Journal ofNeurochemistryRaven Press, Ltd., New York© 1995 International Society for Neurochemistry

Regulation of Cytochrome c Oxidase Subunit mRNA andEnzyme Activity in Rat Brain Reward Regions During

Withdrawal from Chronic Cocaine

*John R. Walker and tKevin A. Sevarino

Division of Molecular Psychiatry, Departments of *Pharmacology and t Psychiatry, Yale University School ofMedicineand Connecticut Mental Health Center, New Haven, Connecticut, U.S.A .

Abstract: A subtractive hybridization and differentialscreening procedure was used to detect up-regulation ofcytochrome c oxidase (CO) subunits I, III, and IV mRNAin the nucleus accumbens (NAc) of rats chronicallytreated with cocaine. Northern blot analyses of mRNAisolated from individual rats confirmed that CO subunit Iwas up-regulated by chronic, but not acute, cocaine intwo brain regions, the NAc (33%) and caudate-putamen(CP) (35%) . CO activity, used as a measure of metabolicactivity, was increased by 88% in the NAc, and de-creased by 20% in the medial prefrontal cortex (mPFC),the day after chronic treatment was terminated . CO en-zyme activity was not regulated in the CP, or in otherbrain regions not involved in drug reward . CO activity inboth the NAc and mPFC showed unique time-dependentpatterns of regulation during the week after chronic co-caine treatment. Key Words: Cytochrome c oxidase-Medial prefrontal cortex-Nucleus accumbens-Cau-date-putamen-Cocaine-Withdrawal-Metabolism .J. Neurochem. 64, 497-502 (1995) .

Cocaine addiction is a problem of significant socialand medical concern (NIDA, 1992) . Acutely, cocaineaffects dopamine, norepinephrine, and serotonin reup-take, thereby increasing concentrations of these trans-mitters at their target sites (Jaffe, 1990) . Mesolimbicdopamine neurons of the ventral tegmental area (VTA)and one of its projection sites, the nucleus accumbens(NAc), are strongly implicated in cocaine reinforce-ment (Koob, 1992) . Animals will self-administer co-caine into the VTA, whereas lesions of the VTA orNAc, or administrations of dopamine receptor antago-nists into these regions, prevent drug self-administra-tion (Le Moal and Simon, 1991) .

Chronic cocaine produces effects different fromthose seen acutely . Rodents develop locomotor sensiti-zation to repeated doses of cocaine, whereas humansdisplay increased drug craving and paranoid reactionsafter long-term use (Post and Rose, 1976; Gawin andKleber, 1986) . Lesions of the VTA or NAc, or dopa-mine receptor antagonist infusions into the NAc of

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rats, abolish the sensitizing effects of cocaine (Robertset al ., 1977 ; Maldonado et al ., 1993) . Studies examin-ing synaptic dopamine concentrations, or density ofdopamine receptors, in the NAc after chronic treat-ment, have not provided a mechanistic explanation forbehavioral sensitization (Akimoto et al ., 1989 ; Hurdet al ., 1989 ; Kalivas and Duffy, 1990 ; Segal and Kuc-zenski, 1992) . Regulation of the cyclic AMP systempartly explains the enhanced effects of cocaine in thisregion after chronic treatment, but clearly there aretargets of chronic cocaine action that remain to beelucidated (Nestler, 1992) .

Using a subtractive hybridization screening ap-proach, we have detected a novel target of chroniccocaine action . Chronic cocaine increased cytochromec oxidase (CO; EC 1 .9 .3.1) subunit mRNA levels inthe NAc and caudate-putamen (CP) . CO activity, mea-sured spectrophotometrically, was increased in theNAc, and decreased in the medial prefrontal cortex(mPFC), after an 18-h withdrawal period . With a morecomplete characterization of CO activity through thecocaine withdrawal period, we observed unique time-dependent patterns of CO activity regulation in thesetwo important drug reward regions .

MATERIALS AND METHODS

Treatments and dissectionsMale Sprague-Dawley rats (150-250 g) were used for

all experiments . Rats were kept on a 12-h light/dark sched-ule with free access to food and water . Chronic cocainetreatment consisted of twice daily 15 mg/kg intraperitonealinjections of cocaine-HCI (Research Triangle Institute,

Received March 31, 1994; revised manuscript received June 9,1994 ; accepted June 13, 1994.

Address correspondence and reprint requests to Dr . K. A. Sevarinoat Division of Molecular Psychiatry, Department of Psychiatry, YaleUniversity School of Medicine and Connecticut Mental Health Cen-ter, 34 Park Street, New Haven, CT 06508, U.S.A.

Abbreviations used : CO, cytochrome c oxidase ; CP, caudate-puta-men ; 2-DG, 2-deoxyglucose ; mPFC, medial prefrontal cortex ; NAc,nucleus accumbens ; VTA, ventral tegmental area .

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NIDA), for 2 weeks. For cDNA library construction, ratswere killed by decapitation 18 h after the last cocaine injec-tion . For other experiments, rats were killed at the indicatedtimes after the last injection. Control animals were injectedwith saline . Block dissections of the NAc, which also in-cluded the olfactory tubercle, islands of Calleja, medial sep-tal nucleus, and the anterior portion of the ventral pallidum,were used for subtractive hybridization and library construc-tion . For northern blot analyses and CO enzyme activitymeasurements, 2-mm-thick punch dissections were takenwith 14-gauge tubing, following the anatomical landmarksdescribed by Deutch and Cameron (1992) for dissectionscontaining both the NAc core and shell. All animal proce-dures were performed in strict accordance with the policiesoutlined in the Guide for the Care and Use of LaboratoryAnimals (NIH publication no . NIH860-23) and were ap-proved by the Yale University Animal Care and Use Com-mittee .

Subtractive hybridizationTotal RNA was isolated by guanidine isothiocyanate ex-

traction and cesium chloride gradient centrifugation (Daviset al ., 1986). Poly (A)' RNAwas isolated on oligo-dT cellu-lose columns. To prepare "driver" mRNA from control rats,photobiotinylation was performed twice with 200 /ug pho-tobiotin acetate and 100 Mg poly (A) + mRNA. Excess pho-tobiotin was removed by 2-butanol extraction . First-strandcDNA from chronic cocaine-treated animals ("target"cDNA) was prepared using a primer containing the T7 RNApolymerase promoter 5' to the oligo-dT sequence (VanGelder et al ., 1990) . Two percent of this reaction was per-formed in the presence of [a 32p]dCTP (3,000 Ci/mmol,New England Nuclear), and this was added back to thetarget cDNA to trace the efficiency ofsubtraction. Hybridiza-tion was performed in 0.75 M NaCl, 25 mM HEPES, pH7.5, 5 mM EDTA, 0.1% sodium dodecyl sulfate, with a 15-fold molar excess of driver mRNA to target cDNA at 68 °Cfor 48 h (Rot = 983) . The biotinylated driver mRNA andmRNA-cDNA hybrids were removed by binding to strep-tavidin and extraction with phenol/chloroform. The re-maining cDNA (17% of starting material) was rehybridizedwith a 15-fold molar excess of driver mRNA to repeat thesubtraction . The subtracted cDNA (4.1% of the starting ma-terial) was converted to double-stranded cDNA using ran-dom hexamers, and amplified antisense RNA was synthe-sized by in vitro transcription in the presence of T7 RNApolymerase and [a32P]CTP (Van Gelder et al., 1990) .

Library screening and clone isolationA directional cDNA library was made from NAc

poly (A) + RNA obtained from chronic cocaine-treated rats .First-strand cDNA was made using a Notl-dT i5 primer (Pro-mega), and second-strand cDNA was synthesized using amodified Gubler-Hoffman strand replacement procedure(Sambrook et al., 1989) . Inserts containing a 5' Notl site,and a 3' blunt end, were ligated into pBluescript KS - (Stra-tagene), and transformed into DH10B cells (GIBCO/BRL)by electroporation . Library screens were performed ac-cording to the manufacturer's protocol for NEN/DuPont ny-lon membranes. Positive colonies were picked and trans-ferred to 96-well microtiter plates with LB media, 100 /cg/ml ampicillin, and 33% glycerol to allow indefinite storageat -70°C. Two further screenings were performed to obtainpure colonies . Clones were transferred from microtiter platesonto duplicate nylon filters (Zeta Probe GT, Bio-Rad) and

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J. R. WALKER AND K. A. SEVARINO

alkaline lysis performed. The filters were probed with anti-sense RNA prepared from nonsubtracted NAc cDNA of sa-line-treated or cocaine-treated rats, and blots were washedaccording to the manufacturer's protocol . Duplicate blotswere analyzed with a Betagen 6-emission analyzer . The nu-cleic acid sequences of clones of interest were determinedby polymerase chain reaction sequencing using 32P-labeledM13 primers (Circumvent, New England Biolabs) . Se-quences were aligned to the GenBank data base using theMacVector sequence analysis program (Eastman KodakCompany) .

Northern blot analysisTotal RNA from NAc block dissections was isolated by

guanidine isothiocyanate extraction and cesium chloride gra-dient centrifugation (Davis et al ., 1986) . When needed,poly (A)' RNA was isolated using PolyATtract mRNA iso-lation kits (Promega), and total RNA from punches wasisolated by briefsonication in RNAzol B and organic extrac-tion (Biotecx Laboratories) . RNA was separated on 1%formaldehyde agarose gels, and blots made by capillarytransfer to Zeta ProbeGT membranes (Bio-Rad) . Blots wereprobed with either random prime-labeled BssHII fragmentsof pBluescript recombinants, containing the entire insert, orantisense RNA probes synthesized from isolated plasmids .Blots were washed as described above, and were exposedto Kodak XAR5 film for image analysis by densitometry(Image 1 .41, NIH) . The signal for cyclophilin was used asan internal standard to normalize for sample loading varia-tion .

CO activity assayTissues were dissected by punch, as described above, and

assayed for CO activity according to Hess and Pope (1953)with some modifications. Frozen tissues were sonicated in0.75% sodium deoxycholate in 0.1 M phosphate buffer, pH7.1 (50 ul/mg of tissue), and centrifuged at 16,000 g at 4°Cfor 15 min. Supernatants were diluted in deoxycholate bufferto give an absorbance change at 550 nm of 0.02-0.07 U/min, using 5-10 /1 of sample with 350 /d of 3 x 10 -5 Mreduced cytochrome c (Sigma) in 0.05 Mphosphate buffer,pH 7.1 . This absorbance change was within the linear rangeof the enzyme assay for each tissue type tested . Dilutedsupernatants were stored on ice between readings andwarmed for 2 min to room temperature just before use. Spec-trophotometry was performed at room temperature . Sampleswere read in duplicate, and slopes for the rate of loss ofcytochrome c absorbance were averaged for each sample .Rates were normalized to the protein content of each samplesupernatant (Lowry et al ., 1952) .

Data analysisDensitometry values from northern blots and enzyme ac-

tivity numerical values for each group of animals were usedto calculate the mean and standard error of the mean . Stu-dent's t tests were performed to evaluate the statistical sig-nificance of differences from control levels .

RESULTS

Library screening and clone selection32P-labeled in vitro-transcribed subtracted probe

was used to screen NAc cDNA library from chroniccocaine-treated animals. Screening 100,000 coloniesyielded 1,563 primary positives (-1.5%) . A second-

CYTOCHROME c OXIDASE ACTIVITY IN COCAINE-TREATED RAT BRAIN

FIG. 1 . Mitochondrially-encoded and nuclear-encoded CO sub-unit mRNAs are up-regulated by chronic cocaine treatment. Ani-mals were treated with cocaine-HCI (15 mg/kg), or saline, twicedaily for 14 days, and killed 18 h after the last injection. Eachbar represents the results from one pair of lanes that each con-tained 20 Mg of total RNA pooled from NAc blocks of either 50control or 50 cocaine-treated rats and probed with the indicatedcDNA probe. Clones analyzed were as follows: I, CO subunit I ;III, CO subunit III ; IV, CO subunit IV ; 16S, mitochondrially-en-coded 16S rRNA, The results are expressed relative to the salinecontrol lanes.

ary screen confirmed 94% (1,469) ofthe primary posi-tives, ofwhich 75% (1,100) were from single colonies .These secondary positives were transferred to micro-titer plates containing LB media and glycerol for stor-age . Four hundred of the secondary positives from sin-gle colonies were transferred to duplicate nylon filtersfor differential screening with probes from nonsub-tracted NAc cDNA of control or cocaine-treated ani-mals . Signals from the duplicate blots were comparedon a,8-emission analyzer, and up-regulated clones (51of 400 = 13%) were chosen for sequencing and north-ern blot analyses .

CO subunit I, III, and IV mRNAs areup-regulated in the NAc and CP

Twenty-five of the 51 up-regulated clones were cho-sen at random and sequenced . It was found, as follows,that 12 were mitochondrially-encoded sequences : COsubunit I (four clones), CO subunit III (three clones),and 16S mitochondrial rRNA (five clones) . In addi-tion, 11 clones of 285 rRNA and two clones of 18SrRNA were found . Analyses on northern blots, usingNAc tissue from rats killed 18 h after the last cocaineinjection, indicated that none of the rRNA species wasregulated (data not shown) . We reason that the highabundance of these sequences leads to artifactual de-tection in the library and differential screening . North-ern blots, using the same mRNA isolated from chroniccocaine-treated or saline-treated rats as used in thesubtraction, were performed to confirm that the COsubunits were up-regulated (Fig . 1) . Subunit I mRNA(1 .7 kb) was up-regulated by 29%, and subunit IIImRNA (0.8 kb) was up-regulated by 15%. Further,to determine if a nuclear-encoded subunit of the COcomplex was up-regulated by chronic cocaine, CO sub-unit IV mRNA (1 .3 kb) was probed using a mousecDNA specific for that subunit (obtained from L .Grossman, Wayne State University) and was foundto be up-regulated by 18% (Fig . 1) . Levels of 16Smitochondrial rRNA (1 .6 kb) did not show up-regula-

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TABLE 1 . Regulation of cytochrome oxidase subunit ImRNA by chronic cocaine treatment

-p < 0.05, by Student's t test .

tion . Northern blot analyses using NAc tissue fromindividual animals confirmed the finding that 165 mito-chondrial rRNA was not up-regulated (78.3 ± 16.6%,n = 6) .We next examined cocaine regulation of CO subunit

I mRNA, which had the largest change in the northernanalyses, in individual animals, and in multiple brainregions (Table 1) . Only the NAc and CP showed sig-nificant changes from saline control levels . The per-centage change for the NAc punches was similar tothat seen above for the pooled NAc blocks . NAc tissuefrom animals treated with a single 15 mg/kg injectionof cocaine, and withdrawn for 18 h, showed no dif-ference from saline control levels (94.4 ± 5 .2%,n = 10) .

CO activity is regulated by chronic cocaine in theNAc and mPFC

Pharmacological insults that change mRNA levelsof mitochondrially-encoded CO subunits have beencorrelated with changes in enzyme activity (Hevnerand Wong-Riley, 1993) . A critical question waswhether an increase in CO activity accompanied theincreased CO subunit I mRNA levels seen in the NAcand CP in response to chronic cocaine treatment. Table2 shows the results of a spectrophotometric assay forCO activity in the NAc, CP, and other regions, inchronic cocaine-treated rats killed 18 h after the last

TABLE 2 . Regulation of CO activityby chronic cocaine treatment

p < 0.02, by Student's t test .

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Tissue% of change from saline

control ± SEM n

NAc 133 .1 ± 10.1° 14CP 135 .1 ± 13 .0° 12Ventral pallidum 98 .8 ± 6 .9 12Frontal cortex 78 .7 ± 10 .1 6Amygdala 104.5 ± 22 .4 4VTA 112 .0 ± 17 .8 6Substantia nigra 65 .3 ± 42.2 6Hippocampus 80.3 ± 9 .2 6Olfactory bulb 124.9 ± 23 .8 6

Tissue% change from saline

control ± SEM n

NAc 187 .9 ± 19 .8° 6CP 93.2±9.9 9Substantia nigra 81,2 ± 10.2 9Ventral pallidum 102 .1 ± 11 .8 6VTA 119.0±11.9 5mPFC 80 .0 ± 1 .7° 11

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injection . CO activity for the NAc was significantlyincreased, by 88%, in rats chronically treated with co-caine . The only other brain region that showed a sig-nificant change was the mPFC, where CO activity wasdecreased by 20% . CO activity in the CP was notregulated, though CO subunit I mRNA in that regionwas up-regulated .

CO activity in the NAc and mPFC variesthroughout the withdrawal period

After chronic cocaine treatment, rats were killed atmultiple time points after the last injection and werecompared with saline-treated animals that were killedat the same times . CO activity in the NAc was notsignificantly increased above control levels at 45 minor 4 h after the last cocaine injection, though there wasa clear trend toward an increase at the latter time point(Fig . 2A) . Peak elevation in CO activity, 88%, oc-curred 18 h after injection . After 3 days, CO activitywas still significantly elevated by 22%, and after 7days, CO activity was back to baseline .

In the mPFC, at 45 min after injection CO activitywas largely unchanged (Fig . 2B) . At 4 h of with-drawal, there was a significant 17.6% increase, whichdropped to 80% of control levels at 18 h . By 3 days,CO activity returned to baseline .

CO activity in chronic cocaine-treated andwithdrawn rats is not hyperresponsive to an acutecocaine challenge

Multiple studies have shown that locomotor sensiti-zation to cocaine develops after chronic treatment andpersists for several months of cocaine abstinence (Postand Rose, 1976; Kalivas and Weber, 1988) . To testwhether enhancement of locomotor activity with anacute cocaine challenge was reflected by increased COactivity in the NAc, a group of rats was treated chroni-cally with cocaine and withdrawn for 7 days. A singlecocaine challenge (15 mg/kg) did not elevate NAcCO activity levels over those of chronic saline-treatedanimals challenged with cocaine, when animals werekilled 18 h after the challenge injection (89 .4 -}- 4.7%,n = 9) . CO activity in the NAc, therefore, is not hyper-responsive after drug treatments that induce locomotorsensitization .

DISCUSSION

Subtractive hybridization followed by differentialscreening, to enrich for and select cocaine up-regulatedmRNAs from rat NAc, yielded several cDNAs repre-senting mitochondrially-encoded genes . Northern blotanalyses demonstrated that the mitochondrially-en-coded CO subunits I and III and the nuclear-encodedsubunit IV were up-regulated by chronic cocaine . Inindividual animals, it was confirmed that subunit ImRNA was up-regulated by chronic cocaine treatmentin the NAc and CP, whereas CO activity from theNAc, but not the CP, was increased in animals chroni-cally treated with cocaine . Regulation of CO activity

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FIG. 2 . CO activity in the NAc and mPFC is dependent on thewithdrawal period . Cocaine-treated and saline-treated rats werekilled 45 min, 4 h, 18 h, 3 days, or 7 days after their last injection .CO activity was measured and each treatment group was com-pared with its corresponding saline group . Results are expressedas percentage of saline control levels ± SEM . Numerical resultsare listed with experimental group sizes in parentheses . A : COactivity in NAc punches . 45 min, 106.3 ± 7.2% (16) ; 4 h, 122 .1± 11 .2% (9) ; 18 h, 187.9 ± 19.8%* (6) ; 3 days, 117.0 -- 2.6%*(6) ; 7 days, 97.2 ± 4.6% (10) . *p < 0.02, by Student's t test .B : CO activity in mPFC punches . 45 min, 95.8 ± 16.3% (6) ; 4h, 118.0 ± 3.2%** (8) ; 18 h, 80.0 -_ 1 .7%* (11) ; 3 days, 98 .1

6.5% (6) ; 7 days, 92 .9 ± 5.2% (9) . **p < 0.05, by Student'st test ; *p < 0.02, by Student's t test .

was also seen in the mPFC, a region not specificallyexamined by northern blot analysis . Other brain re-gions, including several not implicated in drug reward,showed no regulation of CO activity . For both mRNAand CO activity, increases were not seen with acutecocaine treatment . Withdrawal time course studies in-dicated that CO activity recovered to baseline levelsin the NAc and mPFC after several days, though thetiming of peak effects differed for the two regions .The most prominent finding was that an increase in

CO activity in the NAc occurs during withdrawal fromchronic cocaine . This induction is the result of with-drawal and not chronic treatment alone, as chronicallytreated animals killed 45 min after a challenge injection

CYTOCHROME c OXIDASE ACTIVITY IN COCAINE-TREATED RAT BRAIN

had the same NAc CO activity as saline-treated ani-mals (Fig . 2A) . Because dopamine levels are en-hanced after chronic cocaine (Akimoto et al ., 1989 ;Kalivas and Duffy, 1990), and dopamine inhibits NAcneurons (White et al ., 1987), chronic cocaine wouldpresumably result in chronic inhibition of NAc neu-rons . Our results could be explained if NAc neuronsexposed to chronic cocaine recover from this inhibitionby increasing their firing rate above that of saline-treated animals . This "rebound" in neuronal firingwould be similar to what is seen in locus ceruleusneurons during morphine withdrawal (Aghajanian,1978) . Increased firing rate, in turn, would requiremore ATP, and thus, greater CO activity (Brand andMurphy, 1987) .

Relatively few studies have examined the metaboliceffects of cocaine in brain reward regions . Acutely,cocaine increases 2-deoxyglucose (2-DG) uptake, ameasure of metabolic activity, in forebrain reward cen-ters (London et al ., 1986 ; Porrino et al ., 1988) . Fewstudies have examined these effects in chronicallytreated animals, or in animals during drug abstinence .Clow and Hammer (1991) found significant decreasesin 2-DG uptake in the NAc, VTA, and mPFC ofchronic cocaine-treated rats, after a 3-day withdrawalperiod. However, the same group earlier reported in-creases in 2-DG uptake in the NAc after a 1-h with-drawal period, suggesting that, after chronic cocainetreatment, metabolic activity in this region is depen-dent on the withdrawal period (Hammer et al ., 1989) .This finding led us to examine multiple time pointsduring cocaine withdrawal . In contrast to the 2-DGstudies, at no withdrawal time point did we observe adecrease in CO activity in the NAc. Differences be-tween our study and that of Clow and Hammer, inaddition to the withdrawal periods, were that we useda higher dose of cocaine (15 mg/kg twice daily versus10 mg/kg once daily) and that our methods to measuremetabolic activity were different . CO activity, com-pared with 2-DG uptake, appears more related to astable state of metabolic activity and, therefore, maymore accurately reflect drug-induced gene expressionchanges than the latter, which is related to instanta-neous fluxes in glucose consumption (Wong-Riley,1989) . Finally, whereas 2-DG mainly measures glu-cose uptake in terminals (Schwartz et al., 1979), themajority of CO activity is found in dendrites (Wong-Riley, 1989) . It is perhaps surprising that the twomethods have been shown to be positively correlatedin some instances (Di Rocco et al ., 1989) .The mPFC, which is involved in the initiation of

cocaine self-administration (Goeders and Smith, 1983),has efferent connections to the NAc (Beckstead,1979 ; Walaas, 1981) . The mPFC appears to exert aninhibitory influence on NAc function (Le Moal andSimon, 1991) . We found that the time of peak increasein NAc CO activity corresponded with the peak de-crease in mPFC CO activity (Fig . 2) . Therefore, it is

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possible that decreased mPFC activity is at least partlyresponsible for the increased NAc activity observed .

Unlike the well-characterized withdrawal syndromefrom chronic morphine or heroin, there is little physicalevidence for a cocaine abstinence syndrome, though a"crash" phase that persists for up to 48 h has beenobserved in humans after cocaine binges (Gawin andKleber, 1984 ; Satel et al ., 1991) . Few studies havedescribed biochemical changes in the NAc that appearonly during the first few days after cocaine administra-tion is terminated . In the rat, CO activity, in the brainreward regions of the NAc and mPFC, is regulatedonly during the first 1-3 days after chronic, but notacute, treatment . We speculate that regulation of COactivity represents a biochemical marker of a physicalwithdrawal state after chronic cocaine exposure .

Acknowledgment: We thank Eric J . Nestler for guidanceand helpful discussions and Lawrence Grossman for provid-ing us with a mouse cDNA probe for CO subunit IV . Duringthe course of these studies K.A.S . was the recipient of aPfizer New Faculty Scholar Award . Portions of this workwere supported by U.S . Public Health Service grantDA08189 and by the Abraham Ribicoff Research Facilitiesof the Connecticut Mental Health Center, State of Connecti-cut Department of Mental Health .

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