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A key deficit in alcohol dependence is disrupted prefrontal function leading to excessive alcohol seeking, but the molecular events underlying the emergence of addictive responses remain unknown. Here we show by convergent transcriptome analysis that the pyramidal neurons of the infralimbic cortex are particularly vulnerable for the long-term effects of chronic intermittent ethanol intoxication. These neurons exhibit a pronounced deficit in mGluR2. Also, alcohol dependent rats do not respond to mGluR2/3 agonist treatment with reducing extracellular glutamate levels in the nucleus accumbens. Together these data imply a loss of autoreceptor feedback control. Alcohol dependent rats show escalation of ethanol seeking, which was abolished by restoring mGluR2 expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR2 transcripts compared to control subjects suggesting that mGluR2 loss in the rodent and human cortico-accumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR2 function within this brain circuit may be of therapeutic value.
The molecular and neuroanatomical substrates underlying substance use disorders including alcohol dependence remain poorly understood. Imbalances in glutamate neurotransmission and homeostasis are considered to play a central role for the increased propensity to relapse in addicted individuals (Everitt and Robbins, 2005; Kalivas, 2009; Spanagel, 2009). In particular, the glutamatergic cortico-accumbal pathway plays an essential role for reinstating drug-seeking behavior in animal models of relapse (Kalivas, 2009). It has been shown that lesions or inactivation of the medial prefrontal cortex (mPFC) or nucleus accumbens prevent reinstatement of drug-seeking following extinction, while activation of either structure stimulates drug-seeking (Cornish and Kalivas, 2000; Capriles et al., 2003; McFarland et al., 2004). Supporting this notion, human functional magnetic resonance imaging identified a positive correlation between cue-reactivity, craving and activity in prefrontocortical regions in addicted patients (Wilson et al., 2004; Schacht et al., 2012). A dysregulation of central glutamate levels in these areas during withdrawal and protracted abstinence was recently reported as well (Hermann et al., 2011, 2012). Despite these findings on the role of the mPFC-accumbal pathway in relapse, relatively little is known about the molecular and cellular neuroadaptations within this circuit that result in susceptibility to relapse.
Here we set out to elucidate alcohol-induced dysregulation of mPFC function in rats with a history of alcohol dependence, i.e. by exposure to daily cycles of intermittent alcohol vapor intoxication and withdrawal, a paradigm that produces high intoxication with brain alcohol levels above 200mg/dl and induces behavioral and molecular changes relevant for the pathophysiology of alcoholism in both rats and mice (Rogers et al., 1979; Roberts et al., 2000; Rimondini et al., 2002, 2003, 2008; Becker and Lopez, 2004; O’Dell et al., 2004; Hansson et al., 2008; Sommer et al., 2008; Melendez et al., 2012). Animals derived from this procedure are termed ‘post-dependent’ to emphasize the fact that neuroadaptations induced through a history of alcohol dependence remain even in the absence of continued ethanol intoxication. This phenomenon has been consistently demonstrated for a long-lasting behavioral sensitivity to stress and altered amygdala gene expression (Funk et al., 2006; Heilig and Koob, 2007; Sommer et al., 2008; Vendruscolo et al., 2012). In this sense post-dependent animals may model the increased propensity to relapse in abstinent alcoholic patients (Björk et al., 2010; Heilig et al., 2010). We used a multilayered search strategy that started with an unbiased transcriptome screening of multiple brain regions, and converged on a distinct neuronal population that exhibits a profound mGluR2 deficit. This receptor belongs to the class II metabotropic glutamate receptors (mGluR2/3) that are key to regulating glutamatergic neurotransmission in brain regions mediating drug-seeking and incentive motivation, including the mPFC-accumbal pathway (Ohishi et al., 1993; Olive, 2009). mGluR2/3 negatively modulate glutamate transmission as autoreceptors by inhibiting glutamate release and by reducing neuronal excitability at the postsynaptic level (Ferraguti and Shigemoto, 2006). Dysregulation of mGluR2/3 function within the mPFC-accumbal pathway has been found after withdrawal from chronic exposure to cocaine, nicotine and opioids (Liechti and Markou, 2007; Moussawi et al., 2009; Olive, 2009). Here we found that the mGluR2 autoreceptor function is specifically disrupted after a history of alcohol dependence, which allowed us to develop a rescue strategy for restoring behavioral control in alcohol-dependent rats by focal mGluR2 overexpression.
Male Wistar rats, initial weight 220 250g, were used (Charles River, Germany), housed four/cage under a 12h light/dark cycle with ad libitum access to food and water. All behavioral testing was carried out during the dark phase, 5days a week. All experiments were conducted in accordance with the ethical guidelines for the care and use of laboratory animals, and were approved by the local animal care committee (Regierungspraesidium Karlsruhe, Germany). Five batches of animals were uniformly treated with either intermittent alcohol vapor or air exposure: Batch 1, n=10 per group for microarray and n=8 per group for in situ hybridization; batch 2, n=8 per group for LCM study; batch 3, n=8 per group for microdialysis; batch 4 (n=8 per group) and 5 (n=16 per group) for operant self-administration experiments.
Rats were weight-matched, assigned into the two experimental groups and exposed to either ethanol vapor or normal air using a rodent alcohol inhalation system as described previously (Rimondini et al., 2002). Briefly, pumps (Knauer, Berlin, Germany) delivered alcohol into electrically heated stainless steel coils (60°C) connected to an airflow of 18L/minute into glass/ steel chambers (1×1×1m). For the next 7 weeks rats were exposed to five cycles of 14-hr of ethanol vapor per week (0:00a.m.–2:00p.m.) separated by daily 10-hr periods of withdrawal (see supporting material). Twice per week blood (~20µl) was sampled from the lateral tail vein and blood alcohol concentrations were determined using an AM1 Analox system (Analox Instruments Ltd, London, UK). After the last exposure cycle rats remained abstinent for 2–3 weeks before further entering further experiments (3 weeks for gene expression and microdialysis analysis, 2 weeks for resumption of operant training).
Using a withdrawal rating scale according to Macey and colleagues 1, alcohol withdrawal signs including irritability to touch (Vocalization), body tremors, tail rigidity, and ventro-medial limb retraction were weekly scored, 6h after ethanol vapor was turned off. Each sign was assigned a score of 0–2, based on the following severity scale: 0 = no sign, 1 = moderate, 2 = severe. The sum of the 4 observation scores (0 to 8) was used as a quantitative measure of withdrawal severity. For these behavioral observations, animals were individually transferred from their home cages to a quiet observation room to avoid extraneous stimulation and animals were observed in a blind fashion.
3 weeks after the last exposure cycle, post-dependent (alcohol exposed, n=10) and control (air exposed, n=10) animals were killed during the first 4hr of the light cycle by decapitation, and brains were frozen in −40°C isopentane and kept at −80°C. Bilateral samples were obtained under a magnifying lens using anatomical landmarks (Paxinos and Watson, 1998). Amygdala, nucleus accumbens and medial prefrontal cortex (including Cg1+2, PL and IL) according to Paxonis& Watson 1998 were prepared as described previously (Arlinde et al., 2004). Briefly, Amygdala was prepared from a 2mm thick coronal slice, taken in a Kopf brain slicer by placing the rostral blade on the caudal edge of the optic chiasm. For preparation of cingulate cortex and accumbens, the rostral blade was placed 4mm rostral to this landmark, and a second 2mm coronal slice was obtained. Cortical tissue was dissected out with a scalpel, while amygdala and accumbens tissue was obtained using a punch (2mm diameter). Samples were stored at −80°C until RNA was prepared.
Total RNA was extracted with Trizol reagent (Gibco BRL Life Technologies, Baltimore, MD, USA) followed by an RNeasy (Qiagen, Hilden, Germany) column based clean-up step according to the manufacturer's instructions. All RNA samples showed A260/280 ratios between 1.9 and 2.1. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and only material without signs of degradation was used.
Microarray target preparation was done for individual samples and hybridization to RAE230A arrays, staining, washing and scanning of the chips were performed according to the manufacturer's technical manual (Affymetrix, Santa Clara, CA, USA). The Microarray Analysis Suite 5.0 (MAS5) produced CEL-files were inspected for regional hybridization bias and quality control parameters as recently described (Reimers et al., 2005). 48 microarrays (mPFC 9/9, Acb 7/7 and Amy 7/9, post-dependent vs. control rats) passed quality control. The MAS5 recognized approximately 60% of the 15 800 probe sets on the RAE230A array as present in our samples. Robust Multichip Average (RMA) expression values were obtained and tested for differential gene expression using Welch’s two-sample t test, assuming unequal variances at a p<.05 threshold. The microarray CEL-files were imported into GSEA software available at (www.broadinstitute.org/gsea/) and gene set enrichment analysis was performed against gene sets for glutamatergic and GABA-ergig neurons described by Sugino and colleagues (Sugino et al., 2006) (Table 1).
Human brain tissue samples were obtained from the New South Wales Tissue Resource Centre at the University of Sydney, Australia (http://www.pathology.usyd.edu.au/trc.ht). Tissue from 30 male subjects of European descent consisting of 15 chronic alcoholics and 15 control cases was used for this study. Subject affiliation to the alcoholics or control group was confirmed post-mortem using the Diagnostic Instrument for Brain Studies – Revised (DIBS-R) which is consistent with the criteria of the Diagnostic and Statistical Manual for Mental Disorders, 4th edition (DSM-IV) (American Psychiatric Association, 1994). All alcoholics had consumed more than 80 g of ethanol per day while the control cases had an average daily consumption of below 20 g. To reduce the number of confounding factors we tried to not include any subjects were the cause of death was suicide, the post-mortem interval was above 40 h, or blood alcohol or significant amounts of psychiatric medication (conc. > 1.0 mg/L) was detected at the autopsy whenever possible. For each subject we analyzed tissue samples from the anterior cingulate cortex.
RNA extraction and analysis was done as described (Sommer et al., 2010). RNA from brain tissue was isolated using Trizol according to manufacturer’s protocol (Invitrogen, UK). RNA samples underwent a cleanup step using the RNeasy Mini Kit (Qiagen, USA) and were then treated with RQ1 RNase-free DNase (Promega, USA) following manufacturer’s instructions, to eliminate DNA contamination. All RNA samples had acceptable 260/280 ratios (1.8 - 2.1). RNA samples were then analyzed with an Agilent 2100 Bioanalyzer and the RNA integrity number (RIN). 100 ng RNA was used for cDNA synthesis using reverse transcription reagents according to the manufacturer’s protocol (Applied Biosystems Inc., Foster City, CA, USA). For quantitative real-time PCR method, see below. In addition to Gapdh we used AluSX as a second endogenous control. Results were similar for both reference genes.
For stereotaxic injections of the retrograde tracer (n=8 per group), rats were anesthetised (Isofluran) and placed in a Kopf (Tujunga, CA) stereotaxic instrument and 300 nl of rhodamine-labelled fluorescent latex microspheres (Lumafluor, CA) were delivered to the nucleus accumbens shell at 70nl/min, using a WPI microinjection pump through a 33G beveled needle. The stereotaxic coordinates for the injections (Wistar rats, 500g) were A/P +1.8 mm, M/L +0.9 mm, and D/V −7.5 mm, relative to bregma. Following surgery, rats were single-housed for 2 days. After a 7-day recovery period, rats were euthanized for tissue collection as described below.
For Lentiviral injections, rats received 600nl of either Lenti-control or Lenti-mGluR2 to bilaterally into the infralimbic cortex at 70nl/min. The stereotaxic coordinates for the injections (500g wistar rats) were A/P +3.2 mm, M/L ±0.52 mm, and D/V −5.1 mm, relative to bregma.
Rats recovered for one week following stereotaxic tracer delivery. For perfusions, rats were anesthetised (Ketamin : Xylazin, 100mg/kg : 5mg/kg IP) and transcardially perfused with ice cold 50ml PBS followed by 80 ml 0.5% Paraformaldehyd containing 20% sucrose. After perfusions, brains were removed and flash-frozen in −40°C isopentanol and stored at −80°C up to 72h before sectioning.
Frozen brains cut into 12 µm thick coronal sections with a cryostat. Sections were mounted on Palm Membrane Slides® and kept at −80°C and process at the same day. Just prior to LCM, slides were thawed to −25°C, rapidly trimmed of tissue tech, and dehydrated with 75% ETOH (30s), 95% ETOH (30s), 100% ETOH (30s), and xylene (1 min), then air-dried for 5 minutes and immediately used for LCM.
LCM was performed using a Zeiss PALM® laser system. Tracer-labeled cells were identified using a CY3 advanced filter cube (excitation: BP 546/12 emission: BP 575 – 640). The laser focus followed a circular trajectory of 8–10µm in diameter to cut out and separate tracer positive cells from the adjacent tissue, following a final slightly subfocal laser pulse to catapult the cell into an LCM cup. Laser-targeted cells were bonded to adhesive LCM caps by aiming the laser beam at the thin plastic sheet in the cap directly above the target cell. Per animal approximately 70–100 cells were collected.
RNA was extracted with the RNeasy® Micro kit for microdissected cryosections. All steps were performed according to the manufactures recommendations. A speed vac (Vacufuge 2015727, Eppendorf, Germany) was used to dry down the eluted RNA to 3 µl for the further amplification step. Total RNA was amplified using the TargetAmp 2-Round aRNA Amplification Kit 2.0 (Epicentre, USA) according to manufacturer’s recommendations. We typically obtained 2 – 5 µg of amplified RNA after the second amplification round, respectively. Amplifications were performed from six exposed and seven control tracer cell RNA extractions.
100 ng total RNA was reverse transcribed using the High Capacity RNA-to-cDNA Master Mix [Applied Biosystems (ABI), Darmstadt, Germany] following the manufacturer’s protocol. Samples were assayed in triplicate in a total reaction volume of 20 µl using Power SYBR® Green PCR Master Mix (ABI) on an ABI 7900 HT RT-PCR System (40 cycles of 95° C for 15 sec and 60° C for 1 min). A melting profile was recorded at the end of each PCR to check for aberrant fragment amplifications. Primers for each target were designed towards the 3’ end of the coding sequence by considering exon-exon junctions when possible based on NationalCenter for Biotechnology Information (NCBI) reference sequence database. Amplicons were designed with 95–110 bp length and melting temperatures > 75°C to be able to distinguish between amplicons and primer-dimer formations in the melting analysis. For primer sequences see table 2. ABI’s SDS 2.2.2 software was used to analyze the SYBR Green fluorescence intensity and to calculate the theoretical cycle number when a defined fluorescence threshold was passed (Ct-values). Relative quantification was done according to the 2−ΔΔCT method whereby Actin, beta (Actb) was used as internal normalizer for rat tissue and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the human tissue. The 2−ΔΔCt method is defined, that a cycle (Ct) is the cycle at which there is a significant detectable increase in fluorescence; the ΔCt value is calculated by subtracting the Ct value for the endogenous control from the Ct value for the mRNA of interest. The ΔΔCt value is calculated by subtracting the ΔCt value of the control sample from the ΔCt of the experimental sample. For graphical interpretation, the ΔΔCt values were transformed (−x), thus downregulated genes show ΔΔCt< 0 and upregulated genes ΔΔCt> 0. The −ΔΔCt values were compared by an unpaired t-test for each gene (p < 0.05 = significance). Actb and GAPDH Ct-values were not different between groups. Statistical testing was done by t-test on the ΔΔCT values. The software and ΔΔCT method was used to determine statistical significance. Melting curves for all primers used in this study exhibited single fluorescence change peaks at the appropriate melting temperatures. This indicates the absence of primer–dimer formation.
Riboprobes and in situ hybridizations were performed as recently described (Hansson et al., 2008). In a parallel batch of animals to the microarray, post-dependent (alcohol exposed, n=8) and control (air exposed, n=8) animals were killed by decapitation during the first 4 hours of the light phase, and brains were frozen in −40°C isopentane and kept at −80°C. 10µm coronal brain sections were cryo-sectioned at forebrain bregma levels +3.0mm and +2.0 mm. The rat specific riboprobes for all genes have been generated based on gene reference sequence in PubMed database (http://www.ncbi.nlm.nih.gov/Entrez).
Egr-1: Position 1384 bp to 1851 bp on rat cDNA (gene reference number: NM_012551.1), mGluR2: Position 1327 bp to 1620 bp on rat cDNA (gene reference number: XM_343470.1), mGluR3: Position 314 bp to 662 bp on rat cDNA (gene reference number: XM_342626.1), NMDA receptor 2a: Position 434 bp to 876 bp on rat cDNA (gene reference number: RATNMDA2A), NMDA receptor 2b: Position 205 bp to 591 bp on rat cDNA (gene reference number NM_012574.1).
Phosphor imager (Fujifilm Bio-Imaging Analyzer Systems, Japan) generated digital images were analyzed using MCID Image Analysis Software (Imaging Research Inc., UK). Regions of interest were defined by anatomical landmarks as described in the atlas of Paxions & Watson and illustrated in Figure 2. Based on the known radioactivity in the 14C standards, image values were converted to nCi/g.
Three weeks after ethanol exposure, rats weighed 450–550g for surgery, were housed in groups of four before- and individually after surgery. Rats were anesthetized (isofluran, 1.5–2%) and placed in a stereotaxic frame (Kopf Instruments). CMA11 guide cannula (20 guage, 14 mm, CMA Microdialysis) were unilaterally implanted 2.0 mm above the nucleus accumbens shell (A/P, + 1.6 mm; M/L, ± 0.8 mm and D/V, 5.6 mm). Coordinates were based on bregma, midline and dura, respectively (Paxinos and Watson 1998). Cannulas were anchored with three stainless steel screws and dental acrylic. Animals were allowed to recover from surgery for one week.
Microdialysis experiments were conducted in conscious, freely moving rats, 3weeks after last ethanol vapor exposure. Dialysis probes (CMA11 11/2, CMA Solna, Sweden) with 2 mm active membrane were introduced into the guide cannula 12h prior to the beginning of the dialysis experiments in order to minimize damage-induced release of neurotransmitters and metabolites. Each animal participated in only one microdialysis experiment. Samples were collected every 15 minutes at a flow rate of 1.5µl/min. After 3 baseline samples, rats were injected with a saline solution as a control. 30 minutes later rats were injected i.p. 3mg/kg mGlur2/3 agonist LY379268 and sampling continued for the remaining time of the experiment.
8 µl of OPA/IBLC solution (ortho-pthaldialdehyde, Calbiochem; N-isobutyryl-L-cysteine, Fluka) were added to 20 µl microdialysate or standard volume. After three times mixing and a reaction time of 3 minutes, 14 µl were injected (CTC PAL autosampler, Axel Semrau GmbH, Germany) onto a HPLC column (Waters Xbridge C18 3.5 µm 10/2.1mm Guard Cartridge and Waters Xbridge C18 3.5 µm 100/2.1 mm). The mobile phase consisted of 50 mM Na2HPO4, 1 mM Na-EDTA, 20 % Methanol, pH 6.5 with phosphoric acid. Flow rate was set to 0.3 ml/minute (Rheos flux pump, Axel Semrau GmbH, Germany). Between every single injection the system was flushed with 20 µl acetonitrile. Glutamate was measured via a fluorescence detector (L-7480 Merck, Germany). The system was calibrated by standard solutions of glutamate containing 10pmol/10µl/injection. Glutamate was identified by its retention time and peak height with an external standard method using chromatography software (Chrom Perfect®, Justice Laboratory Software).
Rats were killed by transcardial perfusion with 0.9% saline (wt/vol) followed by 4% paraformaldehyde (PFA, wt/vol) in 1 × phosphate-buffered saline (PBS). Brains were postfixed in 4% buffered PFA at 4 °C for 12 h, dehydrated in 1×PBS-Sucrose(10%) solution for 3–7 days and flash-frozen at −80°C. Sections (14µm) were cut with a cryostat, cycled with an Immuno-pen, washed 1x 5 min (200 µl 0.01 M PBS pH 7.4 on the sections) and air dried. Sections were incubated with primary antibody in diluted 0.01 M PBS pH 7.4 + 0.3% Triton at 4°C over night, followed by appropriate secondary antibodies (diluted with 0.01 M PBS (pH 7.4) + 0.03 % Triton) for 1 h at room temperature. All antibodies were tested for optimal dilution, the absence of cross-reactivity and nonspecific staining. To detect the enhanced green fluorescent protein, we used rabbit eGFP, diluted 1:500 (Invitrogen, Germany) as primary antibody and donkey-anti-rabbit Alexa 488 diluted 1:600 (Invitrogen, Germany) as secondary antibody. For visualization of the mGluR2 we used a mouse-mGluR2 diluted 1:500 (SantaCruz, Germany) as primary antibody and the donkey-anti-mouse 594 diluted 1:800 (Invitrogen, Germany) as secondary antibody.
All alcohol-seeking experiments were carried out in operant chambers (MED Associates Inc., St. Albans, VT) enclosed in ventilated sound-attenuating cubicles. The chambers were equipped with a response lever on each side panel of the chamber. Responses at the appropriate lever activated a syringe pump that delivered a ~30µl drop of fluid into a liquid receptacle next to it. A light stimulus (house light) was mounted above the right response lever of the self-administration chamber. An IBM compatible computer controlled the delivery of fluids, presentation of stimuli and data recording.
The reinstatement protocol used in the present report is the one that is used by Ciccocioppo and Weiss (Ciccocioppo et al. 2002) with a slight modification, i.e., a syringe pump delivered a ~30µl drop of fluid into a liquid receptacle as opposed to 100µl drop of fluid in the Ciccocioppo protocol. This modification markedly increased responding for alcohol (~5-fold) that allowed us to better monitor animal’s motivation to receive alcohol.
All animal training and testing sessions were performed during the dark phase of their light cycle. Animals were trained to self-administer 10% (v/v) ethanol in daily 30-min sessions using a fixed-ratio 1 (FR 1) schedule using Samson’s sucrose-fading procedure (Tolliver et al., 1988). During the first three days of training, animals were kept fluid deprived for 20 hours per day. Responses at the left lever were reinforced by the delivery of 0.2% (w/v) saccharin solution. For the next three days, animals underwent the same procedure without fluid deprivation. Following acquisition of saccharin-reinforced responding, rats were trained to self-administer ethanol. During the next three sessions, responses at the left lever resulted in the delivery of 0.03 ml of 5% (v/v) ethanol +0.2% saccharin solution. Responses at the left lever were recorded but had no programmed consequences. Thereafter, the concentration of ethanol was increased first to 8% and then to 10% v/v and the concentration of saccharin was decreased until saccharin was eliminated completely from the drinking solution.
The purpose of the conditioning phase was to train the animals to associate the availability of ethanol with the presence of specific discriminative stimuli. This phase started after the completion of the saccharin-fading procedure. Discriminative stimuli predicting ethanol (10%) availability were presented during each subsequent daily 30-min session. An orange flavour extract served as the cue stimulus (S) for ethanol. This olfactory stimulus was generated by depositing six drops of an orange extract into the bedding of the operant chamber before each session. In addition, each lever press resulting in ethanol delivery was accompanied by a 5-s blinking conditioned light stimulus (CS). The 5-s period served as a "time-out", during which responses were recorded but not reinforced. At the end of each session, the bedding of the chamber was changed and trays were thoroughly cleaned. The animals received a total of 10 ethanol conditioning sessions. Throughout the conditioning phase, responses at the right lever were recorded but not reinforced (inactive lever). After the final conditioning phase, rats were sorted into two balanced experimental groups of which one was exposed to alcohol vapor (resulting in the post-dependent group) and the other received normal air (control group).
Following 2-week abstinence phase all animals were re-conditioned to self-administer 10% ethanol in 10 daily conditioning sessions. After completing the re-conditioning phase, rats were subjected to daily 30-min extinction sessions for 12 consecutive days, which in total were sufficient to reach the extinction criterion of less than 10 lever responses/session. Extinction sessions began by extending the levers without presenting olfactory discriminative stimuli. Responses at the previously active lever activated the syringe pump, without resulting in the delivery of ethanol or the presentation of response-contingent cues (stimulus light).
For reinstatement, animals were divided into 2 groups per condition (control and post-dependent) on the basis of their performance during the last four re-training sessions. After the last extinction trial, animals received bilateral stereotaxic injections in the infralimbic cortex (for details see section stereotaxic injections). Reinstatement began seven days after the final extinction session. In these tests, rats were exposed to the same conditions as during the conditioning phase, except that the ethanol was not available. Sessions were initiated by the extension of both ethanol-associated and inactive levers and the presentation of the discriminative stimulus predicting ethanol (S). Responses at the ethanol-associated lever were followed by the activation of the syringe pump without any ethanol delivery and the presentation of the CS (light).
The mGluR2 cDNA was amplified using the IMAGp998E1215366Q clone as template (Imagenes, Berlin, Germany). After purification, the cDNA was cloned into the pCDH-MCS-T2A-copGFP vector (Biocat, Heidelberg, Germany). The vector containing the mGluR2 insert was purified, sequenced, tested in cell culture and finally used for lentiviral production. Active lentiviral particles were produced by System Biosciences, CA 94043, USA.
Microarray, PCR and in situ hybridization data were compared by t-test. Data from the microdialysis experiment were analyzed using 2-way repeated measures ANOVA. Behavioral experiments were analyzed by 2-way ANOVA or t-test where appropriate. Post-hoc testing was done with Fisher LSD. The withdrawal scores were compared using a Mann-Whitney test. Statistical significance was set at a p<0.05. Statistica 10.0, Software for Windows was used (StatSoft, Inc., Tulsa, OK).
We started with an unbiased transcriptome analysis to determine potential targets of alcohol-induced neuroadaptations, classified the affected cell-types in the region, and identified candidate genes for further experiments. Microarray based transcriptome analysis revealed that chronic intermittent alcohol exposure had long-term effects on gene expression in three brain regions implicated in drug dependence, namely mPFC, nucleus accumbens and amygdala (Koob and Volkow, 2009)(Figure 1A). We used gene set enrichment analysis (GSEA) (Subramanian et al., 2005) to test the hypothesis of functionally related post-dependent neuroadaptations in GABA-ergic or glutamatergic neurons. For this purpose we employed two marker gene sets previously described as extremely divergent between GABAergic and glutamatergic neurons (Sugino et al., 2006). Results indicate a highly significant enrichment of downregulated glutamatergic marker genes (p<0.01) in the mPFC of post-dependent rats (Figure 1B,C and table 1). We selected a number of candidate genes for corroborative analysis by quantitative PCR (Table 3). Among the confirmed candidates was Grm2, the gene coding for metabotropic glutamate receptor subtype 2 (mGluR2), which was robustly downregulated in the mPFC of post-dependent rats compared to controls. We next used in situ hybridization to address the question whether or not a specific subregion of the mPFC is preferentially affected in post-dependent rats. Several genes derived from the transcriptome study, i.e. members of the activity-dependent Egr-family (Egr1 and Egr2) and glutamate receptors (Nr-2a, Nr-2b, Grm2) showed significant downregulation only in the infralimbic cortex, with the most profound effect again the gene for mGluR2 (Figure 2A and B). In contrast, the expression of the pharmacologically highly similar mGluR3 was not altered in this region (MEAN nCi/g±SEM; infralimbic cortex: control: 40.80 ± 2.13, post-dependent: 41.59 ± 2.19, NS; PL: control: 51.49 ± 1.75, post-dependent: 53.06 ± 1.09, NS). Together, these findings suggest that the IL is a hot spot within the mPFC for alcohol dependence induced alterations.
Together, these experiments lead to the conclusion that glutamatergic neurons in the infralimbic cortex are highly sensitive to alcohol-induced neuroplasticity. To identify the specific neurocircuitry involved, we utilized a strategy that allows labeling pyramidal neurons within the infralimbic cortex via their projections to the nucleus accumbens shell subregion. We performed retrograde tracing by infusing rhodamine-labelled fluorescent latex microspheres into the nucleus accumbens shell (Katz and Iarovici, 1990; Reynolds and Zahm, 2005), isolated the labeled cell population (~70–100 cells) within the infralimbic cortex through Laser-Capture-Microscopy (LCM), and extracted the RNA for expression analysis (Figure 3A and B).
We tested 8 candidate genes from the mPFC microarray experiment (Table 3). Among these, Grm2 as well as the Egr-family genes Egr2 and Egr4 were identified as significantly downregulated in the infralimbic cortex neurons of the post-dependent group. Expression differences detected within the purified neuronal population were markedly enhanced compared to the analysis carried out on tissue homogenates. Expression of Grm2 and Egr2 was about 10 respectively 500-fold altered in enriched infralimbic projection neuron populations from post-dependent rats (Figure 3C), while these differences were <2-fold when applying the same PCR analysis to tissue homogenates. The experiment reveals the extent to which major dysregulation can be disguised in heterogeneous samples and emphasizes the importance of studying well characterized cell populations in the brain. In conclusion, we demonstrate that infralimbic-accumbal glutamatergic projection neurons are highly sensitive to alcohol dependence-induced neuroadaptations, and identify mGluR2 receptor downregulation in this pathway as a candidate mechanism for observed behavioral deficits.
mGluR2 function in the cortico-accumbal pathway was assessed by in vivo microdialysis. We measured extracellular glutamate levels in the nucleus accumbens shell of freely moving rats (Figure 4A). Given its role as a presynaptic autoreceptor, stimulation of mGluR2 is expected to downregulate glutamate release, resulting in reduced glutamate overflow in the dialysate. Accordingly, systemic administration of the mGluR2/3 agonist LY379268 (3mg/kg, i.p.) resulted in a robust and sustained decrease of extracellular glutamate levels in the nucleus accumbens shell of control rats. In contrast, no such effect was seen in post-dependent rats (Figure 4B). Basal glutamate levels were not different between post-dependent and control rats (Figure 4B). These data are consistent with the interpretation that the downregulation of Grm2 expression could lead to a lack of mGluR2 autoreceptor function at the terminals of the infralimbic projection neurons. Such a deficit would impact on activity-dependent glutamatergic neurotransmission in the corticostriatal pathway and presumably also on behavioral output.
We next examined the role of mGluR2 receptors in infralimbic neurons projecting to the nucleus accumbens shell for cue-induced reinstatement of alcohol-seeking behavior, an established animal model of relapse (Epstein et al., 2006; Sanchis-Segura and Spanagel, 2006). First, rats were trained to self-administer alcohol before alcohol vapor exposure (experimental design see Figure 5A–E). After the last exposure cycle, post-dependent rats showed clear signs of withdrawal (about 5 out of maximal 8 points from a global withdrawal score (Macey et al., 1996)), whereas this rating for control rats was close to zero (p < 0.004; Mann Whitney, Figure 5B). After two weeks of recovery, all rats were retrained to self-administer ethanol until stable response rates were achieved once more. Control rats regained self-administration rates that were similar to their pre-exposure rates (90% of pre-exposure). In contrast, post-dependent rats rapidly escalated their self-administration rates in more than 155% (Figure 5A,D). Motivation to obtain alcohol was further assessed by a progressive ratio (PR) reinforcement schedule (Hodos, 1961). Post-dependent rats showed a significantly higher break point for alcohol self-administration than controls (t=3.09, p< 0.01; Figure 5C) and a significantly steeper slope of the correlation between response rates during ethanol self-administration under an FR1 schedule, and progressive ratio breakpoints (correlation equations test, p<0.05, Figure 5F). This shows that escalated alcohol self-administration in post-dependent rats is associated with an increased motivation to obtain the drug reinforcer, a key characteristic of addictive behavior (Deroche-Gamonet et al., 2004). These data are consistent with a recent report that also showed increased motivation to obtain ethanol following a history of experimenter imposed alcohol dependence (Kufahl et al., 2011; Vendruscolo et al., 2012).
Alcohol associated cues are potent triggers of relapse in alcoholic patients. This pathological behavior is typically modeled in the reinstatement procedure (Epstein et al., 2006; Sanchis-Segura and Spanagel, 2006). Following stable lever responding accompanied by discrete cues predicting alcohol availability (CS+) post-dependent and control rats underwent extinction (Figure 5D) followed by a cue-induced reinstatement test. Post-dependent rats displayed significantly higher reinstatement of alcohol-seeking than control rats (p<0.01) (Figure 5E). To directly assess the role of mGluR2 in mPFC for cue-induced reinstatement of alcohol-seeking, we generated two lentiviral vectors expressing either the mGluR2 receptor together with the green fluorescence protein (EGFP) or the EGFP (lenti-mGluR2 and lenti-control, respectively; Figure 6A). Following alcohol/cue training all rats went through extinction training, resulting in fewer than 10 responses (Figure 6F). After completion of extinction training, rats were bilaterally injected with the respective lentiviral constructs into the infralimbic cortex (Figure 6B–F), allowed to recover, and examined for cue-induced reinstatement of alcohol-seeking. Notably, immunohistochemistry confirmed the co-expression of the lenti-mGluR2 construct for all studied animals exclusively in the infralimbic cortex and in their neuronal projection target, the nucleus accumbens shell, EGFP positive axon terminals were clearly visible (Figure 6D and E). Presentation of the ethanol-associated cues resulted in significant resumption of operant responding in animals receiving the control lenti construct (paired t-test; control: t7=4.157, p<0.01; post-dependent: t7=4.211, p<0.01; Figure 6F), with highly increased mean (± SEM) number of responses in post-dependent (87.6±8.9) compared to control (51.9 ± 8.1) rats. Lenti-mGluR2 did not significantly alter drug-seeking behavior in controls. However, lenti-mGluR2 showed significant reduction in post-dependent animals, such that their lever pressing behavior declined for about 40% to control levels. These effects were confirmed by two-way ANOVA with significant main effects of ethanol dependence history [F1,24=9.947, p<0.01] and virus treatment [F1,24=7.932, p<0.01], but no significant interaction [F1,43=2.032, p=0.163]. We did not find time-dependent spontaneous recovery of lever pressing when re-exposing the animals to the operant chamber after the one week time delay between last extinction trial followed by sham operation and reinstatement test (Extinction 8.9 ± 1.3, spontaneous recovery 10.9 ± 2.5, NS). We also did not find evidence for behavioral abnormalities (for example, weight loss, agitation and self-injury) in any experimental group. Lenti-control and lenti-mGluR2 rats did not differ in locomotor activity or their responding for natural rewards under the same reinforcement schedule used for ethanol self-administration (Figure 7A–C), demonstrating that effects of mGluR2 overexpression on reduced ethanol-seeking behavior were not secondary to alterations in task performance.
To translate these animal findings to humans, we determined GRM2 expression in post-mortem brain tissue samples from alcohol dependent patients and controls matched for age and post-mortem interval (Sheedy et al., 2008).
A human brain region that is anatomically and functionally related to the rodent mPFC is the anterior cingulated cortex (Uylings et al., 2003). However, it has to be pointed out that a one-to-one relationship between human and rodent prefrontocortical regions does not exist and functional elements of rodent distinctions including of the infralimbic cortex can be found in various areas of the enlarged human prefrontocortical volume. Within the anterior cingulate cortex we found a significant, 2.6-fold decrease in GRM2 transcript levels in alcoholics compared to controls (Figure 8A and B).
The data presented here provide a fundamentally new insight into the molecular basis by which a prolonged history of alcohol dependence causes a substantial and long-lasting reorganization of the medial prefrontal cortex. To the best of our knowledge, the present data from gene expression and functional studies constitute strong experimental evidence of anatomical and molecular pathway specific plasticity in the mPFC as a sequel to alcohol dependence and establish a key pathophysiological mechanism for the increased propensity to relapse. In particular, we discovered a locally restricted but profound molecular pathology, namely the infralimbic cortex specific expression deficit of mGluR2 as a critical component for excessive alcohol seeking in post-dependent rats and that restoring this receptor function is sufficient for regaining control over this addictive behavior.
The infralimbic cortex shows a unique pattern of alcohol dependence induced alterations, as evidenced by the regional specific downregulation of transcription factors Egr1 and Egr2 known to be involved in neuronal plasticity, as well as on the glutamate receptor genes Grin2a, Grin2b and Grm2. Importantly, the downregulation of Grm2 and Egr2 is much more pronounced in purified infralimbic-accumbens shell projection neurons, about 10-, respectively 500-fold, highlighting these genes within this cell population as functionally relevant for the pathological process. Notably, the Grm2 promoter contains transcription factor binding sites for the Egr-family as well as a unique Egr2 binding site (according to the DECODE database at http://www.sabiosciences.com/chipqpcrsearch.php?app=TFBS), which provide a potential substrate for regulation of Grm2 expression by Egr2. On the other hand, mGluR2 may regulate Egr2 expression as suggested by experiments in Grm2 knockout mice which show a lack of Egr2 activation following drug application (Moreno et al., 2011). Whether or not the downregulation of these genes is functionally related is yet unknown, but both seem to be involved in dependence-related plasticity of glutamatergic neurons, mGluR2 directly at the level of the synapse, Egr2 via stimulus-transcription coupling.
Importantly, we found a lack of mGluR2 receptor function in the terminal fields of the infralimbic projections which became evident as an inability of these neurons to modulate nucleus accumbens shell glutamate levels in response to receptor stimulation with an mGluR2/3 agonist. This effect is consistent with the pronounced reduction in mGluR2 expression – with no change in mGluR3 expression - in the infralimbic cortex of post-dependent rats. However, a recent study found no differnces in mGluR2/3 functional activity within the mPFC in post-dependent rats after 4 weeks of repeated cycles of vapor exposure (Kufahl et al., 2011). How can this discrepancy been explained? We have previously demonstrated that in the alcohol vapor exposure procedure a temporal threshold for induction of escalation of alcohol consumption and concomitant neuroplastic changes occurs (Rimondini et al., 2003). Hence, post-dependent rats which were exposed to alcohol vapor for 7 weeks displayed a marked increase in alcohol self-administration whereas post-dependent rats exposed for shorter periods (2 and 4 weeks) did not show such an escalation (Rimondini et al., 2003). Here we report that post-dependent rats exposed to alcohol vapor for 7 weeks show an augmented reinstatement response of alcohol-seeking behavior and that this dependence -like phenotype is directly linked to a mGluR2 deficit in the infralimbic cortex. In the Kufahl et al. study (2011) rats were exposed to alcohol vapor for 4 weeks and did neither differ in their reinstatement response nor in mGluR2/3 functional activity from controls which supports the definition of a temporal threshold for induction of escalation in alcohol consumption and alcohol seeking and the herewith associated neuroplastic changes. Taken together, a careful, cell-type specific investigation of the group II mGluRs shows a highly restricted mGluR2 downregulation in sparsely distributed glutamatergic neurons located in the ventral part of the mPFC, the infralimbic region.
With our viral rescue experiment we could further show that mGluR2 receptors in infralimbic neurons are necessary for the control exerted by this region on alcohol seeking. Consequently, infralimbic neurons in post-dependent animals are capable of eliciting a sufficient glutamate response to drug cues, but in the absence of feedback provided by mGluR2 receptors, the information transmitted by this signal cannot be properly processed thereby disrupting adequate behavioral control. On the other hand, adding extra mGluR2 autoreceptors to normal infralimbic neurons does not seem to disrupt glutamatergic signaling and behavioral output in a task controlled by this brain structure. This concept is further supported by electrophysiological evidence from long-term cocaine exposed rats., Using in vivo stimulation from the prefrontal cortex of long term cocaine exposed rats revealed a mGluR2/3 deficit in the nucleus accumbens (Moussawi et al., 2011). In another model of cocaine induced addiction-like behavior there was a lack in mGluR2/3-mediated long-term depression in mPFC neurons that was associated with a strong downregulation of mGluR2/3 receptors (Kasanetz et al., 2012). Thus, both alcohol and cocaine dependence are associated with medioprefrontal mGluR2 deficits that may lead to an inflexible state of the brain. Although we did not provide electrophysiological evidence, our study substantially extends the findings from the cocaine models by demonstrating that an addiction-like behavior, here excessive alcohol seeking, can be rescued through restoring mGluR2 levels in the mPFC.
Impairments in executive control over behavior are known risk factors for drug addiction (Everitt and Robbins, 2005). Alcohol dependent patients have severe deficits in many aspects of prefrontocortical functions encompassing emotion, cognition and behavior, whereby medial subdivisions of the prefrontal cortex are of particular interest here because of their role in motivation, control of emotions, salience attribution and decision making (Goldstein and Volkow, 2011). These functions have been established not only in humans but also in rodents (Uylings et al., 2003). Typical behaviors seen in patients with damage to the ventromedial PFC are social inappropriateness, impulsivity and poor judgment (Bechara et al., 1994). Enduring medioprefrontal grey matter losses were found in alcoholic patients and are associated with severe functional deficits in the ability to control reward predicting stimuli (Duka et al., 2011). Interestingly, these deficits increase with the number of detoxifications experienced by the patients, which resonates with previous observations in experimental animals that the number of withdrawals, rather than the mere level of intoxication, is important for the occurrence of long-lasting behavioral and neural symptoms, i.e. a post-dependent state (Roberts et al., 2000; Stephens et al., 2005; Sommer et al., 2008; Heilig et al., 2010). A previous fMRI study in alcoholics found increased mPFC activation in response to alcohol cues, which was positively correlated with relapse risk (Grüsser et al., 2004). In experimental animals cue presentation of conditioned stimuli predicting a drug reward results in a significant increase in glutamate levels in the nucleus accumbens (Hotsenpiller et al., 2001). Most likely, this input derives from prefrontal areas, given that the mPFC-accumbal glutamatergic pathway is necessary for reinstating drug-seeking behavior. Likewise, our observed deficit in mGluR2 autoreceptor function within the infralimbic cortex of post-dependent rats may lead to increased accumbal glutamate levels after cue-presentation with subsequent excessive drug-seeking behavior.
Importantly, we also find a reduction in GRM2 expression in the anterior cingulate cortex from alcohol dependent patients, which suggests that the deficits found in our animal model may be a feature in alcoholism in at least some patients. It remains to be clarified whether or not the reduced GRM2 expression found in the present sample is functionally linked to the progressive reduction in prefrontal neuronal density which was seen in an earlier study on post-mortem brain tissue from alcoholics (Miguel-Hidalgo et al., 2006). However, the reduction in GRM2 expression and number of neurons may together lead to an absolute deficit of mGluR2 receptors in the mPFC of alcoholics. This may have important implications for the development of treatments for relapse prevention because absolute deficits cannot be efficiently targeted by agonist treatment. Indeed, this may be one of the reasons for the relatively narrow therapeutic window for reducing alcohol-seeking in experimental animals by mGluR2/3 agonists (Kufahl et al., 2011). Thus, instead of focusing on the development of more specific mGluR2 ligands, novel therapeutic strategies should attempt to overcome the blockade of mGluR2 expression. Focal virus-mediated gene therapy, although potentially feasible (Kaplitt et al., 2007) is unlikely to be applied for the treatment of addictions.. Alternatively, pharmacological approaches targeting key proteins involved in glutamate homeostasis, such as glutamate transporters or mGluRs, could potentially be effective treatments in relapse prevention. In conclusion, the present study illustrates the feasibility of a structured discovery strategy, that starting with an unbiased screening over progressively narrowing experimental approaches allows identifying a specific pathological mechanism and can point towards new directions for therapeutic development.
The authors would like to thank Elisabeth Röbel and Fernando Leonardi-Essmann for assistance in laboratory experiments. Funding was obtained by the Bundesministerium für Bildung und Forschung within the frameworks of NGFN plus (NGFN Plus; FKZ: 01GS08151, 01GS08152 and 01GS08155, see under www.ngfn-alkohol.de and (Spanagel et al., 2010)) and ERA-Net TRANSALC (FKZ: 01EW1112), the European Commission FP-6 Integrated Project IMAGEN (PL037286), by Deutsche Forschungsgemeinschaft (center grant SFB636, project grant to ACH, HA 6102/1-1). MH was supported by the Intramural Research Program of the NIAAA.
Conflict of Interest
We declare no conflict of interest