Journal oI Studieson Alcohol• Vol. 39, No. 1, 1978

Effect of EthanolPreexposure on EthanolInduced

Conditioned

Taste Aversion x

Lewis M. Barker and ThomasJohns SvMMastY.In a taste-aversiontest pairing saccharinand ethanol, rats that had recentl•t ingestedethanol showed attenuated aversionto saccharin.

REVIOUS RESEARCH hasindicated thatassessment oftol-

erance to ethanol canbe confounded by the typeof task

employedto reflectit and the degreeto which prior training in the taskis necessary (2-5). An ideal task for the assessment of drug tolerancewould be one that is sensitiveto small changesin tolerance,doesnot require extensiveprior training, and can provide for measurement of the dependentvariablewhen the animal is not drugged.The tasteaversionparadigm(6, 7), which fulfills theserequirements,was used in the presentstudy to assessthe development of differentialethanoltolerancefollowingseveralpretreatmentconditions.The build-up and loss of ethanol tolerance havebeenreported(2) to follow a fairly specificcourseover time andtheseconditions werereplicatedin the presentstudy.However, toleranceis not the only mechanism by which preexposure to a drug attenuatessubsequent conditioning(8). Therefore,various schedules of ethanolpreexposure were employedin the present studyto separateethanol-specific effectsfrom other more general effects.For instance,conditioned tasteaversions inducedby lithium chloridehave been found (9) to be more attenuatedin rats that werewater-deprivedthan in thoseallowedwater ad libitum during preexposure to the drug. In the presentstudy, restrictedand ad xFrom the Departmentof Psychology,Baylor University, Waco, TX 76703. This researchwas basedon a thesis(1) submittedby the secondauthor to the Graduate School,Baylor University, in partial fulfillment of the requirementsfor an M.A. in Psychology.The researchwas supported by an Encouragementof Scholarship Award by Baylot University to the first author. Receivedfor publication:3 May 1976. Revision:6 April 1977. 39

40

I•. 1¾[.BARKERAND T. JOI-INS

libitum water was scheduledfor groupsof rats allowed one of severalschedulesof preexposureto ethanol,and these treatment effectswere measuredby a conditionedtaste-aversion procedure. METHOD

The subjectswere 48 male Holtzman albino rats weighing350 to 450 g at the beginningof the experiment.All had 2 weeks of habituation to the laboratory prior to the 37-day experiment.All animals were individuallyhousedin standardcagesin which Purina Lab Chow was available ad libitum throughoutthe experiment.The animal colony was maintainedat 21øC on a 12-hr light-dark cycle (lights on from 0730 to 1930hr). All animalswere weighed daily for the first 8 days of the experimentand thereafteronly at the 3- to 4-day intervalswhen the intubation solutionor dosagewas changed. Each of the 48 rats was randomlyassignedto i of 6 groups.Five of the groups received different schedulesof ethanol intubation and accessto water during a 21-day preconditioning phase,while a control groupreceiveddaily placebointubationsof a 50g sucrose(w/v) solution in dosesdesignedto producea caloricintake equal to that of the animalsreceivingethanolduring the preconditioning phase (Chart 1). The groupsdifferedalongtwo main dimensions, namely,the sequence of ethanoland sucroseintubationsand the availabilityof water during this period. For groups1, 3, 5 and the control group, a 23•-hr waterdeprivationschedulebeganon day i and lastedthroughoutthe experiment. Groups2 and 4 were allowedwater ad libitum during days 1 through 18 and a 30-min watering period each day thereafter. For all groupsthe 30-minwateringperiodsoccurred4 to 5 hr after intubation. All intubationswere performedbetween 1000 and 1200 hr.

The groupsreceivingethanolwere intubatedwith a solutionof tap water containing30• (w/v) ethanol,daily dosesbeing 3 g per kg for the first 4 days,4 g per kg for days5 through7, 5 g per kg for days C•RT Group I

Intubations

1.--PreconditioningSchedules Da•ls

Water

Da•ls

30 min per day

1-21

2

Ethanol Sucrose Ethanol

1-14 15-21 1-14

ad libitum

3

Sucrose Sucrose

15-21 1.-7

30 min per day 30 min per day

Ethanol Sucrose Ethanol Ethanol

8-21 1-7 8-21 1-2

4

5

ad libitum

1-18

19-21 1-21 1-18

30 min per day 30 min per day

19-21 1-21

30 min per day

1-21

4-5...

Control

Sucrose

3, 6 . . .

Sucrose

1-21

ETHANOL-INDUCEDTASTE AVERSION

41

8 through10, and 6 g per kg for days11 through14. This intubation procedure, usedin groupsi through4, hasbeenshown(2) to produce ethanoltolerancein the rat. For group5, this intubationsequencewas interruptedeverythird day by sucrose intubations, requiringa 21-day insteadof a 14-dayperiodfor the completion of the full scheduleof ethanol intubations.

All rats were intubatedwith No. 8 Fr polyethyleneinfant feeding tubes(Cutter) connected to syringesvia a 3-way stopcock.Following an intubationwith ethanolor sucrose,the feeding tube was flushed with a sufficientamountof water to clear the tube, thereby eliminating the taste of the treatment

substance when

the tube was withdrawn

from the stomachthroughthe oral cavity.

The second phaseof the experiment beganon day 22, the conditioning day, when all groupswere allowed30 min of accessto a 0.15gsaccharin sodiumsolutioninsteadof water, followed immediatelyby intubation with 6 g per kg of ethanol.The sameprocedurewas followedon five test days (days 25, 28, 31, 34 and 37). On the interveningdays, all rats were allowed 30 min of accessto tap water. Fluid intakeswere measuredby weighing the drinking bottles before and after presentation and recordingconsumptionto +--0.5g. The data were analyzedby a split-plotfactorialanalysisof variance of the saccharinintakeson the five test days and separate,completely randomizeddesignson the samedays (10). The multi-comparison rate of error was controlledby Scheff•'smethod,a = .05. RESULTS

Three animals(two in group3 and one in group4) died during the firstweekof the preconditioning phase,apparentlyfromcauses other than the experimentaltreatment.All animalsbecameprogressivelyeasierto handle and to intubate during the preconditioningphase.Nonsystematic observationof individual rats' behavior following ethanol intubationrevealed that even though the dosesof ethanol were increasing,the rats manifestedless ataxiaand somnolence in responseto the ethanol. Withoutexception,everyrat intubatedwith ethanollostweight. Following 2 weeks of ethanol intubation while water-deprived, the rats experienceda mean weight lossof 46 g comparedwith a meanlossof 30 g in rats intubatedbut allowedwater ad libitum. By contrast,there was no changein the mean body weight of the controlgroup,which was intubatedfor 3 weekswith sucrose. Analysisof the saccharinintakeson day 22, i.e., the intakesof the conditionedstimulus(cs) on the conditioningday, revealed no significant intergroupdifferences. The meancs intakesranged

42

L.M. BARKER ANDT. JOHNS

from6.1 to 11.8g. The effectof the pairingof cs and the unconditionedstimulus(vcs, i.e., ethanol) on day 22 and following days is shownin Figure 1, where mean saccharinintakesof all groupswere plotted as a functionof successive treatmentdays. The meanintakesof the five groupspretreatedwith ethanolshow a similarpattern,an initial rise followedby a gradualdeclinein consumption. The controlgroup (C in Figure 1), which experiencedethanolfor the first time on day 22, showedsignificantly greateraversionto saccharinas the test continued.A significant over-alltreatmenteffectacrossdays(F = 14.831,5/39 dr, p (.01) can be accountedfor by differencesbetween the experimental groupsand the controlgroup (F -- )20.0, 1/39 dr, p ( .01 for all individual comparisons of groupsI through 5 with the control group). The main findingsof this experimentoccurredon the first test day (Figure 1). An omnibusF on this day was significant,indicating intergroupdifferences.The mean intakesof saccharinon this day by all groupspretreatedwith ethanol were compared with eachotherand the controlgroupby singledegree-of-freedom contrasts,controllingType I rate of error at .05 usingScheff•'s method(10). A pretreatmenteffect was found in groups3, 4 and 5 in comparison with the controlgroup,all F valuesbeing greater than the criticalScheff•F of 12.275(p--• .05) at 5/39 dr. Groups

• 24

3

•/

--_

z_ 2o

1•

!

! /

! !

• 16

2/'

12

I

I

I

I

I

1

2

3

4

5

TEST

DAYS

F•ct•v, 1.-Mean Intakes of Saccharinby GroupsI through5 (Days 25, 28, 31, 34 and 37). Brokenlines indicate groupshaving accessto water ad libRum during preconditioning.

ETHANOL-INDUCED TASTE AVERSION

43

1 and 2, however,whose14-dayethanolpreexposure was followed by 7 days of sucroseintubationprior to the conditioningday, did not showthe preexposure effect, as inferred from the finding that their saccharinintakeson the first test day did not differ from thoseof the controlgroup(F: 11.569and 0.829 for groups I and 2, respectively).GroupsI and 2 combined,moreover,were significantly differentfromgroups3, 4 and5 combined(F -• 15.687, p < .05). On the secondthroughfourth test days,all preexposure groups differed from the controlgroup. On the fifth test day, groups1 and 2 did not differ significantlyfrom the controlgroup(F = 8.712 and 10.791,respectively, p ), .05).Hence,at leastin the early and later stagesof conditioning,both groups1 and 2 showedsignificantly more conditioningthan did the remainingexperimental groups.

Additionalanalysesindicatedthat on the remainingtest days there were no significantintergroupdifferencesin saccharinaversions(for all, p > .05). The orderingof mean intakesof saccharin acrosstest days, however,showsdegreesof aversionconsistent with predictionsbasedon assumptions aboutthe developmentand lossof ethanol toleranceduring preconditioning;i.e., the control groupshowedthe greatestaversion,groupsI and 2 slightlyless, and groups3 through5 the least aversionto saccharin.Groups2 and 4, which had water availablead libitum during preconditioning, did not differ significantlyin saccharinconsumption from groupsI and 3, respectively, on any test day (for all, p > .05). DISCUSSION

Conditionedtaste aversionsare formed by pairing flavored ingestantswith the aversiveconsequences of administereddrugsor

toxins(12, 13). The effectiveness of theseaversiveconsequences hasbeenshown,asin the presentstudy,to be reducedby presenting the drugsor toxinsprior to the conditioningsituation.In most studiesof this preexposure phenomenon, whichhavebeenreviewed recently(8, 14),the levelof preexposure doesnot lead to tolerance. However,the developmentof drug tolerancehas been proposed as an explanationfor the attenuationof conditionedtaste aversionsobservedfollowingpreexposure to ethanol(6) and morphine (15). Thesetheorieshave beenreviewed(16). The findingsof the presentinvestigation concernethanol-specific

44

L. 1v[.BARKER A1VD T. JOHI•S

effects(ethanoltolerance)versusnonspecific effects(generalized preexposure to a drug). GroupsI through4 were exposedto an experimentalprocedurewhich has been demonstrated(2) to produceethanoltolerancein rats.Prior exposureto ethanolmarkedly reducedthe association of ethanolwith saccharinin rats in groups I through 4, resulting in little conditionedaversioncompared with that of the controlgroup.Furthermore,groupsI and 2, which receivedsucrosefor 7 days after they had developeda tolerance to ethanol,showedlessof this preexposure effect on the first and fifth testdays.Sincethis7-dayinterruptionof preexposure produces a lossof tolerance (2), the reduced preexposureeffect seen in groupsI and 2 may be attributed to a generalizedpreexposure effect,not specificto ethanoland presumablynot attributableto elevatedblood alcohollevelsat the time of conditioning.Whether the reappearance of the preexposure effect on the secondthrough fourth test daysin groupsI and 2 can be attributedto a resumed buildup of toleranceor to a sensitization-like single-trialpreexposure effect is unclear. Since attenuated conditioninghas been reportedfollowinga singlepreconditioning exposureto ethanol (7), presumablyin the absenceof tolerance,a nonspecificpreexposureeffect is the more likely explanation. In the studyby Cannonet al. (7) the preconditioning exposure was found to be effectiveonly within a 24-to-96-hrperiodprior to conditioning,and was ineffective in attenuating conditioning if presentedmore than 96 hr before the cs-vcs pairing. In the presentstudythe attenuatedconditioningof groupsI and 2 suggeststhat the developmentof tolerance(as opposedto a single preexposure to ethanol)may yield morepermanenteffectswhich exist(presumably in memory)followingthe lossof tolerance. This suggestion issupported by therelativelytemporaryeffectsongroups I and 2 of the 7-dayinterruptionin ethanoladministration. These groupswere similarto groups3, 4 and 5 on the secondthrough fourthtestdays.It is unclearwhy both groupsi and 2 apparently beganto be conditionedafter five saccharin-ethanol pairingsso that comparedwith the other experimentalgroupsthey drank significantly lesssaccharin solutionon the fifth test day. A similarlydesigned studyof the effectof preexposure to lithium chloride on lithium-inducedtaste aversions(9) found that the preexposure-attenuation phenomenon was state-dependent with respectto waterdeprivation. Ratspreexposed to lithiumandtrained and tested while water-deprivedshowedgreater attenuationof

ETHANOL-INDUCED TASTE AVERSION

45

conditioningthan did rats preexposedto lithium while provided water ad libitum but trained and testedwhile water-deprived.Although a similar state-dependency effect was not found in the presentstudy,the saccharinintake of group 2 (preexposedwhile water was providedad libitum) was nearestthat of the control group on the first test day and was, therefore,in the predicted direction. That is, the more dissimilar the animal's milieu interne during preexposure and on the conditioningday (day 22 in the presentstudy),the greaterthe predictedlevel of conditioning.Rats maintainedon restrictedfood and water have been shown (17) to have a reducedrate of ethanolmetabolism.It may be that the group2 rats receivingfood and water ad libitum during preconditioningshowedlesspreexposure effect becausethey were better able to metabolize the intubated

ethanol and were therefore more

susceptible to ethanolwhen it was presentedon day 22. The doseof ethanol(6 g per kg) intubatedinto the controlgroup was of sufficientmagnitudeto produceunconsciousness of several hours'duration.Despitethis apparentlystrongvcs the conditioning was markedlyineffective.The controlgroupdrank 10.2 g of a 0.1%saccharinsolutionprior to treatment with any vcs, and after five specificpairingswith ethanolshowedno decrement(the mean amountingestedon the fifth test day was 13.5 g). The conditioningeffect mustbe inferredfrom the controlgroup'sfailure to developduring the 30-min test trial the elevatedsaccharin intakeswhich occurredfor the five groupspreexposedto ethanol. The conditioningwhich resultsfrom pairingsof a novel taste with ethanolis poorerthan that which resultsfrom pairingsof a novel taste with other drugs.This phenomenonhas been noted (6, 7, 18, 19) as has the difficulty of inferring an aversivestate from clinicalsignsfollowingtreatment(20).

REFERENCES

1. JoHNs,T. Assessment of toleranceto ethanol in the rat by taste aversionconditioning. M.A. thesis, Baylor University; 1976. 2. LEBLANC,A. E., KaLANT, H., Gm•NS, R. J. and BE•tMAN,N. D. Acquisition and lossof toleranceto ethanol by the rat. J. Pharmacol. 168: 244-250, 1969. 3. CHEN, C. S. A study of the alcohol-tolerance effect and an introductionof a new behavioral technique. Psychopharmacologia,Berl. 12: 433-440, 1968. 4. LEBLANC,A. E., GIBBINS,R. J. and KALANT,H. Behavioral augmentationof toleranceto ethanolin the rat. Psychopharmacologia, Berl. 30: 117-122, 1973. 5. MosKow•Tz, H. and WaeNEa, M. Studies on the acquisition of behavioral toleranceto alcohol.Quart. J. Stud. Alc. 25: 619-.626, 1964.

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L. 1•. BARKER ANDT. JOHNS

6. BE•M•,N, R. F. and C•,NNON,D. S. The effect of prior ethanol experienceon ethanol-inducedsaccharinaversions.Physiol. & Behav., Elmsford, NY 12: 1041-1044, 1974. 7. C•,•o•, D. S., BE•sf,•N, R. F., B,•r,•l•, T. B. and ATKINso•, C. A. Effect of preconditioningunconditionedstimulusexperienceon learned taste aversions. J. exp. Psychol.Anim. Behav. 104: 270-284, 1975. 8. G•,sfzv, E. The multifaceted nature of the post-ingestionalconsequence-is there a single common factor? In: B,•r,s•, L. M., BEST, M. and DOMJAN, M., eds. Learning mechanismsin food selection.Waco, TX; Baylot University Press; 1977. 9. Sv,taEZ,E. M. and B,•, L. M. Effects of water deprivationand prior LiCI exposurein conditioningtaste aversions.Physiol.& Behav.,Elmsford,NY 17: 555-559, 1976.

10. KINK, R. E. Experimentaldesign;proceduresfor the behavioral sciences.Belmont, CA; Brooks/Cole; 1968. 11. Boy,z,R. J. and Km•z, R. E. A general method of partitioning sumsof squares of treatmentsand interactionsin the analysisof variance. Educ. psychol. Measmt, 1977. [In press.] 12. G,•cI,•, J. and KOEX,I•N•,R. A. A comparisonof aversionsinduced by x-rays, toxins,and drugs in the rat. Radiat. Res., Suppl. No. 7, pp. 439-450, 1967. 13. B,•m•r.•, L. M., Br.s% M. and DOMJ,•N, M., eds. Learning mechanismsin food

selection.Waco, TX; Baylor University Press;1977. 14. Baxvv.•,•N, N. What studiesof preexposureto a pharmacologicalagent tell us about the nature of the aversion inducing treatment. In: B,•I•g•, L. M., Br.s% M. and DOMJAN, M., eds. Learning mechanismsin food selection. Waco, TX; Baylor University Press; 1977. 15. LEB•,,•,Nc,A. E. and C,•,•,E•,•,, H. Attenuationof punishingeffects of •norphine and amphetamineby chronicprior treatment.J. comp. physiol. Psychol.87: 691-698,

1974.

16. C,•,•,gx,•, H. and LEB•,,•Nc,A. E. Gustatoryavoidanceconditioningby drugs of abuse;relationshipsto general issuesin researchon drug dependence.In: M•,a•,•, N. W., K•,•Es, L. and A•,•ow,•r, T. M., eds. Food aversion learning. New York; Plenum; 1977. 17. L• B•groN, E. Influence du je6ne sur la vitessed'oxydationde l'alcool •thylique chez le rat blanc. C. R. Soc. Biol., Paris 122: 330-332, 1936. 18. EcIc•m•r, M. J. Alcohol-inducedconditionedtaste aversionin rats; effect of concentrationand prior exposureto alcohol.J. Stud. Alc. 37: 334-346, 1976. 19. LEsrE•, D., N,•c•,•N, M. and LE M,•aNEN,J. Aversiveconditioningby ethanol in the rat. Quart. J. Stud. Alc. 31: 578-586, 1970. 20. B,•r,•, L. M., S•Ir•, J. C. and Sv•,•,•z, E. M. Sicknessand the backward conditioningof taste aversions.In: B,•m•, L. M., BEst, M. and DOMJAN, M., eds. Learning mechanismsin food selection.Waco, TX; Baylor University Press; 1977.