level and the phosphorylation state of the NR2B subunit in synaptic membranes of the DMS, and in increased function of the NR2B-NMDARs.

Repeated administration of ethanol and withdrawal leads to long-lasting activation of Fyn kinase in the DMS

We, and others, previously showed that Fyn phosphorylates the NR2B subunit of the NMDAR, which results in an increase in channel function [START_REF] Yaka | NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1[END_REF][START_REF] Yaka | Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol[END_REF][START_REF] Prybylowski | The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2[END_REF][START_REF] Hayashi | Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking[END_REF]. Furthermore, we found that acute ex vivo and in vivo exposure of the dorsal striatum to ethanol leads to increased activity of Fyn [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. We also showed that Fyn is required for the acute ethanol-mediated LTF in the dorsal striatum [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. Therefore, we were interested in determining whether the observed long-lasting increase in NR2B subunit phosphorylation induced by repeated ethanol exposure correlates with an increase in the activity of Fyn in the DMS. To do so, total homogenates were prepared from the DMS of rats 16 hrs after the 7 th injection of saline or ethanol. Fyn was immunoprecipitated, and Fyn activity was detected using phospho-specific antibodies that recognize the active (pY420[Fyn]) or inactive (pY531[Fyn]) form of the kinase. We found that the levels of active kinase were elevated (Mann-Whitney Rank Sum test, P = 0.02) whereas the levels of inactive Fyn were decreased (Mann-Whitney Rank Sum test, P = 0.02) in the DMS of ethanol-treated rats compared to saline-treated animals (Figure 5). Together these data suggest that repeated daily exposure to ethanol results in long-lasting robust activation of Fyn kinase, which could account for the sustained upregulation of NR2B phosphorylation and the increased synaptosomal localization of the subunit.

High levels of ethanol consumption result in increased NMDAR activity in the DMS of adult rats

We reasoned that the daily systemic exposure of rats to ethanol resembles the intermittent access 2-bottle choice procedure in which rats have access to a high concentration of ethanol (20%) every other day [START_REF] Wise | Voluntary ethanol intake in rats following exposure to ethanol on various schedules[END_REF], We previously showed that this procedure results in excessive level of ethanol consumption (1-2 g/kg in 30 min), leading to an average BEC of approximately 20-30 mM [START_REF] Carnicella | Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor[END_REF], which corresponds to BEC induced by binge drinking in humans (NIAAA, 2004). We therefore examined whether the activity of the NMDAR in the DMS is enhanced as a result of the repeated cycles of ethanol exposure and withdrawal.

Rats underwent an intermittent access to 20% ethanol in a 2-bottle choice drinking procedure as described in Materials and Methods. After achieving a baseline of high levels of ethanol consumption (Supplemental Table 1), NMDAR activity was measured in the DMS 1 day after the last ethanol drinking session, and compared to the activity of the channel in the DMS from a group of rats that were consuming only water. We found that excessive ethanol-consumption induces higher amplitude of NMDA-induced currents in the DMS as compared to water controls (Figure 6A) (t 35 = -3.02, P < 0.01) and of NMDAR-EPSCs (Figure 6B) [main effect of stimulation intensity: F 4, 128 = 55.41, P < 0.001; main effect of ethanol treatment: F 1, 128 = 4.57, P < 0.05; interaction between both factors: F 4, 128 = 6.50, P < 0.001] as compared to water onlydrinking rats. These results suggest that repeated cycles of excessive ethanol intake cause an upregulation in the activity of NMDARs in the DMS, at least in part, at the synaptic site.

To determine whether the increase in NMDAR activity is long-lasting, rats underwent the same voluntary ethanol intake procedure and the activity of NMDAR was measured 9 days after the last ethanol consumption session. We observed the NMDA-induced current in the DMS was still higher after 9 days of ethanol deprivation, compared to rats drinking water only (Figure 6C)

(t 60 = -3.98, P < 0.001). The amplitude of NMDAR-EPSCs was also higher in the ethanol group than in water group (Figure 6D) [main effect of stimulation intensity: F 4, 208 = 149.57, P < 0.001; main effect of ethanol treatment: F 1, 208 = 27.13, P < 0.001; interaction between effects of ethanol treatment and stimulation intensity: F 4, 208 = 15.44, P < 0.001]. These results suggest that repeated cycles of voluntary ethanol intake and deprivation cause a persistent increase in the activity of synaptic NMDARs in the DMS.

High levels of ethanol consumption lead to an increase in the phosphorylation level of NR2B in the DMS

Next we determined whether the phosphorylation level of the NR2B subunit is elevated after repeated excessive ethanol drinking and withdrawal. Rats experienced an intermittent access to 20% ethanol two-bottle-choice procedure as above, and the level of NR2B phosphorylation was measured 24 hrs after the end of the last ethanol drinking session (i.e., 24 hrs withdrawal). As shown in Figure 7A, the phosphorylation level of NR2B was markedly increased in the DMS of rats consuming ethanol as compared to rats that consumed only water (t 13 = -2.67, P < 0.05),

suggesting that ethanol drinking results in a long-lasting increase in NR2B-NMDAR activity. In addition, similar to what we observed using the repeated systemic administration of ethanol (Figure 4B), the increase in the phosphorylation levels of NR2B did not correspond to increases in total protein level of the NR2B subunit (Figure 7B, t 13 = 0.19, P = 0.85), NR1 or NR2A (data not shown).

Inhibition of Src PTKs or NR2B-NMDARs in the DMS, but not in the DLS, significantly decreases operant ethanol self-administration in rats

Finally, we investigated the possible behavioral consequences of the long-lasting increase in NR2B-NMDAR function in the DMS induced by repeated ethanol exposure. We therefore tested whether inhibition of NR2B-NMDAR and/or Fyn activity in the DMS alters operant ethanol self-administration in rats following a history of high voluntary ethanol consumption. We found that intra-DMS infusion of the NR2B-NMDAR inhibitor, ifenprodil, or the Src PTKs inhibitor, PP2 (which inhibits Fyn), significantly reduced operant responding for ethanol (Figure 8A, significant effect of treatment: F 4, 20 = 2.91, P < 0.05) and, therefore, ethanol intake (Figure 8B, significant effect of treatment: F 4, 20 = 2.98, P < 0.05). Next, we determined the specificity of ifenprodil and PP2 actions in the DMS by examining their ability to attenuate operant selfadministration of the natural, non-drug reinforcer, sucrose. As shown in Figure 8C (no significant effect of treatment: F 4, 28 = 0.43, P = 0.78) and Figure 8D (no significant effect of treatment: F 4, 28 = 0.48, P = 0.75), intra-DMS infusion of ifenprodil or PP2 did not alter operant responding for sucrose or sucrose intake, indicating that the decrease in ethanol self-administration induced by NR2B-NMDAR or Src family kinase inhibition in the DMS, is not due to a general decrease in motivation or to locomotor activity impairment. Importantly, similar treatment into the DLS did not significantly affect operant ethanol self-administration (Figure 8E, no significant effect of the treatment: F 4, 24 = 1.27, P = 0.31, and Figure 8F, no significant effect of the treatment: F 4, 24 =

1.01, P = 0.38). These data suggest that long-term activation of Fyn and NR2B-NMDAR within the DMS are important players in the mechanism that underlies excessive ethanol-drinking behavior.

Inhibition of Src PTKs or NR2B-NMDARs in the DMS reduces reinstatement of ethanolseeking in rats

To test whether the decrease in operant ethanol self-administration induced by the inhibition of Src PTKs activity or NR2B-NMDARs is due to a decrease in the motivation of rats to seek ethanol, instrumental performances were evaluated during a single session of extinction.

As shown in Figure 9A, intra-DMS infusion of ifenprodil and PP2 at a dose that efficiently reduced operant ethanol self-administration (Figure 8A), did not decrease the number of lever presses on the ethanol lever during the extinction session (no significant effect of treatment: F 2, 12 = 0.075, P = 0.93). We also tested whether intra-DMS infusion of ifenprodil or PP2 affects ethanol-priming-induced reinstatement of ethanol-seeking as described in Materials and Methods.

The initiation of operant responding during the reinstatement test, as assessed by the latency to the third press (i.e, the first reward), was reduced in vehicle-treated rats compared to the last day of extinction (Figure 9B) (significant effect of treatment: χ 2 = 10.35, P < 0.02), indicating that the oral ethanol prime efficiently reinstated ethanol-seeking. However, this priming effect was prevented by intra-DMS infusion of ifenprodil or PP2 (Figure 9B). Consistent with the result above, reinstatement of ethanol-seeking was significantly reduced by intra-DMS infusion of ifenprodil or PP2 (Figure 9C) (significant effect of treatment: F 3, 21 = 8.15, P < 0.001). Therefore, these data indicate that the NR2B subunit and Fyn are major components of the mechanisms controlling reinstatement of ethanol-seeking by an ethanol prime.

Discussion

Here we demonstrate that repeated systemic administration of ethanol as well as intermittent excessive ethanol consumption, produce a robust long-lasting increase in the activity of the synaptic NMDAR in the DMS. We further provide evidence to suggest that Fyn-mediated phosphorylation of the NR2B subunit, which in turn results in higher levels of the NR2B-NMDARs at the synaptic membrane, could be the mechanism that underlies the long-lasting upregulation of NR2B-NMDAR activity in response to ethanol. Finally, we show that the inhibition of NR2B-NMDARs or the Src family of tyrosine kinases (which includes Fyn) in the DMS decreases operant ethanol self-administration and reinstatement of ethanol-seeking in rats that consume high levels of ethanol.

Long-term facilitation of the NR2B-NMDAR function upon ethanol exposure and withdrawal is centered in the DMS

Ethanol-mediated LTF of NMDAR was not centered in the DLS, although the protein levels of NR2A and NR2B subunits and Fyn (data not shown) in the DMS and DLS, and synaptic responses in the two subregions of naive rats were similar. An alternative mechanism for the subregion specificity may be linked to whether or not Fyn is associated to the NMDAR-NR2B complex. Previously, we observed that ex vivo and in vivo exposure to ethanol leads to phosphorylation of NR2B subunit in the dorsal striatum where the NR2B subunit and Fyn are colocalized, but not in the ventral striatum where NR2B and Fyn are not compartmentalized within the same complex [START_REF] Yaka | Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol[END_REF][START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF]. Therefore, the compartmentalization of Fyn near the NMDAR complex in the DMS might account for the enhanced LTF in the DMS compared to the DLS.

The activity of DMS NMDARs following ex vivo acute withdrawal from ethanol was also examined by Yin et al. but no facilitation was observed following ethanol withdrawal [START_REF] Yin | Ethanol reverses the direction of long-term synaptic plasticity in the dorsomedial striatum[END_REF]. While the discrepancy between our studies and Yin's is unclear, the studies are difficult to compare since Yin et al. relied only on ex vivo slices electrophysiology experiments, and the experimental conditions were different. For example, Yin et al. used a higher concentration of EGTA in the intracellular solution, which may buffer the Ca 2+ rise that is required for acute ethanol withdrawal-induced facilitation of NMDAR activity [START_REF] Li | Protein kinase Cgamma mediates ethanol withdrawal hyper-responsiveness of NMDA receptor currents in spinal cord motor neurons[END_REF]. Another possibility is the inclusion of a tyrosine phosphatase inhibitor during recording in our study.

Long-lasting upregulation of NMDAR activity in the DMS upon voluntary and passive exposure to ethanol

We found that repeated systemic administration or voluntary consumption of ethanol results in a long-lasting upregulation of the activity synaptic NMDAR in the DMS. This result is particularly striking as the increase in synaptic NMDAR activity remains high even 9 days after the last episode of ethanol drinking. We further showed that repeated exposure to ethanol leads to an increase in the contribution of the NR2B subunit to the overall activity of the channel.

Together, our results suggest that the NR2B-dependent LTF may underlie the persistent increase in the function of the NR2B-NMDAR induced by repeated in vivo ethanol exposure. Based on these results we suggest the following model: the first administration of ethanol induces LTF of the NR2B-NMDAR activity that decays over time (Figure 10). However, repeated cycles of ethanol exposure and withdrawal results in a cumulative enhancement of the NR2B-NMDAR activity (Figure 10).

Interestingly, the upregulation in the activity of the channel in the DMS upon multiple ethanol exposure correspond with a striking long-lasting increase in the activity of Fyn, as well as in the phosphorylation of its substrate the NR2B subunit, and a corresponding increase in the localization of the subunit in the synaptosomal compartment. Importantly, voluntary consumption of ethanol also produced an increase in the phosphorylation of NR2B, which was not due to an increase in the protein level of the subunit. Fyn phosphorylation of the NR2B subunit was shown to enhance the activity of the channel [START_REF] Yaka | NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1[END_REF][START_REF] Yaka | Scaffolding of Fyn kinase to the NMDA receptor determines brain region sensitivity to ethanol[END_REF], to promote the trafficking of the receptor from internal pools and to increase its stability at the cell surface [START_REF] Nakazawa | Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor[END_REF][START_REF] Dunah | Dopamine D1-dependent trafficking of striatal N-methyl-D-aspartate glutamate receptors requires Fyn protein tyrosine kinase but not DARPP-32[END_REF][START_REF] Prybylowski | The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2[END_REF][START_REF] Hayashi | Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking[END_REF].

Therefore, a long-lasting and cumulative elevation in Fyn activity in vivo may underlie the increased NR2B-NMDAR function in the DMS induced by repeated ethanol exposure.

NR2B-NMDARs and Fyn in the DMS contribute to mechanisms that underlie excessive ethanol consumption and relapse

The intermittent access 2-bottle-choice drinking procedure induces a progressive increase in ethanol intake and preference in rats [START_REF] Carnicella | Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor[END_REF], that resembles binge drinking in humans (NIAAA, 2004). We found that intra-DMS, but not intra-DLS infusion of the NR2B-NMDAR antagonist ifenprodil significantly reduces operant ethanol self-administration in rats with a history of excessive voluntary ethanol consumption. Similar results were obtained upon the inhibition of Src PTKs activity in the DMS with PP2. Furthermore, infusion of ifenprodil or PP2 in the DMS does not alter operant sucrose self-administration, indicating that the inhibition of NR2B-NMDAR or Src PTKs interferes with processes specifically induced by ethanol that are not shared with non-drug reinforcing substances. We previously reported that LTF of NMDARmediated EPSCs in response to ex vivo exposure of DS slices to ethanol is not observed in Fyn

Wang et al.

29

deletion mice [START_REF] Wang | Ethanol induces long-term facilitation of NR2B-NMDA receptor activity in the dorsal striatum: implications for alcohol drinking behavior[END_REF], and that Src activity is inhibited after acute treatment of ethanol in hippocampal slices [START_REF] Suvarna | Ethanol alters trafficking and functional N-methyl-D-aspartate receptor NR2 subunit ratio via H-Ras[END_REF]. Therefore, although PP2 is not a selective Fyn kinase inhibitor, it is highly likely that the inhibitory actions of PP2 on ethanol drinking behaviors are due to the inhibition of Fyn. Therefore, these results suggest that the long-lasting adaptive upregulation of Fyn and NR2B-NMDAR activity in the DMS observed upon repeated ethanol exposure contributes to mechanisms underlying excessive ethanol-drinking and -seeking behaviors.

We found that infusion of ifenprodil or PP2 into the DMS decreases reinstatement of ethanol-seeking induced by re-exposure of rats to the exteroceptive properties of ethanol. This finding is reminiscent of the role of the DMS in the processing of A-O actions, and the control of instrumental performance by the outcome that mediate goal-directed behaviors [START_REF] Yin | The role of the basal ganglia in habit formation[END_REF][START_REF] Balleine | The integrative function of the basal ganglia in instrumental conditioning[END_REF]. In line with these findings, Vengeliene et al. showed that systemic administration of ifenprodil attenuates the alcohol deprivation effect, another model of relapse, in rats [START_REF] Vengeliene | The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect[END_REF]. Importantly, we also show that the NMDAR activity remains elevated in the DMS 9 days after the last ethanol drinking session, which corresponds with the reinstatement test day. Together, these results suggest that the DMS is a critical substrate for relapse mechanisms involving the NR2B-NMDAR.

NR2B-NMDARs in the DMS and mechanisms that underlie ethanol consumption, seeking and relapse

Increasing evidence suggests that aberrant neuroadaptations induced by drugs of abuse in the dorsal striatum result in compulsive drug-seeking and taking [START_REF] Gerdeman | It could be habit forming: drugs of abuse and striatal synaptic plasticity[END_REF][START_REF] Everitt | Neural systems of reinforcement for drug addiction: from actions to habits to compulsion[END_REF][START_REF] Everitt | Review. Neural mechanisms underlying the vulnerability to develop compulsive drug-seeking habits and addiction[END_REF][START_REF] Belin | Parallel and interactive learning processes within the basal ganglia: relevance for the understanding of addiction[END_REF]. However, little is known about the molecular and cellular mechanisms implicated in such modifications. NMDARs are crucial for synaptic plasticity [START_REF] Malenka | NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms[END_REF], and our results suggest that aberrant synaptic plasticity in the DMS resulting from ethanol exposure contributes to the development and/or maintenance of ethanol-drinking and -seeking behaviors. Several possibilities may underlie the contribution of the NMDAR in the DMS to ethanol consumption and relapse. First, LTP of AMPARs in the DMS is NMDAR-dependent [START_REF] Partridge | Regional and postnatal heterogeneity of activitydependent long-term changes in synaptic efficacy in the dorsal striatum[END_REF], and very recently Foster et al., showed that the NR2B subunit is essential for LTP in the hippocampus [START_REF] Foster | Distinct Roles of NR2A and NR2B Cytoplasmic Tails in Long-Term Potentiation[END_REF].

Therefore, it is plausible that the facilitation of NR2B-NMDAR activity reduces the threshold of AMPAR-LTP induction. LTP in turn may strengthen goal-directed behaviors associated with ethanol. Another possibility relies on the observation that NMDAR activity contributes "UP state" firing of striatal neurons [START_REF] Pomata | NMDA receptor gating of information flow through the striatum in vivo[END_REF]. Specifically, the medium spiny neurons (MSNs), which are the principal cells of the striatum, alternate between two states: the depolarized UP state and more hyperpolarized DOWN state [START_REF] Nicola | Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumbens[END_REF]. MSNs fire action potentials only during the UP state [START_REF] Mahon | Relationship between EEG potentials and intracellular activity of striatal and cortico-striatal neurons: an in vivo study under different anesthetics[END_REF][START_REF] Tseng | Cortical slow oscillatory activity is reflected in the membrane potential and spike trains of striatal neurons in rats with chronic nigrostriatal lesions[END_REF][START_REF] Kasanetz | Functional integration across a gradient of corticostriatal channels controls UP state transitions in the dorsal striatum[END_REF]. Therefore, it is possible that repeated cycles of ethanol exposure and withdrawal during the intermittent-access procedure induce increased NR2B-NMDAR activity, which in turn leads to increased UP state potentials of the neurons in the DMS. This change in neuronal activity results in a reduction in afferent threshold for neuronal firing action potentials, and consequently contributes to increased ethanol-seeking and intake. Lastly, NMDAR activity may also contribute directly to the fast synaptic transmission, which is mediated by AMPARs under presynaptic stimulation [START_REF] Miwa | Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS[END_REF], and can directly contribute to the information flow through the striatum.

In summary, our results suggest that the Fyn/NR2B-NMDAR pathway in the DMS plays an important role in neuroadaptations that lead to excessive alcohol consumption and to the development of compulsive alcohol-seeking. Our study also puts forward an important role of the NMDARs in the DMS in the propensity to relapse.

  repeated measures, followed by the Student-Newman-Keuls (SNK) post hoc test (two-way RM-ANOVA, Figures 1A, 2B, 2D, 6B, and 6D) or using the unpaired, two-tailed t-test(Figures 2A, 2C, 3, 6A, and 6C). Specifically in Figure1A, the brain region (DMS and DLS) was analyzed as a between-subject factor and the ethanol treatment as a within-subject factor, which consists of three time periods: baseline, EtOH (during ethanol application), post-EtOH (30 min after ethanol washout), which correspond to the time periods 1-3 in Figure1Ab, respectively. At each time period, 10 min EPSCs were averaged to construct one data point for each subject. To closely examine the interaction between the two factors, only two time periods (EtOH and post-EtOH) were analyzed. In Figures2B,

  middle panel, left bar graph, although the protein level of the NR2B subunit in the synaptosomal fraction was only marginally increased after repeated administration of ethanol and withdrawal (two-tailed t-test: t 10 = -1.92, P = 0.08), the levels of NR1 or NR2A in synaptosomal fractions (data not shown), as well as the total protein level of NR2B (Figure4B) (t 16 = 0.31, P = 0.76), NR1 and NR2A subunits were also unaltered (data not shown), suggesting a specific increase of the NR2B subunit in membranes. Tyrosine phosphorylation of the NR2B subunit onWang et al. 20

  Zapata A, Gonzales RA, Shippenberg TS (2006) Repeated ethanol intoxication induces behavioral sensitization in the absence of a sensitized accumbens dopamine response in C57BL/6J and DBA/2J mice. Neuropsychopharmacology 31:396-405.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Repeated ethanol administration leads to an increase in NMDAR activity in the

Figure 3 .

 3 Figure 3. Repeated ethanol administration results in an increase in contribution of NR2B

Figure 4 .

 4 Figure 4. Repeated administration of ethanol increases the phosphorylation level of the

Figure 5 .

 5 Figure 5. Repeated administration of ethanol increases Fyn activation in the DMS. Rats

Figure 6 .

 6 Figure 6. Intermittent-access to ethanol results in an increase in NMDAR activity in the

Figure 7 .

 7 Figure 7. Intermittent access to ethanol results in increased phosphorylation of NR2B

Figure 8 .

 8 Figure 8. Inhibition of NR2B-NMDARs or Src PTKs in the DMS but not in the DLS

Figure 9 .

 9 Figure 9. Inhibition of NR2B-NMDARs or Src PTKs in the DMS reduces ethanol-priming-

Figure 10 .

 10 Figure 10. Hypothetical model of the mechanism leading to increased NR2B-NMDAR

(

  Leica, Nussloch, Germany) in an ice-cold solution containing (in mM): 40 NaCl, 85 choline chloride, 4 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 0.5 CaCl 2 , 7 MgCl 2 , 10 glucose, 1 sodium ascorbate, and 3 sodium pyruvate, saturated with 95% O 2 and 5% CO 2 . Slices were then incubated

	in the same solution at 32°C for 45 min before being transferred to a chamber at room
	temperature that contained an external solution composed of (in mM): 125 NaCl, 2.5 KCl, 2.5
	CaCl 2 , 1.3 MgCl 2 , 1.25 NaH 2 PO 4 , 25 NaHCO 3 , and 10 glucose, saturated with 95% O 2 and 5%
	CO 2 .

Acknowledgements

This research was supported by NIAAA (R01AA/MH13438) (D.R.) and by funds provided by the State of California for medical research on alcohol and substance abuse through the


Marju Medar
email: marju.medar@ut.ee

Kandela Õun
email: kandela.oun@ut.ee

Merle Looring
email: merle.looring@ut.ee

Economic growth versus development of social welfare structures in Europe

Keywords:

Artikli eesmärgiks on pakkuda välja metoodika heaoluriikide kontseptuaalse raamistiku

INTRODUCTION

Well-being is one of the most important aspects of people´s lives both as individuals and as societies. However, despite unprecedented economic prosperity in the last 35 years people do not necessarily feel better individually or as communities. According to [START_REF] Miller-Keane | Encyclopedia and Dictionary of Medicine, Nursing and Allied Health[END_REF] the Centre for Well-being at NEF (New Economics Foundation) seeks to understand measure and influence well-being. In particular, people ask the question: "What would policymaking and the economy look like if their main aim were to promote well-being?" (Well-being… 2011) Although assessment tools are available to evaluate physical and social dimensions, an individual's general sense of well-being or satisfaction with the attributes of life is more difficult to evaluate. Well-being is often measured as happiness or satisfaction with life, but well-being is about having meaning in life, about fulfilling one's potential and feeling that our lives are worthwhile [START_REF] Eckersley | Well & Good: How We Feel & Why It Matters[END_REF]. The discussion of well-being draws on several recent reviews of literature, including [START_REF] Bond | The pursuit of happiness[END_REF], [START_REF] Hamilton | Growth Fetish[END_REF], [START_REF] Diener | Beyond Money: Toward an economy of wellbeing[END_REF], [START_REF] Eckersley | Well & Good: How We Feel & Why It Matters[END_REF], Myers (2004), [START_REF] Shah | A wellbeing manifesto for a flourishing society[END_REF] and measuring well-being (Feasibility study…, 2010). While a number of individual nations and international organisations have already used (or are in the process of setting up) some measures of well-being, currently no nation or organisation regularly and systematically collects a full spectrum of measures of subjective well-being currently. This state of affairs has led several researchers and public figures to express the need for developing and systematically using national accounts of well-being in recent years [START_REF] Diener | Subjective well-being: The science of happiness and a proposal for a national index[END_REF]. [START_REF] Diener | Beyond Money: Toward an economy of wellbeing[END_REF], in an article titled "Beyond money: Toward an economy of well-being", also argued that nations should create ongoing assessments of well-being to complement existing economic indicators (e.g. GDP, savings rates, consumer confidence) and social indicators (e.g. crime rates, longevity, infant mortality rates) used by country leaders to make governmental decisions. They emphasised that monitoring well-being at a national level will alert the citizenry to important information beyond economic growth that should help guide policy, and which can help improve the quality of life in societies. The above publication seeks to discuss how we think about national progress, and especially about how we equate progress with economic growth. Increasing scientific interest in well-being is contributing to this re-evaluation. Discussions of well-being seek to consider other dimensions such as people's perceptions of social well-being or national quality of life; patterns and trends in physical and mental health; and alternative measures of progress. It is also discussed how different qualities such as people's social welfare and social reforms, structure of economy, fiscal and structural reforms, innovation, research, innovative activities and coordination of industrial policies and transition to a socio-ecological model in the future (best practices across welfare states, strengths, weaknesses, unregulated areas) affect personal and social well-being and influence territorial intelligence. Socio-economic analysis of welfare states seeks to answer this question: how welfare states intend to resolve a dilemma of preserving or enhancing their commitments whilst ensuring budget consolidation and innovation which enable the application of green economy principles under budget deficit conditions in the forthcoming years. This article explains the concept of welfare from three aspects: social, economic and ecological, and aims to draw in related indicators available in the EUROSTAT database. It is planned to use data from the 27 Member States of the European Union as well as candidate and EFTA (European Free Trade Association) countries in the analysis. Additionally we suggest applying the cluster analysis method for comparison of the different countries and bringing them together into territories which will serve as a basis of territorial intelligence research. The authors intend to use the EUROSTAT databases (a major quality indicator for research data) because they contain data collated using a common methodology across all countries included in the research. The authors will now explain what is understood under these three concepts.

THE MAIN CONCEPTS 1.The approach of social welfare and welfare policy

As portrayed in the preceding discussion, welfare of people and institutions is clearly linked to welfare regimes of the country they are living in. Welfare regimes are primarily targeted at improving people's welfare and this is directly connected to the concept of ensuring quality of life.

Modern welfare policies play an important role in guaranteeing a basic level of equality of life chances and social integration. In the process of modernisation, the meritocratic triad of educational degree, occupational standing and level of workrelated income became the leading principle of social stratification. [START_REF] Siegrist | Work, health and welfare: new challenges[END_REF] Conversely, adult healthy people who deviate from this norm of economic independence and individual autonomy, depending on public welfare or informal subsidiary support, are confronted with negative attitudes of disregard, often irrespective of reasons of their dependence. [START_REF] Sennet | Respect in a World of Unequality[END_REF] The way welfare regimes are organised in modern states varies considerably. A welfare state is a "concept of government in which the state plays a key role in the protection and promotion of the economic and social well-being of its citizens. It is based on the principles of equality of opportunity, equitable distribution of wealth, and public responsibility for those unable to avail themselves of the minimal provisions for a good life. The general term may cover a variety of forms of economic and social organisation" (Britannica Online Encyclopaedia).

The economic approach

There are differences in welfare structures and economic growth between European countries and territories. At a national level welfare states can be divided into four welfare regimes by using economic, ecological and social indicators. These indicators are used in welfare state modelling, a longstanding strand within the comparative social policy literature [START_REF] Cutright | Political structure, economic development, and national social security programs[END_REF][START_REF] Ichimura | Social Foundations of Post-Industrial Economics[END_REF][START_REF] Therborn | Welfare State and Capitalist Markets[END_REF][START_REF] Titmus | Social Policy[END_REF][START_REF] Wilensky | The Welfare State and Equality[END_REF][START_REF] Wilensky | Industrial Society and Social Welfare[END_REF], and a strong focus on mapping welfare regimes [START_REF] Pierson | Beyond the Welfare State[END_REF] with ongoing research into welfare state regimes and welfare state typologies [START_REF] Bambra | The words of welfare: Illusory and gender-blind?[END_REF].

Theories considering welfare states provide an adequate framework for the understanding of levels of measurable effects engendered by political orientations, and especially the key relationships between market institutions and social spending and their effects on levels of poverty. However, theories of welfare states should not be considered ultimately unique with respect to importance attributed to relationships between political variables and poverty, on the one hand, and then labour markets and welfare state institutions, on the other. In general, it is possible to distinguish between two approaches: (i) structural -an approach that emphasises the importance of structural-functional factors, and (ii) a political-economic approach that emphasises roles played by political factors. The latter underlines the key role of political orientation in determining the level and dynamics of poverty in developed democracies. Market and welfare state institutions are strongly influenced by the political processes. Leftwing parties are often associated with the strengthening of the welfare state institutions while the right-wing parties give emphasis on the deregulation i.e. the implementation of market mechanisms in solving social problems. On the basis of this it is possible to further distinguish between two basic approaches in the implementation of social policy: (i) liberal -primarily favoured by conservative and right wing parties and (ii) the redistributive -closer to social democratic and leftist parties. The division of liberal welfare into the state and redistributive models represents a rough theoretical framework within which specific types of welfare states are profiled, also recognisable in the specific practices of individual countries. Having in mind the diversities present in the sources, the EU is characterised by four main models: the Socialdemocratic, Corporatist, Mediterranean and the Liberal welfare state regime. [START_REF] Josifidis | The European welfare regimes: Political orientation vs[END_REF]2) Total welfare economics is, thus, more of a hypothesis as it requires perfect market structure-like conditions to be implementable. However, some or the other form of welfare economics mechanisms are always at play, be it in the regulation of market forces or the fixing of remunerations and prices of various factors of production and utilities, respectively. Most economic mechanisms are of an automatic nature and they exist in the economic system whether or not we deliberately interfere. Welfare economics is no exception (Shukla, 2011: 2).

The ecological approach

Eco-efficiency links economic efficiency with environmental efficiency. The main purpose of the concept is to identify and implement activities to enable production that is both economically more efficient and cleaner. This means that parameters with a high indicative value have to be used [START_REF] Wursthorn | Economic-environmental monitoring indicators for European countries: A disaggregated sector-based approach for monitoring eco-efficiency[END_REF]. Eco-efficiency is a key concept which can help individuals, companies, governments or other organisations become more sustainable. It brings together the essential ingredients -economic and ecological progresswhich are necessary for economic prosperity to increase with more efficient use of resources and lower emissions of substances that can have adverse environmental consequences. [START_REF] Verfaillie | Measuring ecoefficiency. A guide to reporting company performance[END_REF] Sustainability indicators are essential in illustrating to policymakers and the public alike the relationship and trade-offs among the three dimensions of sustainable development. It is also crucial in monitoring progress and performance in terms of achieving economic, social and ecological goals over time, and in evaluating the future implications of existing decision and policy direction. However, capturing the dynamics of sustainable development and presenting them in terms of measurement indicators that could be unambiguously interpreted and easily communicated to policymakers for public policies remain a challenging task. (ESCAP, 2009:6) Measuring sustainability needs to review every aspect of economic, environment and social linkages but it also requires simple measures and an adequate framework that informs policymakers about major trends and issues as well as support in-depth analysis and identify concrete policy options. For Asia and the Pacific, as one of the most ecologically vulnerable regions on the planet, there is a pressing need to present an easy tool clearly showing the direction for economic growth with less resource consumption and pollution, a key ingredient of and prerequisite for sustainable development. In this region particularly, eco-efficiency indicators have been used in the analysis of efficiency of the use of resources and measuring environmental impact of economic activities. (ESCAP, 2009:7) Politics commonly focuses on certain key economic indicators, such as gross domestic product (GDP) and employment, for the quantification of economic prosperity. Widely accepted eco-efficiency indicators could fulfil a comparable function by additionally incorporating the environmental impact of economic activities. Furthermore, the concept of eco-efficiency, if adequately defined, could also be used to assess the efforts to decouple economic activities from emissions. To achieve this, ongoing monitoring of macroeconomic eco-efficiency is necessary [START_REF] Wursthorn | Economic-environmental monitoring indicators for European countries: A disaggregated sector-based approach for monitoring eco-efficiency[END_REF]. EEI (eco-efficiency indicator) presents a set of indicators that demonstrate the linkage between economic activity, resource usage and environmental impact in order to evaluate economic policies more effectively and thereby assist policymakers in improving the ecoefficiency of economic growth. Rather than presenting a single index, EEI provides a range of economy-wide and sector-wide indicators that clearly establish the pattern and relationship between economic activity and environmental issues. (ESCAP, 2009:7) EEI is defined as:

Eco-efficiency = Environmental Cost/ Economic Output, where environmental costs can be: pollution emissions (CO 2 or SOx emissions, biochemical oxygen demand, etc.), resource-use (energy or water used), cost associated with an environmental burden (traffic congestion costs), and where economic outputs can be: value added of benefit (GDP per capita), unit of product or service (per km, per m 2

RESEARCH METHOD

), and cost associated with an environmental burden (traffic congestion costs). (ESCAP, 2009:3) A number of measures have been suggested in recent years, such as the ecological footprint, sustainability and other indicators and indices, but none has clearly shown the path to economic growth with less resource consumption and pollution, a key ingredient and prerequisite of sustainable development. The authors will investigate relevant indices in more detail in their research but in Table 1 they show some indices what they think to be useful.

The presentation describes a suggested methodology for analysing a conceptual framework for welfare states including economic structures and financial and social reforms of the states. The wider goal of the methodology is to analyse socio-economic indicators, structural reforms and fiscal policies in European countries which preserve or enhance income redistribution in Europe, and also the coordination of research, innovation and industrial policies in preparing society for a transition to a socio-ecological model in the future. The authors propose using cluster analysis because despite having many obvious benefits for welfare state classification, it is a surprisingly underused approach in comparative social policy [START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF]. In cluster analysis, countries are classified on the basis of the combination of predetermined selection criteria (defamilisation factors) so that each country in a cluster is similar to the others in that cluster and different from countries in the other clusters [START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF][START_REF] Grimm | Reading and understanding more multivariate statistics[END_REF]. Clusters thus represent different regime types. There are two more commonly used forms of cluster analysis: hierarchical and K-means [START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF][START_REF] Pitruzzello | Decommodification and the Worlds of Welfare Capitalism[END_REF]. Hierarchical cluster analysis locates the closest pair of countries and combines them to form a pair; this (joining cases into pairs or joining two pairs) continues until all cases are in one cluster. Once countries are joined in a cluster, they remain joined throughout the rest of the analysis [START_REF] Cramer | Advanced Quantitative Data Analysis[END_REF][START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF]. Thus the clusters emerge from the data, facilitating the emergence of welfare state taxonomies. However, hierarchical cluster analysis is rather theoretical and so it is often conducted alongside the K-means cluster analysis [START_REF] Cramer | Advanced Quantitative Data Analysis[END_REF][START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF][START_REF] Grimm | Reading and understanding more multivariate statistics[END_REF][START_REF] Pitruzzello | Decommodification and the Worlds of Welfare Capitalism[END_REF]. The K-means analysis enables the a priori specification of the number of clusters to be formed [START_REF] Gough | Social Assistance regimes: A cluster analysis[END_REF]. This has a benefit for the classification of welfare states, as it enables the testing of the number of different types of welfare state regimes (3, 4 or 5) suggested by the welfare state modelling literature [START_REF] Bambra | The words of welfare: Illusory and gender-blind?[END_REF].

DISCUSSION

The research and methodology are currently under preparation and thus one cannot describe the course of study or research results yet. This is a research topic in its initial stage and further discussion aims to analyse earlier studies into socio-economic and ecological data of the target countries. Through the discussion the authors expect feedback and recommendations regarding indicators which could best cover socio-ecological sustainability of the countries and which could form a platform for further studies into the topic of ´territorial intelligence´ deriving from the concept of welfare regimes of the countries.

Table 1 .

 1 EUROSTAT indices to describe different countries social, ecological and economic situation

	social indicators	ecological indicators	economic indicators
		Components of domestic material
	Total long-term unemployment rate	consumption 1000 tonnes	Employment rate
	Healthy live years	Final energy consumption 1000 toe	GDP
	Suicide death rate	Total fresh water abstraction millions m³	Consumption patterns
	Religion and cultural background	Built-up areas	Production patterns
		Greenhouse gas emissions, Kyoto base
	…	year (source: EEA)	Development of services
			Networks of
		Generation and discharge of wastewater	entrepreneurships
		…	…