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Benzodiazepine Receptor Ligands with Different Intrinsic Efficacies Alter Ethanol Intake in -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 (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 Alter Ethanol Intake 57

\iously tested for free.d imml>clia tely prior to injec­ tions and administcn.."CI intrapcritonea l1y (lr) in a volume P.J.lJlability-lnduced Ethanol Intake by NP Rats of I ml / kg body weight. l\vccn-SO vehicle was admin­ The rats were acclimah...oci over 2 weeks to 22-hour fluid istered as the control injection for all drug conditions. depriva ti on schedule, while all fluid access was re­ Drug treatments were administert"CI in a random order. stricted to the dark phase of the cycle between 1000 and To control for any residual dmg carryover effects, pre­ 1200 hours. Food (Tekl ad Diet #700 1, Harlan Industries, trea tments with Tween-SO vehicle were adminiSlcn."CI Indianapolis, IN) was available ad libitum. Animals re­ every 3 to 4 days, after baseline d rinking patterns were mained on this schedule throughout the remainder of rt.."t.'Stablished. At the conclusion of the study, the EtOH the experi ment. This experimental appTTh'lch was used was removed from the cocktail and animals were pro­ because of the reported short half-life (Mandema et al. vided access to the polycosc solution and water 2 hours 1992) and short duration of action (d' Argy et al. 1987) of daily for an additional 2 weeks. Drug treatments that some of the BDZ ligands and the ability to measure showed selectivity in attenuating EtOH intake were eval­ drug actions on both the cocktail and water intake uated for their ability selectively to alter the 3,... polycosc when food was available ad libitum. The position of the solution in the absence of EtOH and water. bottles was alternated daily to avoid the development of any position preferenct.>s. Method of Analysis During the initial depriva tion phase, animals were provided water in two 100-011 gradualt:.'d Richter bOllk'S Two analyses were conducted on the data; the fi rst for for 2 hours daily. After 2 wl.'t!ks of acclimation to the EtOH, and the second for water intake. The analysis Auid-deprivation schedule, a modification of the proce­ consisted of a 16 X 4 repeated-measures analysis of dures of Ku lkosky (1978) was implemented to initiate variance (ANOVA). The first factor represented treat­ EiOH intake (see Caito ct al. 1990, for complete details ment condition (Tween-SO vehicle; 0.02, 0.04, 0.08, and of the procedure). Briefly, before EtOH was added, the 0.1 5 mg/ kg ROI9-4603; 1, 4, 8, and 16 mg/ kg FG 7142; "cocktail " consisted of 3% (w I v) polycose, 0.5'>'0 (w I v) 8, 16, and 32 mg/ kg ROI6-6028; I, 4 and 8 mg/ kg NaCl, and 0.125'>'0 s.,ccharin (w I v). EtOH was added to DMCM; and a combination condition comprising 16 the cocktail in 2% (v I v) increments evcry third day un­ mg/ kg FC 7142 + 0.08 mg/ kg ROI9-46(3). The second til a 10% (v / v) EtOH concentration was attained. Once factor represented consumption interval (15, 30, 60, and the drinking patterns of both the EtO H cocktail and wa­ 120 minutes). The combination condition was adminis­ ter had stabilized, Tw(..oen-SO veh icle injections wcre ad­ tered to evaluate whether an inverse agonist with 11 ministered prior to the drinking sessions to acclimate [3 -c.uboline structure would N.'Semble the imidazoben­ the animals to the injection procedure. Intakes were zodiazcpine ROI5-451 3 Qune et al. 1993) in its ability to measured at 15-, 30-, 60-, and 120-minute intervals dur­ attenuate R019-4603's suppressant effects on EtOH Ing the 2-hour period o f fluid availabili ty. Subjects were self-administration. The Newman-Kellis a posteriori weighed twice per week to monitor body weight gain. test was conducted on painvise comparisons of group Animals were given daily access to the two Auids for I means. month prior to the experimental dmg treatments. Previously, we (June et al. 1994b) demonstrated that in P rats mai ntained on 24-hour free-c.hoice access to 100/.. (v I v) unflavored EtO H and wilter, ROI9-4603 se­ Drugs lectively suppressed EtOH intake up to 32 hours post­ Drugs were prepared as no emulsion by powder agita­ drug administration. ik.ocause of R019-4603's potentiill tion (Scientific Mixer) in 1'>'0 Tween-SO vehicle (Sigma thera peutic usefulness in the development of less toxic Chemical Co., St. Louis, MO) and mixed with a 0.90% agents in reducing ilbnormal alcohol-seeking behavior, NaCi solution to a fixed volume. ROI9-46OJ (tert-butyl our p revious work with P rats was replicated in the NP 5, 6-dihydro-5-methyl-60x0-4 H-imidazo-ll,5-a]-thieno- line. Hence, to examine the time course of R019-4603, 12,3-flll,4 l-diazcpine-3-carboxylatc) from Hoffmann-L..t separate two-way ANOVAs (drug condition x time) Roche (Nutley, NJ) was tested at doses of 0.02, 0.04, were conducted on the EtO H and water data 24 hours and 0.15 mg / kg. FC 7142 (N'-methyl-l3eta-carbo­ postdrug administration for the four R019-4603 dose Jine-3-carboxamide) from Sigma Chemical Co. (51. Louis, conditions (0.02, 0.04, 0.08, and 0.15 mg/ kg) compared MO) was testl.>d at I, 4, 8, and 16 mg / kg. R016-6028 with the vehicle control. 58 II .L cl al.

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 pretreement with a previous report (s..,mson et al . The l"CSults of the pre.ent study further confirm Ou 1989b), the current study demonstrated thai, unlike P et al. 1993) thai a single injection of R019-4603 OIl tht (Murphy el al. 1986) and nonl'oCit.octLxI stock rats (s..,m­ 0.15- and O.OS-mg/ kg dose levels arc capable of exert­ son 1986), NP rats distributed thei r EtOH drinking in ing prolonged attenuation of EtOH intake; at. 24-hour smaller bouts over the length of the session. Specifically, postdrug administration, the O.1S-mg/ kg dose of ROI9- animals maintained a moderate level of intake over 60 4603 significantly reduced EtOH intake during the 15- minules, with negligible intake occurring during the minute inlerval, and the O.08-mg/kg dose completely 120-minute interval (sec Figure IA). Gatto et al. (1990) supprt.>SSed intake at the 6O-minute interval (Figure suggested that NP rats I\.--du ce their drinking of the 3% Water consumption was signific .. ntly decreased at thr Polycose + EtOH solution when 8ACs reach a point IS-minute inten'al 24 hours after the 0.04- and O.08-mg/ where the aversive effects of the EtOH begin to counter kg dose of ROI9-4603, but it was increased at the 30- the highly palatable sweet taste of the polycosc solution. minute inten'al for the 0.02-mg/ kg dose, and at the This hypothesis is consistent with previous research in­ minute interval for the 0.02-, 0.04-, and O.tS-mg/ kg dicating that, in contrast to P rals, the postingestional dOSL"S (Figure 2B). Recently, we demonstrated thai when pharmacological properlies of EtOH al comparati vely r rats were provided 24-hour continuous access to un­ moderate BACs arc aversive the NP rat (Waller et al. navored EtOH and WOller in their home cage, EtOH in­ 1984; Froehlich et al . 1988; Uice and Kiefer 1990). take during the 8 hours afler R019-4603 (0.075-0.]) The primary purpose of the prt..'SCnt study, however, mg/ kg) was substantially reduced; however, during thr was to examine the ability of BDZ rc-ceptor ligands with 9-to 24-hour interval, EtOH intake did not differ from different intrinsic efficacies selectively to alter palatabil­ control values. At the 24- to 32-hour post-ROI9-460J ad· ity-induced EtOH intake. The major findings of the ministration, significant suppression was again SCttI study are that some compounds acting via the GABA­ with the higher R019-4603 dose (0.30 mg/kg). Water BDZ rc-ceptor complex can modify drinking patterns in drinking in that study was not altered or showro in­ the NP rat line. creases at the 8-, 24- and 32-hour consumption inter­ Consistent w ith our previous work in P rats under vals. The differential eff.xts obsen'ed with ROI9-4603 limited Oune et al. 1993) and continuous 24-hour access 24 houTS after administration bctwL'e1l animals under Oune et al . 1994b), the present shldy demonstrates thai continuous acct$S in the previous study and limited ac­ a single injection of the polent BDZ inverse agonist, cess drinking in the present study may be relaled to thr R019-4603 (0.04 and 0.15 mg/ kg) given acutely, mark­ pattern of EtOH drinking under the two tyPt.'s of sched­ Lodly suppressed EtOH intake during the initial 15 min­ ules on the one hand, and the manner in which EtOu utes of the 2-hour consumption period in NP rail.. In intake is measured on the other (for review s..."C Murphy addition, the highest dose continued to suppress intake etal.1986;alsoJunectal. I99-1b). al the 3O-minute interval. Water intake showed no dif­ Unlike the profile of effects seen with ROI9-4603, the ferences from vehicle control intakes or showed in­ f3.-<:arboline partial inverse agonist, FC7142, did not re• creases across two of the consumption intervals (e.g., 30 duce EtOH intake at any of the doses testL"Ci at the IS. and 120 minutes), although the highest dose (0.15 mg/ minute and JO-minute intervals, although reductions \ElllOl'SYOIOI'UARMACOlOGY 14, NO. 1 Bcnzodiazepincs Alter Ethanol lnt.lke 63

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_'