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Benzodiazepine Receptor Ligands with Different Intrinsic Efficacies Alter Ethanol Intake in Alcohol-Nonpreferring (NP) Rats Harry L. JUlie, PhD., James M. Murphy, PhD., Robert L. Hewitt, M.S., Terri L. Grewe, B.S., Mtlrgaret Lill, B.S., Jackie J. Mellor-Burke, B.S., Lawrellce Lumellg, MD., alld Tillg-Kai Li, MD.
Bellwtiiaul'illc (8DZ) rcecIJlar ligullds willi varying EtOH drillkillg at ti,e 6O-mirillte interval witll the 1-mg/kg III/ril/sic efficacies IR019-4603, 0.02-0.15 "'glkg; FG 7142 dose, and ti,e 16-mg/kg dose reduced water illfake at thc 15- 1-161118Ik8; DMCM, 1-8 IIIg/kg; R016- 6028 (bretaulli/), mimlle interval. DMCM, tllc full {3-carboli"c i"verse . 8-32 IIIg/kgJ ill modulating GABAergic activity were agol/ist, significantly reduced water intake at 15 minlltes (4 l'Xalllillcd for tile ability to alter palatability-induced etllUllol and 8 mg{kg), alld /l,e SlIme doses cal/sed a Sllbstantial (EtOI-1) jll/ake ill the afco/toi·llollprqerrillg (NP) lille of il/crcase ill f.tOI-J drillk/llg al Ille 120-mimlle intervat. 'file rals. NP rats all a 22-l/ollr fluid-deprivation schedule were mlxiolytic agent brclazcllil (16 alld 32 IIIg/kg) increased given 2-llOlir daily access to Q 10% (v/v) f.tOH/3% (gM EtOH collsllmptioll dllrillg tile initia/IS minlltes to 270% polycose soilltioll and water. Average EtOH ill lake was to 425% of call trot levels, and water intake i"creased by ti,e 2.1 :!. 0.2 g/kg/2/10Ilr5, and water jlltake was 17. 1 :!: 0.9 ella of tllC2-1I01lr sessioll to as mllel, as 210% of cOlltrol ml{2 hours. During tile illilial 15 lIIil/llles of file 2-1I01lr follawi"g aallli,ll'stralio" of /lw 32-lIIg/kg dose. These sessiOll , R019-4603, the imidawtllicllodiazepillc partial findillgs sIIpport existing evidell ce sIIggestillg tllat BOZ if/verse ngollist reduced £/01-1 ill lake /0 19% of COll/rol rcceptor ligands lIIay modify IICI/rollal processes tllal values at 0.04 mg/kg alld coml}lctely sllPI}ressed drillkillg of mediate some reillforcillg alld/or aversive prolJCrties of liJl' £tOH so/lliion at 0.15 mg/kg. Twolty-follr-Ilollr alcohol. Tiley f"rtller demOllstrate a potelltial illl110rtmlce of pasldrug admillislratio", the D.D8-mg/kg dose of R019- ti,e CABA,,-BDZ receptor complex ill mediatillg 4603 comple/ely slIPIJresscd drillkillg of /lIC EIOI-I soluliotl Ixl/atabilily- (ctivirollll/ctitally) il/allccd EfOH drillkillg at tile 6O-mimde illt(!rval, and the O.1S-mg/kg dose n'iiuced eve" ill rats selectively bred for law alcolwl preferel1ce. iI/lake to 20% of cOlltrol lewis at tllC 1S-milllltc illierval. fNellropsycilOplurrmucology ]4:55-66, 19961 FG 7142, /lIC parliall3-caroolille illverse agollist red/Iced
KEY WOROS: BCllzodiazcpillc iI/verse agollisl; Etlrauol; From the 1A·p.lrtmcnt of Psychology (HLJ; JMM; RLH; TLC, A1co/rol-llollpre[errillg (NP) rals; CABAII-BOZ )),,"'1·8), /'urdue School ofScicmx. Program In Nl'Urubl' oIot,'Y (HL); JMM; MLl. Dep.utm('nts of Ml'didnl' (LL; Identifying both the environmental and biological vari T·J(L). and Biochi'mistry (T·KL), India",1 Univ(,T!li ty School of Ml'di· ables that promote and maintain high alcohol-seeking a ll(' VA "'looie.ll Cent .. r (LL), Indi.l",1 University·Purdue Uni· versity, l lltli.u\ilpohs.IN. or alcohol self-administration behavior is key to under Addn.'Ss rorn.'Spondcncc to: J-Llrry L June, Ph.D .• Assistilnt Pro standing the disorder of alcohol abuse and alcoholism fl'SSOr of rsychology Neurobiology, Department of (Li et al. 1991). Selective breed ing methodology has Psychology, to 3124, IUI'UI,.JD2 North Blackford Stl'('('l, lis. IN 46202-3275. demonstrated convincingly thai there is genetic int1u Rluwl'd FcbruM)' 18, 1994: October 6, 1994; iloxptcd ence on alcohol-seeking beh.wior ( Li et aL 1991). Cur Dro>mb 1996-1101.. NO.1 rublfshcd 1996 by Sacncc Inc. OS93-I33X/%/S15.00 65S AvC'nu(' of the AmeriGls, New York, NY 1001 0 SSOl 0893-13JX(9-IXX) IU-M 56 H.L June et al. N CUROI'SYCHOI'11AItMACOUJCY 19'J6-VOl. 1-1, NO. I 1991). These pharmacogenetica lt y different animallincs review, sec June et al. 1994a). Previous research with P provide useful tools for investigating alcohol's neuro and NP rats has shown that ROI5-4513, a partial BDZ chemical and neurophMmacological efrects (McBride ct inverse agonist, selecti vely attenuates EtOH intake with· 011. 1992). The alcohol-preferring (P) and -nonpreferring out affecting water consumption during limited fluid (NP) rats are one pair of rats that h;;we been devetopt..--d availability (McBride et al. 1988). Recentl y, we Qune l'I through selective breeding from a Wistar stock for their al. 1995; also sec June et al. 1993) compared the ability of divergent preference for EtOH (Lumeng et al. 1977; Li et several BDZ inverse agonists IROI5-350S (s.1rmazcnil), at. 1987). With food and water ad libitum, P rats volun Fe 7142, ROI9-4603, DMC MI with a bro.,d spectrum of tarily consume at least 5 g l kg body weightl day of an intrinsic efficacil.'S (1·laefely 1990; Richards et OI l. 1991) unflavored EtOll solution (10% v I v), whereas NP rats with that o f ROI5-4513 in modifyi ng the reinforcing consistently consume less than 1 g / kg body weight I properties of EtO H in the P rat. Besides exerting pro day. ResponSt.'"-Contingent EtOH intake without the use longed supprt.'Ssion of EtOH intake, R019-4603 was of sweeteners has also been demonstrated in P rats with proximately 10 to 20 times more potent than ROl5-4S13 concentrations as high as.3{)o/" (v / v) (Penn et a1. 1978; in attenuating EtOH intake. The results with DMCM, a Wa ller et al. 1984; Murphy et a1. 1989); howevcr, NP rals full negative a ll osteric modulator of CI\BAergic activity rt'Spond very little for alcohol concentrations exct.'t.>d ing (Richards et al. 199 1), showed that, although it was ca· 5% (v I v) (Murphy et a1. 1989), unll.'SS EtOH initiation pablc of decreasing EtOH intake, the reduction ap procedures are employed (Samson ct al. 1989a). Thus, rats peared to be part of a general overall dciicit on moti· of the P line may be used as a model for studying envi vated behaviors, insofar as reductions were also ronmental variables that mediate alcohol consumption. observed in the control fluid and food intake. 5,;1fmazc Some information is available on the environmental nil did not alter EtOH intake, and FC 7142 demon· variables that CM initiate EtOH self-administration in strated selective antagonism only at moderate to hi gh the N P rat. Silmson and his colleagues (1989b), as well doses (16 and 32 mg/ kg), presumably reflecting the as others (Ras!'mick et al. 1993), have successfully em lower affi nity of these compounds at the UDZ receptor ployed the sucrose-fading technique to initiate EtOH (Sieghart et a l. 1987; Lister and N ull 1988). drinking to phannacologically relevant levels in NP The objective of the pn.'SCllt study was to determine rats. Blood alcohol concentrations (SACs) have been re the gencraliz.'lbility of our previous findings with P rats by ported in the range o f 30 to 110 mg/ dl fo llowing a 30- showing that inverse agonists with different intrinsicef· minute operant session (5..,mson et al. 1989b; also SLoe ficacies effecti vely antagonize p.1 Iatability-induced EtOH Rassnick et nl. 1993). Previous l"CSCarch from our labora intake of NP rats. Specifica ll y, the BDZ inverse agonists tory has emploYI.-d it modification of the palatability-in FG 71 42, ROI9-4603, and DMCM were evaluated for duced polydipsia method (Kulkosky 1978) to initiate their ability to selectively suppress EtOH intake, follow 24-hour continuous EtOH consumption in r rats ing initiation using a palatability-induced polydipsia (Caito et al. 1990). This procedure consists of presenting method (Kulkosky 1978; Gatto ct al. 1990). In addition, animals a two-bottle choice of \\fater and a "cocktail " to examine the abi li ty of a BDZ receptor ligand with vcry comprised o f 3% (w I v) polyc05e, 0.5% (w I v) NaC!, and weak intrinsic efficacy (Haciely 1984), but at the agonist 0.125% (w / v) s.,ccharin, with EtOH added in 2% incre end of the continuum, ROI 6-6028 (bretazenil), a UOZ ments every third day until a concentration of lO'Yo (v / v) partial agonist, was also investigilted. Bretazenil, li ke is obtained. Although the amount of the cocktail con many congeners of flumazenil (e.g., ROI5-45J3, ROI9- sumed declines .1S EtOH concentration increases, EtO,., 4603, sarmilzcnil), exerts high uffini ty for 8DZ receptors intake levels stabilize and remain mensurable. 61\Cs (Ri chards et a1. '1991) and is currently under cli nica l in were approximately 31 and 61 mg 'Yo at 30 and 60 min vestigation as a pharmacotherilpcutic agent for the treat utes, respecti vely, into the dark cycle (see Methods), in ment of anxiety disorders in humans (Moreau et al. d icating that pharmacologically relevant EtOH levels 1991). information on the ability of this drug to were obtained. Gatto and cO l1eagues (1990) further indi modify the reinforcing properties of EtOH would be of ca too that this modified palatability-induced polydipsia clinica l interest. method was ca pable of mainta ining high EtOH drink ing in NP rats fo r several months. EtOH self-administration in rodents can be alten:.>d METHODS by a variety of neuropharmacological agents that affect Subjects different neurotransmitter systems (Weiss and Koob 1991 ; McBride et al . 1992), suggesting that several neurotrans Experimenta ll y naive adult male rOll.. (II = 7) of the se mitter systems may interact to mediate EtOH reinforce lecti vely bred NP li ne (5-37 generation) were individu ment. Considerable n...osearch implica tes the CABAA all y hOUSl.>d in wire-mesh stainless steel cages in a telll BDZ rt.'Ceptor complex as one neuroreceptor that plays peratufe-Controlled (21"<:) room on a 12:12 reversed an important role in the reinforcing actions of EtOH (fo r light:dark cycle (lights off al 0900 hours). Rats were pre- \WIIOI'SYOIQI'HARMACOlOGY 19'J6.-VOL. 14, NO. 1 Benzodiazepines Alter Ethanol Intake 57 \iously tested for free. RESULTS val interaction emergL-d from the data IH 4s,225) - 4.44, P < .0001 1. As illustrated in Figure 1A, following vehicle Consumplion of the polycose solution dL'Cr£aS(.od from injl..'ctions, the NP rats consumed 4.4 :t 104 ,3.1 ::!: 0.4,3.1 .!: approximately SIlO :t 1.3 ml during days 1-3 (no EtOH) 0.5, and O.SS ::: 0.2 ml of EtO H (mean::!: SEM) during 10 approximately 23.8 ::: 2.8 ml when the EtOH concen the 15-, 3Q., 60-, and 120-minute consumption in tervals, tration (v / v) WilS 2% (days 4-6). It then decreaS(.'(i fur n.'Spe<:tively. Compared with the control condition, the ther to 14.3 ::: 0.64 mJ, when EtOH was 4% (days 7- 9); highest R019-1603 dose (0.1 5 mg/ kg) completely sup only additional small decreilses of intake occurred when pressed EtOH intake during the 15-minute interval (p < the EtCH concentration was 6% or highcr. When the s0- .05), but when the animals were given the O.O-I-mg/ kg lution contained 100/0 EtCH, intakes were 11.2 ::: 1.1 ml dose, drinking of the EtOH solution decreased to about (2.1 ::: 0.20 g/ kg/2 hours) prior to beginning vehicle 19% of control va lues (p < .OS). In addition, the highest and drug injections. dose (0.15 Illg/ kg) continued to suppress EtOH intake Figures 1/\ - SA show EtCH int.:lke for the fi ve dnlg during the 3O-minute interval (p < .OS). At the treatment conditions compared with the Tween-SO ve minute interval. ROI 9-4603 generall y elevated EtCH hicle condition across the four consumption intervals. A consumption compared with the vehicle cond ition, significant treatment condition-by-consumption inter- with statistica ll y significant effects S(.'Cn only wit h the 0.02-,0.0-1·, and O.t5-mg/ kg dose conditions (p :s .OS). Figures I B-s B show water intake for the fi ve drug treatments compared wi th the Tween-SO vehicle condi .v_ tion across the four consumption intervals. A significant A. B .I1...,-11a .Ot""'*l t o ...... Ol""'*l tre"tmcnt condition-by-i nterval interaction emerged from o .. -.'\I.OI""'*l the data IF(4s,225) = 2.57, P < .000 11 . Figure 1 B shows IJ .15 ...... Ol""'*l water intake fo r the Tween-SO vehicle control compan.>d with the R01 9-4603 treatment condit ions. Following ve hicle injections, the Prats cOllSumL>d approximately 9.9 .::!: 0.8,3.2 ::: 0.7, 3.2 :t 0.6, and 0.8 :t 0.2 ml across the 15-, 3().-, 60-, and 120- minute consumption intervals, re spectively. Post hoc analyS(.'S showed that a statistically Significant dL'Crease in water consumption occurred at the l5-minute interval with the O. IS-mg/kg dose (p < .01). Significa nt increaS(.'S in water consumption were fo und for the 0.02-mg/kg dose condition at the JO- and 120-minute intervals and for the 0.04- and O.Q8..mg/kg ' X) " " .. conditions at the 120-minute intcrval (p < .01). E((ects of R0 19-4603 on Consumption of 3% Ilolycose and EtO H a nd Water 24 Hour I'ostdrug Administration Figures 2A and 28 show EtO H and water intOlke fo r the four R019-4603 dose conditions (0.02, 0.0-1, O/ OS, and 0. 15 mg/ kg) compared with the vchicle condition 24 hours after injection. A significan t 1Te00tment condition-by consumption interval interaction was found IF(12,60) "" 3.78, P < .00031. Post-hoc analyses showed that the O.fJ8.. and O.I5-mg/ kg dose conditions significantly rcdua.>d EtOH drinking OI t the 60- and 15-minute intervals, re• specti vely (p < .05). '''' Figure 2B shows waler intake following the four R019- 4603 dose conditions compan..'CI with the control condi Figure 1. Effects of the IP administration of vehicle ilnd tion. A Significant treatment condition-by-consumption four doses of ROI9-4603 on the intake of CA) 3% polycosc + interval interaction emerged IF(12,60) = 2.64, P < .0061. 1{)'''''' EtOH and (8) H:P by NP rats across four consumption Post-hoc analyses indicated Ihat sta tistically signjficant intervals. D.Ha are the mean ::: SEM (II - 7 rats e,lch). tp < reductions in waler intake were found for the O.().l.. and .01 and .p < .05 \'s. control vehide val ues by A IOVA and O.OS-mg/ kg dose conditions at Ihe 15-minule inlerval post hoc Newman-Keuls tLos t. (p < .01), while signi(icOl nt elevations were found for \f.LIlOi'SYCI IOPI-IARI>IACOLCX;Y 1996-VOL. 14, NO. I Alter Elhanol lntakc 59 A. flO A. : Vehide ! 9 I "'IIk& fG 1141 , •8 .02 mcJta: ROlf.46IJ o .ftlll"q fG71.1 o ' ...... n ::11. 1 0 .04 ROIJ...46O) •• • 0 .08 mcfka: ROI'-46OJ • IOl 16 ...... r C 7l41 { • .1 5 ROIJ..46O.) " •:; 0• 2 " •• <• ¥ •c • "t t ., " '" "' '''' " '" "" COlKumption tntHu t Imln) Consumption Interval (min) Figure 3. Effl'Cts of the IP administration of vehide and Figure 2. Effects of the II' administration of ve hicle and four d oses of FG 7142 on the intake of 1M 3% potycosc + four doses of R019-4603 24 hours postdrug administration 10% EtOH and (8) I-I;!O by NP ra ts aems!> four consumption on the intake of (A) 3% polycosc + 10% EtOH and (8) intervals. Data nrc Ihe mean :! SEM (II -: 7 ra ts each). t p < by NP rals across four consumption inlenra ls. Dala arc the .01 and ." < .05 vs. cont rol vehide va lues by ANOVA and mean ::!:: SEM (II = 7 rats each). tp < .0 1 and · p < .05 vs. con post hoc Newman-Keuls lest. trol vehicle by ANOVA and post hoc Ncwmnn-Keuls "' .. and 12O-mi nute intervals, no significa nt differenn.'S were the 0.02-mg/ kg d ose condition at the 30- and 120- observed. minu te intervals (p < .05 and p < .01 , res pl.'Cl: ively). Sta Figure 4A shows consumption o f the EtOH solution tistica ll y significa nt elevations were also found at the for vehicle injections compared with the full nO-mi nute interval for the 0.04- and 0.1S-mg/ kg dose inverse agonist, DMCM. Significant suppression of EtOH cond itions (p < .01). intake was apparent at the 6O-minute interval with the Figure 3A shows intake of the EtOH solution after 8-mg/ kg dose of DMCM (p < .05). In contrast, marked veh icle injections compared with the partial j3-carboline elevati ons in intake were observed at the 120-minute in inverse agonist, FC 7 142. At the IS-minute interval, the terval fo llowing the and B- mg/kg d ose conditions 1-,4-,8-, and 16-mg/ kg d oses did not alter EtOH intake (1' < .0 1). Figure 48 shows that both the 4- and the B- mg/ significantl y (p > .05). Only the t -mg/ kg d ose signifi kg doses of OMCM significa ntly suppressed water in cantl y supprt..>SSed intake at the 6O- minute interval (p < tilke at the 15-minute interval (p < .05). No significant .05). Figure 3B shows that at the IS-minute interval, the effects were observed with any of the dose conditions at 16-mg/kg dose significantly reduced water consump the 30-, 60-, or 12O-minute intervals (p < .05). tion (" < .05), whereas the 1-, 4-, and 8-- mg/kg doses The data in Figure SA show intake of the EtOH solu did not al ter water intake (p > .05). During the 30-, 60-, tion after injection of the partial BOZ agonist, bretazenil 60 H.L. June cl ill. NEUII.Of'SYCHOf'IIAKMACOlOGY l A. • VdUde I ...... DMCM t .v..... I 1; t a o o4.11l&'fl1 DMCM o • o o 3Z ROl6-6fll , t + 20 s. t ! " • j l • • i I,. " '" .. Consumption Inltry.1 (min) u 30 .. llO Figure 5. Effects of the IP administration of thrt.'C doses of CG nsumplion 1"lt n.1 (min) brctazenil (I{OI6-6028) on the intake of (A) 3% polycosc + Figure 4. Err...'Cts of the IP administration of vehicle and 100/" EtCH and (8) H:P by N P rats across four consumption three doses of DMCM on the inl;;J kc of (AI 3% polycosc + intervals. O"til arc the mean .= SEM (II = 7 rats each). tp < 10% EtOl-I and (H) H20 by N I' rats across four consumption .01 and ' 11 < .05 \'S. control vehide values by ANOVA and intervals. Data arc the mean ;!; SEM (II = 7 rats each), tI' < post hoc Ncwllliln-Kculs test. .01 and "I' < .05 \'5. control vchicle values by ANOVA and post hoc cwman-Kculs !Los t. l6-mg/ kg dose of FG 7142, and the O.08-mg/kg dose of RO I9-4603, while also showing the resul ts of EtOH con (ROI6-6028). A different profile of effects cmerg<..od for sumption for a combination of the h vo drug doses. Ap brctazcnil relati ve to the other lesK-d inverse agollislS parent in this figure is a failure of the 16-mg/kg dose of (above) \Vh"'n compared with the control condition. Spe FG 7142 to reverse the suppressant actions of ROI9· cifically, the lower dose (8 mg/ kg) did not alter drink 4603 on EtOH consumption. As shown in Figure tA, ing of the EtOH solution at the initial IS-minute con EtOH intake was not significantly reduced (p > .05) sumption interval (p > .05), but the two higher doses with the O.08-mg/ kg dose of R019-4603. EtOH intake (16 and 32 mg/ kg) markedly elevated EtOH intake (p < for the animals under the combination cond ition was .01). As seen in Figure 58, only the lowest d ose o f brem nearly identical to that of animals given 0.08 mg/ kg zenil (8 mg/kg) reduced watcr intake at the IS-minute ROI9-4603 a lone across each consumption interval (p > interval (.I' < .01). The highest dose of bretilzenil (32 .05). Compared wi th the R01 9-4603 condition, the com mg/kg) significantl y increased water consumption at bination condition was without effect on water intake at the 120-m inute interval (p < .01). the 15-, 30-, and 6O-minule intervals. However, water Figun'S 6A and 6B rcitef<'lte the consumption o f the intake was substil nlially suppressed at the 120-minutc EtO H and water d ata, respecti vely, for the vehide, the interval oom pan:.xl with the ROI 9-4603 condition (p < .0 1). '\n.'ROf'SYCU()f'IIARMACOWCY 1996-Vot• • 14, /'.0. I Bcnzodialepines Alter Ethanol Intake 61 ! A. v...... •B 16 -.Ika: t'C 7till • v...... i o .18 "'II'kI R01,..., 8 ...... ! o Hi.., Fe '7142 ...... R01J..46t3 o .15 -.'\I ROIt-46e3 • !'" • i -: 2 • f • " •C i' [ l ,., " '" .. " .. '>I ,. o. f ,1, " • !'" •• ;; • • 3•• .. ,., "C onsumption'" Tntf.rY. ' (min) .. ,>I 6. Effects of the IP administration of vchide, 16 ms! leg FG11 42. 0.08 mg/ kg R019-46O), and a combination dose Consumption tnlu".1 (min) of Mo drugs on the inta ke of (A ) 100/" EtCH and (8 ) these Figure 7. Effects of the II' administration of vehide and liP by NP rats across fo ur consumption intervals. Data Me IwO doses of RO I9-4603 on the intake of (AI 3% polycosc the mean :: SEM (II = 7 r.lts c on environmentally EtOH drinking. Because kg) nonscl.xtively reduced intake of both nuids at I NP rats do not readily consume EtOllto pharmacologi IS-minute interval. MOI\.'Over, when rats were given cally relevant levels, the palatability-induced polydip choice between water and the polycose solution, wi sia method (Kulkosky 1978; Callo el al. 1990) and nuid out EtOJ-I. only the O.t5-mg/ kg dose supprcssc..>d wa deprivation were used 10 initiate EtOll intake. Simil.u intake during the IS-minule interval (see Figure to previous studies (s..,mson el al. 1989b; Gallo el al. Given the reported consistency of EtOH drinking' 1990; Rassnick et al. 1993), the dala reported here cle;uly NP rats (Mc Bride et al. 1988; s',mson et al. I 989b) un show that environmental processes can be manipulated limited access protocols in the absence of dnlg Ire to enhance EtOH intake in rats that normally avoid sub ments, one possible interpretation of the results sl.. ntial EtOH consumption. Average EtOH intake was served in the current study during the 30- to 120-minu 1.95 and 2.1 :::!;. 0.2 g/kg/2 hr during the 60 and 120 posldrug administr,ltion time inten'als may be that i minute intervals, respectively. Blood alcohol concentra creases in EtOH or water drinking renect elevations d tions (BACs) were nol determined in the present study, to pharmacologically induced effects on Auid inta howe\'er, Gatto et al. (1990) showed that NP rats at during the 1S-minute interval (Weiss and Koob 1991 tained BACs of approximately 60 mg% 1 hour into the However, because the pretre were noted fo r the l-mg/ kg dose at the 6O-m inute inter and water in stock rats, found that chlordiazepoxide nl (see Figure 3A). These findings with FG7142 could (3-12 mg/ kg) generally incrcaSL"'dure, demonstrated thot chlordiazepox potent Ihan ROI5-451 3 in blocking the EtOH-induced ide generally reduced EtOH-reinforCf.od n.>spondi ng and depressant action (2 g/kg) on exploration. However, re intake. Despite the consistent decreoscs o f EtOH-rein rent work (June et al. submitted) in our laboratory has forced I"\.>s ponding and intilke, no overall statistically shown that a 16- and 32-mg/ kg dose of FG7142 effec significant compensatory increase in the alternative ti\·clyantagonized EtOH intake in P rats. The findings fluid (e.g., sucrose, water) was observed for either of of the present study also contrast with previolls work tiu.'Se measures. by Samson et al. (1989a) in outbred rats, who found that Several reports have suggt'Sh_'diazepam, lient differences is the maintenance of the NP rats in the and it produces anticonnict effccts at much lower dQS('S present study on the polycose + EtOH regimen while and over a wider dose range (Richards et al. 1991). The the animals in our previous work (June et 011. 1995), and potent dipsogenic effects of bretilzenil on EtCH drink the study by 5.:1mson et al. (1989) were maintained on ing at IS minutes and water drinking at 120 minutes unflavored EtOH. Samson ct al. (1989a) have demon with the high dose (32 mg/ kg) (sec Figures SA and 13) strated that EtOH-reinforced responding is consider may be related to its ability to exert relatively high BDZ ably more sensitive to the attenuating effects of BDZ in receptor occupancy. Hence, the increase in EtOH intake \·c!"SC agonists (ROI5-4513 and r-c n-l2) than to sucrose in the current study may simply be an indirect reinforced I"\.>sponding. Thus, because of the purported quence o( bretazenil generally enhancing ingestive be-• low affinity of FG 7142 (or the BDZ rL-'Cepto r, along with haviors. An alternative interpretation of the findings the simultancous usc o( the polycose polydipsia me with bretazeniJ on EtOH intake is that brelazenil acts as thod and a fluid-deprivation schedule, it is possible that an anxiolytic in NP rats by blocking some of the aver higher doses of FG 71-12 may have attenuated EtOH sive properties associated with EtCH consumption, intOlke. the l"\.>by enhancing the reinforcing effects of EtOH (Rass Results with the full p-carboline inverse agonist nick et al. 1993). As mentioned, the aversive properties showed that, with the exception of the lowcst of EtOl1 may be related to its postingestional effects in dose (I mg / kg), there was a generalized behavioral sup· the NP rat (Bice and Kiefer 1990; W.lller et al . ]984). NP pl'l.'SSclnt effect on fluid int.lke and level of activity. rats are reported to have a lower threshold for the aver Moreover, with the highest dose, 500k of the animals ex sive effccts of EtOH in conditioned taste aversion stud hibited seizure activity evidenced by myoclonic jerking it..>s (Froc.hlich et oJ. 1988). of the head and forelimbs. These effects persisted for Un like our previous findings with ROI5-4513 Qune approximately S minutes. This generalized behavior.. l et al . 1993), FG 7142 (16 mg/ kg) did not alter the actions effect in NP rats CM possibly be altribuh:..>d of R019-l603 on EtOH intake (sec Figures 6A and B). toa greater n.>duction in GABAergic transmission caused These findings and its wcak potency in blocking the re by the full inverse agonist. inforcing actions of EtOH further confinn the weak af The UDZ partial agonist, bretazenil (RO I 6-6(28), did finily of FG 7142 ot BDZ receptors compared with not alter EtOH intake with the lowest dose (8 mg/ kg); ROI5-4SI3. FG 71 42 also doc'S not antagonize the intox however, the higher doses (16 and 32 mg/ kg) substan icating action of EtCH (Suzdak et al. 1986; Lister and tiallyelevated EtCH intake during the initial IS-minute Durcan 1989). A related hypothesis regarding the weak consumption period. A significant reduction in waler potency of FG 7142 to ontagonize EtOH's octions per in take occurn:.od at the IS-minute interval for the 8-mg/ tains to Ihe completc failure of FG 7142 to bind at diaz kg dose, and an increase occurred at the 120-minute in epam-insensitive (01) sitcs in the cerebellum (Tu rncr cl lerval for the 32-mg/ kg dose (see Figures SA and B). al. 1991). Although the significance of 01 sites are not These findings contrast with the recent work by Rassnick well understood, Turner and his colleogucs (1991; also and her colleagues (1993) who showed that chlordiaz Olsen and Tobin 1990) Sllggt'Stl' binding at diazcpamsensitive (OS) and 01 sites (Turner fluid deprived and initiated EtOH intakc in the horr.r et al. 1991; Wong and Skolnick 1992). At 0 1 sites R019- cage. 11ll1s, the rt'Sults of the present study may nOi 4603 binds at K, - 2.6 nM and at K, - 0.2 nM at OS sites gencrali :;o..e to subjects in ad libitum drinking and EtOH (Wong and Skolnick 1992). ROI5--451 3 binds with the reinforced responding sihlations. Given the preS('11t same affinity (Kd - 4.5 nM) to both OS and 01 sites findings with N P rats, and our previous work with (Turner ct al. 1991). Others (Wong and Skolnick 1992) ROlS-451 3 (McBride et al. 1988; June et al. have also confirmed the ROI5--4513 DI/DS potcncy ra ROI9-4603 Qune et al. 1993, 1994b), and the p-carbolinr tio of about I. Similarl y, the partial agonist bretazcnil inverse agonists FG 7142 and DMCM Qune et al. 1995) binds at 01 sites with a compara ble affinity (K, 10 in P rats, it appears that the property of the ligand nM), but c1assicill BOZ agonists such as diilzepam and (ligand stnacture, e.g., annelated 1, 4-diazepine) may bt midazolam posst--'SS an ICso > 10,000 nM (Wong and important in modifying EtOH intake.. Similarly, bretazt' Skolnick 1992). These findings suggest the possibility nil, at the agonist end of the BOZ continuum, also binds that the pharmacophore for annelated 1, 4-diazepines to 01 sites (Wong and Skolnick 1992) and increases that possess high afftnity to 0 1 BDZ receptors is funda EtOH intake to a greater degree than classical agonists mentally different at the OS BDZ n..>ccptors (Wong and (e.g., chlordiazepoxide, diazepam) that do not confer DI Skoln ick 1992). The identification of high-affinity (5.1mson and Grant 1985; Rassnick et al . 1993). Thus, the ligands at 01 BOZ receptors may assist in elucidating data of the present study with both anxiogenic ligands the pharmacological importance of these sites in the be• and the anxiolytic bretazenil, along with previous re• havioral actions of EtOH. search from our laboratory Ouncet al. 1993, J994b, sub Although food intake was not detennilU'!.. 1 in the mitted) and others (Samson and Grant 1985; Rassnick et present study, with the exception of bretazenil at the al. 1993) appear consistent with a qualitative (i.e., imi highest doses (16 and 32 mg/ kg) and at the 8-mg/ kg dazobenzodiazcpinc-sch::ctive) as well as a quantitative DMCM dose condition, nonc of the drug treatmcnts (relative receptor affini ty) difference in thc interaction have been obscrv(.od to exert any significant alteration of these ligands (affinity for 01 sites). Together, the on food intakc in f"->ely feeding P rats provided 4-hour present findings support existing evidence in P and oul limit(.>d access to lO'Yo EtOH and S<1cc harin solutions bred rats (for review, Sl.."'C June et 31. 1994a, 1994b) sug (June et al. 1993). It is also worth noting that lxxiy gesting that BOZ receptor ligands may modify neuronill weights for the animals in the present study were con processes that mediate some reinforcing and/or aver sistent with weight gain profiles for male NP rats in sive properties of alcohol. They further suggest a potcn previous studies, suggesting that the caloric balance tial importance of the GABA,,-BOZ receptor complex in was maintaim..od throughout the experiment. mediating palatability· (environmentally) induced In summary, the present findings confirm that envi EtOH drinking even in rats seiL>ctivcly bred for low al ronmental processes can be manipulated to initiate cohol preference. EtOH intake in rats of the P line (Samson et al. 1989b; Galto et al. 1990). The results also confirmed (June et al. 1993, 1994b) that doses of ROI9-4603 (0.08 and 0.15 ACKNOWLEDGEMENTS mg/ kg), may be capable of reducing EtOH intake 24 hours after administration, allhough some effects on WOller We wou ld like to express our gratitude to Dr. Peter Sort('r drinking may also occur. Moreover, compared with the (Hoffmann-L.1 Roche, Nutley, NJ) for kindly providing the other anxiogcnic ligands (e.g., Fe 71 42, DMCM) ROI9- ROI9-4603 and ROI6-6028. n,is l\.'SCarch was supported in 4603 was the most potent agent to block EtOH intake. part by grants AA08553 and AA07611; Craduate (RLH) and However, it should be noh..'CI that a scledivc suppres SPUR rrLC, JJM-B) Aw.u"d:. from the Department of Psychol .. sion of EtOH intake with all ROI9-4603 dOSl.'S was not ogy; and, Undergraduate Mentorship Rl.'search Fellowship obscrv(.od in the present study with NP rats, as havc (fLG) from the Office of Faculty Ot-velopmcnl. been observed during ad libitum drinking situations with Prats Oune et OIl. 1993, 1994b). The anxiolytic agent brctazenil ( 16 and 32 mg/ kg) markedly increased EtQI-1 REFERENCES consumption during the initial 15 minutes compan..od with control levels, although the increased consump Beaman OM, Hunter GA, Dunn LL, Reid LD (1984): Opioids, tion may result from a general dipsogcnic effect on flu bcn.t.lXIiazepincs and inl.lke of ethanol. Alcohol 1:39--42 ids. Although caution should be used in extrapolating Sicc I'J. Kiefer SW (1990): Taste reactivity in alcohol-prefer the findings of the current study to human situations, ring (P) and non-preferring (N!') r.lIS. Alcohol elin Exp there is a suggestion that moderate to high doses of ){es 14:72 1- 727 bretazenil may potentiate EtOH intake via an indirect Bracstrup e, Nielsen T. Honore T (1983): Bcnzodiazcpine interaction on ingestive behaviors.. It is important to l\.'CCpl or ti gands with positive and negative efficacy. 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