Neuropeptide Y+Alcohol+GABA+Y2

N co A n T de Ni , Ma M Ba euro me itory (Ce Me exce alc hole GA ed in of Re info gra onde GA t of i act Co sign tar uron ne pend Ke for A pre consequences, compulsive alcohol-seeking behavior, loss of con- tro alc sta pro inv str ute wi see ex me dra co po are he rel pe Ne dan spe am be Recently, it hasbeen shown thatNPY suppresses alcohol drinking in Fro Ad Rec 00 do l limiting intake, andanegative emotional state in theabsenceof ohol (1). Amajor goal of basic research on alcoholism is to under- nd the neural underpinnings of alcohol use and the pathological gression to alcohol dependence (2). The transition from casual drinking to alcohol dependence olves numerous neuroadaptive changes in brain reward and ess systems. The recruitment of brain stress systems contrib- s to the aversive aspects of alcohol withdrawal and, along th dysregulation of brain reward systems, promotes alcohol- king behaviors and relapse (3). Brain stress systems in the tended amygdala (4) appear to be especially important in diating motivational deficits associated with alcohol with- wal and excessive alcohol drinking. The extended amygdala ntains the central nucleus of the amygdala (CeA), the lateral rtion of the bed nucleus of the stria terminalis, and a transition a in the nucleus accumbens shell. These three regions are avily interconnected and have high concentrations of stress- ated neuropeptides. rats (10) via its actions in CeA (11–14). More specifically, NPYmicro- injection into the CeA exhibits an enhanced ability to suppress alcohol drinking in certain subpopulations of drinkers, including rats that are made dependent on alcohol via vapor inhalation. In vitro electrophysiological studies have revealed that acute alcohol facilitates spontaneous and evoked GABAergic transmis- sion in the CeA via presynaptic and postsynaptic mechanisms (15). In CeA neurons from rats exposed to chronic alcohol vapor, base- line GABAergic transmission is increased and CeA neurons do not exhibit tolerance to the facilitatory effects of acute alcohol (16). Recent data have highlighted the effects of various neuropeptides onalcohol-induced facilitationof inhibitory transmission in theCeA of rats. For example, corticotropin-releasing factor (CRF) facilitates (17) and nociceptin opposes (18) GABAergic transmission in the CeA. In thepresent study,we tested thehypothesis thatNPYhas a key role in the development of excessive alcohol drinking during the transition to alcohol dependence. We also examined the effects of NPY on baseline GABA type A (GABAA)-mediated inhibitory trans- mission in the CeA and the interactions of NPY and acute alcohol in alcohol-naive and alcohol-dependent rats, as well as the synaptic mechanisms and NPY receptor subtypes responsible for these ef- fects.We report thatNPYadministrationblocks thedevelopmentof excessive alcohol drinking associated with the transition to alcohol dependence and that NPY opposes alcohol effects on GABAergic transmission inCeA, likely via activationofpresynaptic Y2 receptors. m the Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, California. dress correspondence to Nicholas W. Gilpin, Ph.D., The Scripps Research Institute, Committee on Neurobiology of Addictive Disorders, SP30- 2400, 10550 N Torrey Pines Road, La Jolla, CA 92037; E-mail: nickg@ scripps.edu. eived Nov 29, 2010; revised Feb 4, 2011; accepted Feb 7, 2011. BIOL PSYCHIATRY 2011;69:1091–109906-3223/$36.00 i:10.1016/j.biopsych.2011.02.004 © 2011 Society of Biological Psychiatry europeptide Y Opposes Al minobutyric Acid Release i ransition to Alcohol Depen cholas W. Gilpin, Kaushik Misra, Melissa A. Herman arisa Roberto ckground: During the transition to alcohol and drug addiction, n diate aspects of withdrawal and relapse via convergence on inhib A). thods: This study investigated the role of neuropeptide Y (NPY) in ohol vapor inhalation. This study also utilized intracellular and w BAergic inhibitory transmission in CeA, synapticmechanisms involv alcohol-naive and alcohol-dependent rats. sults: Chronic NPY treatment blocked excessive operant alcohol-re dual increases in alcohol responding by intermittently tested n BAergic transmission and reversed alcohol-induced enhancemen ions at presynaptic Y2 receptors. nclusions: These results highlight NPY modulation of GABAergic get for the treatment of alcoholism. Gamma-aminobutyric acid ne uromodulator systems recruited during the transition to alcohol de y Words: BIBP3226, BIIE0246, central amygdala, negative rein- cement, Y1 receptor, Y2 receptor lcoholism, or alcohol dependence, is a progressive and chronically relapsing disorder. The development of alcohol- ism is characterized by frequent episodes of intoxication, occupation with alcohol and the use of alcohol despite adverse hol Effects on Gamma- Amygdala and Blocks the nce ureen T. Cruz, George F. Koob, and modulator systems in the extended amygdala are recruited to gamma-aminobutyric acid (GABA) neurons in central amygdala ssive alcohol drinking by making rats dependent on alcohol via -cell recording techniques to determine the effects of NPY on these NPY effects, and NPY interactions with alcohol in the CeA rced responding associated with alcohol dependence, as well as pendent control animals. Neuropeptide Y decreased baseline nhibitory transmission in CeA by suppressing GABA release via aling in central amygdala as a promising pharmacotherapeutic s in the CeA likely constitute a major point of convergence for ence. Several neuropeptides have prominent roles in the aversive as- cts of alcohol withdrawal and relapse via their actions in the CeA. uropeptide Y (NPY) is an inhibitory peptide produced in abun- ce in the hypothalamus and phylogenetically conserved across cies (5). Neuropeptide Y is highly co-localized with gamma- inobutyric acid (GABA) in the amygdala (6), which is important cause NPY reduces anxiety (7) via actions in the amygdala (8,9). Methods andMaterials Be Kin sur pla ho an sio gu Use bo (19 rat an lev ing ad com res lize can me mi inf an [aC (no veh ven de No im (21 tim rec (Ha the lef ad (PE Ha de the mu ho gro at vap occ rat Lo tes we 10 tha dri consumption (grams ethanol per kilograms body weight), and eth- anol preference (ethanol consumed/total fluid consumed) using thr wh po wit 11, alc ual fes ho sta Ele or pre (12 cha cut no 30 fus aC po sul ate sol tia ne 8h sha ing mV pC ing me sup din ph nis par toc the ten 1 to at 5 firs fro tud sur sup mi on Po ron fer Ca ter ron 1092 BIOL PSYCHIATRY 2011;69:1091–1099 N.W. Gilpin et al. ww havioral Studies Animals. We used 42 adult male Wistar rats (Charles River, gston, New York). The average body weight before vapor expo- e was 541.7 � 21.4 g. Animals were single-housed in standard stic cages with wood chip bedding under a 12-hour light/12- ur dark cycle (lights off at 8:00 AM) with ad libitum access to food d water throughout except during experimental drinking ses- ns. All procedures were conducted in the dark cycle andmet the idelines of theNational Institutes of HealthGuide for the Care and of Laboratory Animals (National Research Council, 1996). Operant Alcohol Self-Administration Training. Operant xes and training procedures have been described previously ). Briefly, we trainedWistar rats to respond on a continuous fixed io-1 schedule for .1 mL deliveries of supersaccharin (3% glucose d .125% saccharin; see [20]) versus water in a concurrent, two- er, free-choice contingency during 30-minute sessions. Follow- these operant training sessions, 10% (wt/vol) ethanol was ded and sweeteners gradually removed from the solution. Upon pletion of this fading procedure, we allowed rats 25 operant ponding sessions for 10% (wt/vol) ethanol versus water to stabi- operant responding. Rats were stereotaxically implanted with nulae and subsequently divided into four groups based on an intakes during four postsurgery prevapor operant self-ad- nistration sessions: 1) chronic alcohol vapor exposure and NPY usion (dependent-NPY,n� 7); 2) chronic alcohol vapor exposure d vehicle infusion (dependent-artificial cerebrospinal fluid SF]; n � 8); 3) chronic air vapor exposure and NPY infusion ndependent-NPY; n� 7); and 4) chronic air vapor exposure and icle infusion (nondependent-aCSF; n� 8). Stereotaxic Surgeries. We surgically implanted intracerebro- tricular (ICV) cannulae using aseptic procedures as previously scribed (11), after isoflurane anesthesia (Abbott Laboratories, rth Chicago, Illinois). A guide cannula (22 gauge)was unilaterally planted according to the appropriate stereotaxic coordinates ), with a dummy cannula (28 gauge) in the guide cannula at all es except during infusions. We monitored rats for a 1-week overyperiod todetermine that animals resumednormal activity. Microinfusions. We used a Harvard 33 microinfusion pump rvard Apparatus, Holliston, Massachusetts) for all infusions into lateral ventricles (ICV) at a rate of 2.5 �L/min for 2 minutes and t the injection cannula in place 1 additional minute to allow for equate diffusion.We delivered infusions via polyethylene tubing 20; Becton Dickinson & Co, Glencoe, Maryland) connected to a milton 25 �L syringe (Hamilton Company, Reno, Nevada). Operant Tests During Alcohol Vapor Exposure. We infused pendent and nondependent rats with NPY or vehicle and tested m for operant behavior at the 6-hour withdrawal time point on ltiple days during the first 15 days of chronic intermittent alco- l vapor exposure (CIE) (Supplement 1). Rats in the dependent ups were exposed to alcohol vapor 14 hours per day (vapor off 8:00 AM); rats in the nondependent groups were exposed to air or 24 hours per day. Operant tests and NPY infusions never urred on the same day. On days 2, 4, 6, 8, 10, 12, and 14 of CIE, s received either .0 �g or 10.0 �g NPY ICV (Sigma-Aldrich, St. uis, Missouri) in 5.0 �L aCSF. On days 3, 7, 11, and 15 of CIE, we ted rats for operant alcohol responding. On day 16 of CIE, rats re sacrificed and cannula placements histologically verified. The .0 �g NPY dose was chosen based on prior studies that showed t acute ICV infusion of this dose reliably suppresses ethanol nking in rats (10,12). Statistical Analysis. We analyzed operant responses, ethanol w.sobp.org/journal ee-way repeated-measures (RM) analyses of variance (ANOVAs), ere between-subjects factors were vapor exposure (alcohol va- r vs. air vapor) and NPY dose (.0 �g vs. 10.0 �g) and day was the hin-subjects factor (baseline vs. operant tests on vapor days 3, 7, and 15). Because a priori differences were not expected early in ohol vapor exposure,we also analyzedoperant test days individ- ly to determine when vapor effects and/or NPY effects mani- ted during the transition to alcohol dependence. Wemade post c comparisons using the Student-Newman-Keuls test and set tistical significance at p� .05. ctrophysiological Studies Slice Preparation. Following �2 weeks of CIE (14 hours/day) air vapor exposure (Supplement 1), we prepared CeA slices as viously described (14,15). Briefly, male Sprague-Dawley rats 0–300 g; 4–7 weeks old, n � 57) were removed from vapor mbers, anesthetized with halothane (3%), and decapitated. We 400-�m coronal slices on a Vibratome Series 3000 (Leica, Ban- ckburn, Illinois), incubated them in an interface configuration for minutes, then completely submerged and continuously super- ed (flow rate of 2–4 mL/min) them with warm (31°C), gassed SFof the following composition inmmol/L: sodiumchloride, 130; tassium chloride (KCl), 3.5; sodium phosphate, 1.25; magnesium fate heptahydrate, 1.5; calcium chloride, 2.0; sodium bicarbon- , 24; glucose, 10. Drugs were added to the aCSF from stock utions to obtain known concentrations in the superfusate. Intracellular Recording of Inhibitory Postsynaptic Poten- ls in CeANeurons. We recorded in alcohol-free aCSF from CeA urons (n� 64) of alcohol-naive or alcohol-dependent rats for 2 to ours after cutting, aspreviouslydescribed (15).We recordedwith rpmicropipettes (3mol/L KCl) using current-clampmode, hold- potentials near resting membrane potential (mean Vm � �77 ). Data were acquired with an Axoclamp-2A preamplifier and LAMP software (Axon Instruments). We used a bipolar stimulat- electrode to evokepharmacologically isolatedGABAA receptor- diated inhibitory postsynaptic potentials (IPSPs), while erfusing the slices with the glutamate receptor blockers 6,7- itroquinoxaline-2,3-dione (10 �mol/L) and DL-2-amino-5-phos- onovalerate (30 �mol/L) and the GABA type B receptor antago- t CGP 55845A (1 �mol/L). To determine the IPSP response ameters for each cell, we performed an input-output (I/O) pro- ol (14,15). The stimulus strengths were maintained throughout duration of the experiment. We normalized three stimulus in- sities of equal steps (threshold, half-maximal, and maximal) as 3X. We examined paired-pulse facilitation (PPF) using paired stimuli 0-millisecond interstimulus intervals (15). The amplitude of the t IPSP was 50% of themaximal stimulus strength, as determined m the I/O relationship. We calculated the PPF ratio as the ampli- e of the second IPSP divided by that of the first IPSP. All mea- es were taken before the first drug superfusion (control), during erfusion (15–20 min per drug), and following washout (�15 n). To avoid tachyphylaxis, we superfused each drug only once to a single cell. Whole-Cell Patch-Clamp Recording of Miniature Inhibitory stsynaptic Currents in CeA Neurons. We visualized CeA neu- s in brain slices (350–400 �m) using infrared differential inter- ence contrast and CCD camera (EXi Aqua, QImaging, Surrey, BC, nada).Weused aw60water immersionobjective (Olympus, Cen- Valley, Pennsylvania) for identifying and approaching CeA neu- s. Whole-cell voltage-clamp recordings were made with a Mul- ticlamp 700B amplifier (Molecular Devices, Sunnyvale, California), low lec 10 pu ne L): 2; H 5’-t uo con pe �m (1 mi cla tw pro .05 err cor a lo we pa sen Re Be tra (i.e fro vap a g alc att tio CIE rat an CIE ho ma wa an inf 15 do an F (1 (p eff Tw tes op 5.8 NP .07 for exp alc thr tha F (1 chr do rev in a Ele (15 no de cre fig to cou no IPS �m Fig van hol Chr ind also (n � wit (ind on sup vap arti of N pen N.W. Gilpin et al. BIOL PSYCHIATRY 2011;69:1091–1099 1093 -pass filtered at 2 kHz to 5 kHz, digitized (Digidata 1440A; Mo- ular Devices), and stored on a personal computer using pCLAMP software (Axon Instruments). Patch pipettes (4-8M�) were lled from borosilicate glass (Warner Instruments, Hamden, Con- cticut) andfilledwith an internal solution composedof (inmmol/ KCl, 145; ethyleneglycol tetraacetic acid, .5;magnesiumchloride, EPES, 10; adenosine 5’-triphosphate disodium salt, 2; guanosine riphosphate sodiumsalt, .2. Series resistance (�10M�)wascontin- usly monitored with a 10 mV hyperpolarizing pulse. Drugs were stituted inaCSFandappliedbybath superfusion. Recordingswere rformed in the presence of 6,7-dinitroquinoxaline-2,3-dione (10 ol/L), DL-2-amino-5-phosphonovalerate (30�mol/L), CGP 55845A �mol/L), and tetrodotoxin (1 �mol/L) to isolate GABAA receptor niature inhibitory postsynaptic currents (mIPSCs). All cells were mped at�60mV for the duration of the recording. Statistical Analysis. Data were analyzed with one-way and o-way between-subjects ANOVA or RM ANOVA and, when ap- priate, with the Student-Newman-Keuls post hoc test, with p� considered statistically significant. Because there were unequal or variances betweengroups and therewas a significant positive relation betweenmeans and variance of I/O data, we performed g-transformation on all I/O data before analysis. In some cases, used independent-samples or paired-samples t tests for com- ring individual pairs of means. Means reported in figures repre- t measurements at the end of particular drug infusion periods. sults havioral Studies Effects of Alcohol Vapor Exposure onOperant Behavior. To ck the effects of alcohol vapor on operant behavior over time ., the transition to alcohol dependence), we analyzed data only m vapor-exposed and air-exposed aCSF-infused rats. Two-way or history�dayRMANOVAs indicated that over time, therewas lobal increase in alcohol responding, F (3,57)� 3.62, p� .05, and ohol consumption (g/kg), F (3,57) � 4.32, p � .01, which was ributable to the gradual increase in responding and consump- n by CIE vapor-exposed rats (Table S1 in Supplement 1). The -exposed rats consumed more ethanol (g/kg) than air-exposed s across days, F (1,19)� 4.45, p� .05. The ethanol response rates d consumption quantities observed following 11 and 15 days of exposure (in aCSF control animals) reliably produce blood-alco- l levels of approximately 100 mg/dL in alcohol-dependent ani- ls (19). There were no effects of dependence history or day on ter responding in aCSF-treated rats (p .05). Effects of NPY on Operant Behavior. Figure 1A shows oper- t alcohol responding by CIE-exposed and air-exposed rats used chronically with NPY or aCSF and tested across the first days of vapor exposure. A three-way (vapor exposure � NPY se� day) RM ANOVA revealed a significant suppression of oper- t alcohol responding across days by chronic NPY infusions, ,38)� 6.23, p� .017. There was no three-way interaction effect .05), but therewas a significant dependence� day interaction ect on operant alcohol responding, F (3,114) � 3.60, p � .016. o-way (vapor exposure � NPY dose) ANOVAs for each of the 4 t days (vapor days 3, 7, 11, 15) revealed that NPY suppressed erant alcohol responding on day 15 of vapor exposure, F (1,38)� 4, p� .05, and that there was a tendency toward a suppressive Y effect on alcohol responding on days 3 (p� .06) and 11 (p� ) of vapor exposure. A separate two-way (vapor exposure � NPY dose) RM ANOVA cumulative alcohol responding on days 11 and 15 of vapor osure (Figure 1B) indicated a significant suppression of operant ohol responding by NPY, F (1,38) � 5.84, p � .021. Finally, a ee-way (vapor exposure�NPYdose�day) RMANOVA revealed t alcohol-dependent rats responded less for water across days, ,38)� 7.82, p� .008, but water responding was not affected by onic NPY infusions (p .05). Two-way (vapor exposure � NPY se) ANOVAs for each of the 4 test days (vapor days 3, 7, 11, 15) ealed no significant effects of NPY onwater responding (p .05 ll cases). ctrophysiology Studies NPY and Ethanol Effects on IPSPs. As previously described ), baseline GABAA-IPSP input-output curves were higher (data t shown) and basal PPF ratio of IPSPs was lower in slices from pendent rats relative to naive control rats, suggesting an in- ased GABAergic tone via GABA release (statistics below and in ure captions). We first applied ethanol alone, then concomitantly applied NPY CeA slices from vapor-exposed rats and naive control rats (time rse in Figure 2A). As previously demonstrated (14), acute etha- l (44mmol/L) produced robust 35%to45% (p� .001) increases in P amplitudes (Figure 3A,B). Subsequent application of NPY (.5 ol/L) returned IPSPs to baseline levels (p� .025 difference from ure 1. Chronic neuropeptide Y (NPY) administration in a clinically rele- t treatment regimen blocks gradual and cumulative elevations in alco- drinking similarly in alcohol-dependent and nondependent rats. (A) onic NPY treatment blocks the development of alcohol dependence- uced increases in alcohol responding (n�11/vapor-exposedgroup) and blocks moderate increases in alcohol drinking by nondependent rats 10/air-exposed group) over time. Rats were infused in the ventricles h NPY (10 �g) or vehicle on even-numbered days of vapor exposure icated by arrows) and tested for responding at 6- to 8-hour withdrawal days 3, 7, 11, and 15 of vapor exposure. (B) Chronic NPY treatment presses cumulative alcohol responding across operant test sessions on or days 11 and 15. *Significant (p� .05) suppression by NPY relative to ficial cerebrospinal fluid vehicle regardless of vapor history (main