Journal o! Studies on Alcohol, Vol. 39, No. 11, 1978
The Influence
of Ethanol
on Learned
and
ReflexiveHeart Rate Responses of Rats During Classical AversiveConditioning RobertD. Fitzgeraldand GeneL. Stainbrook SVMM.•nY. An unconditioned response wasproducedin rats by electricshockand resultedin a hipbasicdeceleration-acceleration of heartrate a#er 2.4 g of ethanol perkg. A monophasic acceleration occurred with salineand0.8 g of ethanolper kg.
ELATIVELY LITTLEinformation is available concerning
the effects ofalcohol on learned and reflexive responses that occur in Pavlovian conditioning. Most of the experiments bearing onthistopic have been limited totheuseofsingle doses of alcohol (typically ethanol ordistilled spirits) given toa relatively smallnumber of subjects, or multiple doses administered sequen-
tiallytothesame subjects. Moreover, theresults havebeenmixed, andbothfacilitative andsuppressive responses havebeenreported. Doubtless, many ofthese apparent inconsistencies canbeaccounted forbyspecies differences, variations inalcohol doses anddiscrepanciesin thelength oftimethatwasallowed to elapse between the administration of alcoholand the beginningof testing.
Asanexample of theimportance of thelatterfactor, some of theearliest experiments (1-3)revealed that,soon afteradministration,moderate doses of alcohol, estimated to be in therangeof 0.15to0.39gperkgofbody weight, frequently produced increased salivation in dogs toa nonreinforced conditioned stimulus (cs)pre-
sented in a discrimination paradigm without visibly affecting the • Fromthe Department of Medical Psychology, University of Oregon Health Sciences Center, 3181SWSamJackson ParkRoad,Portland, Oregon 97201.This research wassupported byNational Science Foundation GrantBMS74-18542. Gene Stainbrook wasa trainee on a National Institute of Alcohol AbuseandAlcoholism traininggrant(No. 1 T32 AA07074-01).
Received forpublication: 26 August 1977.Revision: 7 June1978. 1916
X-l•.•x-a.•x•
a•svo•s•s
ov a.•xs
1917
reactionsto the reinforcedcs. Similarshort-latency effectswere sometimes observedwhenhigherdosesof alcoholrangingfrom 0.48 to 3.50 g per kg were used,exceptthat in thesecasesincreased salivationwas immediatelyfollowedby sharpreductionsin respondingto both nonreinforcedand reinforced css. A moderate
alcoholdoseof 0.5 g per kg given2 hr beforetestingwasalsofound to blockthe occurrence of the accelerative heart-rate(i•a) conditionedresponse(ca) in somedogsto a reinforcedcs without apparently changingthe responses to a nonreinforcedcs (4). Seemingly paradoxical changes in the vigorof orientingreactions (oR) asopposed to cRshavebeenobserved in both nonhuman and humansubjectsreceivingrelativelyhigh dosesof ethanol.In one instance(2), dogsgiven intoxicatinglevels of alcoholseemedto displayan augmented oR (headturningin the directionof the cs) eventhoughthe performance of the salivarycR was blocked.In
a studyinvolving humans(5), 0.70g of alcoholper kg suppressed the accelerative I•R component of the oR triggeredby a novelcs, whileat the sametimeenhancing the decelerative component. Facilitationof reflexiveI•Rchangesinducedby the Valsalvamaneuver or by a body-tiltmanipulation were alsofoundin humansfollowing ingestion of 0.30or 0.60g of alcoholper kg (6). Severalexperiments haveshownthat amountsof alcoholranging from0.24to 0.57g perkg arecapableof interferingwith eye-blink (7) and galvanic-skin (8) cRsin humans.In anotherstudy (9), however,a relativelyhigh doseof alcohol(approximately 1.60 g perkg) failedto modifyhumaneye-blinkcRsthat wereestablished on the basisof a very strongair-puff unconditionedstimulus(rs). This suggests that the adverseeffectsof alcoholon cRsmight be mitigatedby the useof an intensers. However,studiesof classical conditioning designedto lookspecifically at vs intensityand different amounts of alcohol are not available.
The presentstudyexamined the effectsof ethanolon the •xx•reactionsof restrained rats during aversive classicalconditioning. Particularattentionwas focusedon ethanol-relatedchangesin the oR elicitedby the cs prior to conditioning,in the developmentof the cR and in the unconditioned response(vR) producedby the rs. To provideinformationon the questionof the relationship between vs intensity and conditioningperformanceunder alcohol, two concentrations of ethanol were combined with two intensities
of the us in a factorialdesigninvolvingseparategroupsof rats. A
1918
R.D. FITZGERALDAND G. L. STAINBROOK
preliminarystudywas alsocarriedout in which blood alcoholdeterminations were madein animalsreceivingessentially the same treatmentas that usedin the main experiment. METHODS
Subiects
The animalswere129naivefemaleLong-Evans hoodedrats (SimonsenLaboratories, Gilroy,California),90 to 120daysof ageand ranging in weightfrom250to 325g. Theywerehoused2 per cagewith a 12-hr light-dark cycle and food and water ad libitum. The animalswere tested during the light cycle. Apparatus
An invertedU-shapedplastichousingmanufactured by Narco BioSystems was usedto restrainthe rats.To eliminateunwantedauditory signalsfrom reachingthe animals,the experiment was carriedout in an IndustrialAcousticsound-isolation chamberequippedwith a ventilating fan, a 60-W houselight and a 10.2-cmspeaker.To providefurther
masking of extraneous auditorysignals, whitenoisemeasuring approximately75-dBsoundpressure level (about0.0002dyn/cm 2) was presentedcontinuously throughthe speaker.Two animalswere conditioned
concurrently in identicallyequippedchambers. The electrocardiogram wasrecorded on a Grasspolygraph from20-gauge hypodermic needles insertedthrougha fold of skin oppositeeach other on either side of the rat's thoraciccavity. Heart rate was measuredby meansof an
automatedon-linesystem(10) that provideda punchedpaper-tape tabulation of the totalheartbeats occurring in successive timeperiods of each trial.
The cswasa 6.5-sec, 2.9-kHztonepresented at 85-dBsoundpressure level (about 0.0002dyn/cm") througha 2.5-cmMallory Sonalert mounteddirectlyin frontof the animal.The uswasa 0.5-sec, 60-Hz,AC electricshockproducedby a GrasonStadler E6070B stimulatorand
deliveredto the baseof therat'stail throughtheheadsof No. 6 machine screws thatwereheldin placeapproximately 2 to 3 cmapartwith small piecesof rubbertubing.A delayedconditioning paradigmwasusedin which the 0.5-secvs overlappedand terminatedwith the last 0.5 see of the cs.
Procedure
In the mainexperiment, therewere6 experimental groupsof 16 animalseachand 3 conditioning-control groupsof 5 animalseach.The 6 experimental groupscomprised the cellsof a 3 X 2 factorialdesign, onedimension beingthe drugtreatment(an intraperitoneal injectionof
0.8 g or 2.4 g of ethanolper kg of bodyweightor normalsaline)and
HEART-RATE RESPONSESOF RATS
1919
the otherbeingthe intensityof the us (0.6 or 1.2mA). The ethanoldoses were preparedfrom 95•oethanolmixedwith normalsalineon the basis of I ml of solutionper 100 g of body weight and injected"blind" (the persongiving the iniectionswas unaware of the agent being administered). The 3 controlgroupsreceivedeither saline or one of the two doses of ethanol.
Subsequent to an initial 15-minperiod of adaptation,all animalswere taken out of the sound-isolationchamber and given an iniection of the appropriateagentthrougha hole in the floor of the restrainingdevice. Following a 15-min absorptionperiod, 10 pretest presentationsof the cs alonewere givenat intertrial intervalsof 70, 90 or 110 sec (mean, 90 sec). Upon the completionof thesetrials, animalsin the experimental groupreceived20 paired conditioningtrials at randomlyspacedintertrial intervalsof 160, 180 or 200 sec (mean, 180 sec). Animals in the 3 control groupsreceived20 explicitly unpaired presentationsof the cs and us, the cs followingthe us at randomlyselectedintervalsof 70, 90 or 110 sec(mean, 90 sec). The high-intensity1.2-mAus was usedfor all of the control groups. Samplesof base-levelHa in 6-secperiodswere taken at 3-min intervals during the 16-min preiniectionand 15-min postiniectionabsorption periods.The Ha responses to the cs on the pretest cs-aloneand conditioning trials were measuredby subtractingthe beats-per-minuteHR during the 6 sec before startingthe cs from the rate during the 6-sec interval before the us. The unconditionedHa responsesto shock were also convertedto differencescoresusing the Ha before the cs. The preliminarystudy providinginformationon blood ethanol concentrationsinvolved24 rats of the samestrain, age and weight as those used in the main experiment.These animalswere randomly assignedto I of 4 groupsin a 2 X 2 factorial design,one of the dimensionsbeing ethanoldose (0.8 g vs 2.4 g per kg of body weight) and the other us intensity(0.6 vs 1.2 mA). The pretestingand conditioningprocedures were the same as those in the main experiment. Blood sampleswere taken from the middle caudal vein in the rat's tail with a heparinized microcapillarytube. Prior to conditioning,the rats were anesthetized with ether, and a 2.5-cm midventral incision was made near the base of the rat's tail. The caudal vein xvasexposedand the incisionwas then looselyclosedwith adhesivetape. Procainewas applied topically to the area as a local anesthetic,with repeatedapplicationsbeing made every 30 min. Two hoursafter surgery,the animalswere placed in the plastic restrainer,and allowed to rest for 15 min in the sound-isolationchamber iust as in the main experiment.The first blood sample was taken 10 min after the ethanolwas injected.Additional sampleswere taken at 0.5-, 1- and 2-hr intervals. Blood sampleswere also drawn from randomly selectedanimalsprior to the administrationof ethanol for later use as blanks in the blood ethanol analyses.The analyseswere made on a Beckman Spectrophotometer using an alcohol dehydrogenasemethod (11.)
1920
R.D. FITZGERALD AND G. L. STAINBROOK
RESULTS
PreliminaryStudy
Two groupscorresponding to the 0.8-gand 2.4-gethanoldoses were formed after it was determinedin an analysisof variance that the blood-ethanol levelsof the two rs-intensity groupswere not reliably different.The main finding was that the bloodethanolconcentrations of the2.4-ggroupwerehigherat eachsampling pointthanthoseof the 0.8-ggroup.In bothgroups, peakconcentrationsoccurred approximately 30 min afterthe ethanoliniection. The mean(•sD) bloodethanolconcentrations of the 2.4-gand0.8-g groupsat the 10-,30-, 60- and 120-minsampling pointswere,respectively, 160ñ 36, 185ñ 37, 175ñ 39 and150ñ 32 mg per 100 ml and 59 ñ 28, 68 + 27, 45 ñ 23 and 27 +- 12 mg per 100 mi. A 2 X 4 (ethanoldoseX sampling periods)analysis of variance established that the over-alldifference betweenthe groups wasreliable(F = 217.84,1/22 dr,p < .001) andthat the changein blood ethanollevelsacross sampling periodswasreliable(F = 4.44,3/66 dr, p
The Influence
of Ethanol
on Learned
and
ReflexiveHeart Rate Responses of Rats During Classical AversiveConditioning RobertD. Fitzgeraldand GeneL. Stainbrook SVMM.•nY. An unconditioned response wasproducedin rats by electricshockand resultedin a hipbasicdeceleration-acceleration of heartrate a#er 2.4 g of ethanol perkg. A monophasic acceleration occurred with salineand0.8 g of ethanolper kg.
ELATIVELY LITTLEinformation is available concerning
the effects ofalcohol on learned and reflexive responses that occur in Pavlovian conditioning. Most of the experiments bearing onthistopic have been limited totheuseofsingle doses of alcohol (typically ethanol ordistilled spirits) given toa relatively smallnumber of subjects, or multiple doses administered sequen-
tiallytothesame subjects. Moreover, theresults havebeenmixed, andbothfacilitative andsuppressive responses havebeenreported. Doubtless, many ofthese apparent inconsistencies canbeaccounted forbyspecies differences, variations inalcohol doses anddiscrepanciesin thelength oftimethatwasallowed to elapse between the administration of alcoholand the beginningof testing.
Asanexample of theimportance of thelatterfactor, some of theearliest experiments (1-3)revealed that,soon afteradministration,moderate doses of alcohol, estimated to be in therangeof 0.15to0.39gperkgofbody weight, frequently produced increased salivation in dogs toa nonreinforced conditioned stimulus (cs)pre-
sented in a discrimination paradigm without visibly affecting the • Fromthe Department of Medical Psychology, University of Oregon Health Sciences Center, 3181SWSamJackson ParkRoad,Portland, Oregon 97201.This research wassupported byNational Science Foundation GrantBMS74-18542. Gene Stainbrook wasa trainee on a National Institute of Alcohol AbuseandAlcoholism traininggrant(No. 1 T32 AA07074-01).
Received forpublication: 26 August 1977.Revision: 7 June1978. 1916
X-l•.•x-a.•x•
a•svo•s•s
ov a.•xs
1917
reactionsto the reinforcedcs. Similarshort-latency effectswere sometimes observedwhenhigherdosesof alcoholrangingfrom 0.48 to 3.50 g per kg were used,exceptthat in thesecasesincreased salivationwas immediatelyfollowedby sharpreductionsin respondingto both nonreinforcedand reinforced css. A moderate
alcoholdoseof 0.5 g per kg given2 hr beforetestingwasalsofound to blockthe occurrence of the accelerative heart-rate(i•a) conditionedresponse(ca) in somedogsto a reinforcedcs without apparently changingthe responses to a nonreinforcedcs (4). Seemingly paradoxical changes in the vigorof orientingreactions (oR) asopposed to cRshavebeenobserved in both nonhuman and humansubjectsreceivingrelativelyhigh dosesof ethanol.In one instance(2), dogsgiven intoxicatinglevels of alcoholseemedto displayan augmented oR (headturningin the directionof the cs) eventhoughthe performance of the salivarycR was blocked.In
a studyinvolving humans(5), 0.70g of alcoholper kg suppressed the accelerative I•R component of the oR triggeredby a novelcs, whileat the sametimeenhancing the decelerative component. Facilitationof reflexiveI•Rchangesinducedby the Valsalvamaneuver or by a body-tiltmanipulation were alsofoundin humansfollowing ingestion of 0.30or 0.60g of alcoholper kg (6). Severalexperiments haveshownthat amountsof alcoholranging from0.24to 0.57g perkg arecapableof interferingwith eye-blink (7) and galvanic-skin (8) cRsin humans.In anotherstudy (9), however,a relativelyhigh doseof alcohol(approximately 1.60 g perkg) failedto modifyhumaneye-blinkcRsthat wereestablished on the basisof a very strongair-puff unconditionedstimulus(rs). This suggests that the adverseeffectsof alcoholon cRsmight be mitigatedby the useof an intensers. However,studiesof classical conditioning designedto lookspecifically at vs intensityand different amounts of alcohol are not available.
The presentstudyexamined the effectsof ethanolon the •xx•reactionsof restrained rats during aversive classicalconditioning. Particularattentionwas focusedon ethanol-relatedchangesin the oR elicitedby the cs prior to conditioning,in the developmentof the cR and in the unconditioned response(vR) producedby the rs. To provideinformationon the questionof the relationship between vs intensity and conditioningperformanceunder alcohol, two concentrations of ethanol were combined with two intensities
of the us in a factorialdesigninvolvingseparategroupsof rats. A
1918
R.D. FITZGERALDAND G. L. STAINBROOK
preliminarystudywas alsocarriedout in which blood alcoholdeterminations were madein animalsreceivingessentially the same treatmentas that usedin the main experiment. METHODS
Subiects
The animalswere129naivefemaleLong-Evans hoodedrats (SimonsenLaboratories, Gilroy,California),90 to 120daysof ageand ranging in weightfrom250to 325g. Theywerehoused2 per cagewith a 12-hr light-dark cycle and food and water ad libitum. The animalswere tested during the light cycle. Apparatus
An invertedU-shapedplastichousingmanufactured by Narco BioSystems was usedto restrainthe rats.To eliminateunwantedauditory signalsfrom reachingthe animals,the experiment was carriedout in an IndustrialAcousticsound-isolation chamberequippedwith a ventilating fan, a 60-W houselight and a 10.2-cmspeaker.To providefurther
masking of extraneous auditorysignals, whitenoisemeasuring approximately75-dBsoundpressure level (about0.0002dyn/cm 2) was presentedcontinuously throughthe speaker.Two animalswere conditioned
concurrently in identicallyequippedchambers. The electrocardiogram wasrecorded on a Grasspolygraph from20-gauge hypodermic needles insertedthrougha fold of skin oppositeeach other on either side of the rat's thoraciccavity. Heart rate was measuredby meansof an
automatedon-linesystem(10) that provideda punchedpaper-tape tabulation of the totalheartbeats occurring in successive timeperiods of each trial.
The cswasa 6.5-sec, 2.9-kHztonepresented at 85-dBsoundpressure level (about 0.0002dyn/cm") througha 2.5-cmMallory Sonalert mounteddirectlyin frontof the animal.The uswasa 0.5-sec, 60-Hz,AC electricshockproducedby a GrasonStadler E6070B stimulatorand
deliveredto the baseof therat'stail throughtheheadsof No. 6 machine screws thatwereheldin placeapproximately 2 to 3 cmapartwith small piecesof rubbertubing.A delayedconditioning paradigmwasusedin which the 0.5-secvs overlappedand terminatedwith the last 0.5 see of the cs.
Procedure
In the mainexperiment, therewere6 experimental groupsof 16 animalseachand 3 conditioning-control groupsof 5 animalseach.The 6 experimental groupscomprised the cellsof a 3 X 2 factorialdesign, onedimension beingthe drugtreatment(an intraperitoneal injectionof
0.8 g or 2.4 g of ethanolper kg of bodyweightor normalsaline)and
HEART-RATE RESPONSESOF RATS
1919
the otherbeingthe intensityof the us (0.6 or 1.2mA). The ethanoldoses were preparedfrom 95•oethanolmixedwith normalsalineon the basis of I ml of solutionper 100 g of body weight and injected"blind" (the persongiving the iniectionswas unaware of the agent being administered). The 3 controlgroupsreceivedeither saline or one of the two doses of ethanol.
Subsequent to an initial 15-minperiod of adaptation,all animalswere taken out of the sound-isolationchamber and given an iniection of the appropriateagentthrougha hole in the floor of the restrainingdevice. Following a 15-min absorptionperiod, 10 pretest presentationsof the cs alonewere givenat intertrial intervalsof 70, 90 or 110 sec (mean, 90 sec). Upon the completionof thesetrials, animalsin the experimental groupreceived20 paired conditioningtrials at randomlyspacedintertrial intervalsof 160, 180 or 200 sec (mean, 180 sec). Animals in the 3 control groupsreceived20 explicitly unpaired presentationsof the cs and us, the cs followingthe us at randomlyselectedintervalsof 70, 90 or 110 sec(mean, 90 sec). The high-intensity1.2-mAus was usedfor all of the control groups. Samplesof base-levelHa in 6-secperiodswere taken at 3-min intervals during the 16-min preiniectionand 15-min postiniectionabsorption periods.The Ha responses to the cs on the pretest cs-aloneand conditioning trials were measuredby subtractingthe beats-per-minuteHR during the 6 sec before startingthe cs from the rate during the 6-sec interval before the us. The unconditionedHa responsesto shock were also convertedto differencescoresusing the Ha before the cs. The preliminarystudy providinginformationon blood ethanol concentrationsinvolved24 rats of the samestrain, age and weight as those used in the main experiment.These animalswere randomly assignedto I of 4 groupsin a 2 X 2 factorial design,one of the dimensionsbeing ethanoldose (0.8 g vs 2.4 g per kg of body weight) and the other us intensity(0.6 vs 1.2 mA). The pretestingand conditioningprocedures were the same as those in the main experiment. Blood sampleswere taken from the middle caudal vein in the rat's tail with a heparinized microcapillarytube. Prior to conditioning,the rats were anesthetized with ether, and a 2.5-cm midventral incision was made near the base of the rat's tail. The caudal vein xvasexposedand the incisionwas then looselyclosedwith adhesivetape. Procainewas applied topically to the area as a local anesthetic,with repeatedapplicationsbeing made every 30 min. Two hoursafter surgery,the animalswere placed in the plastic restrainer,and allowed to rest for 15 min in the sound-isolationchamber iust as in the main experiment.The first blood sample was taken 10 min after the ethanolwas injected.Additional sampleswere taken at 0.5-, 1- and 2-hr intervals. Blood sampleswere also drawn from randomly selectedanimalsprior to the administrationof ethanol for later use as blanks in the blood ethanol analyses.The analyseswere made on a Beckman Spectrophotometer using an alcohol dehydrogenasemethod (11.)
1920
R.D. FITZGERALD AND G. L. STAINBROOK
RESULTS
PreliminaryStudy
Two groupscorresponding to the 0.8-gand 2.4-gethanoldoses were formed after it was determinedin an analysisof variance that the blood-ethanol levelsof the two rs-intensity groupswere not reliably different.The main finding was that the bloodethanolconcentrations of the2.4-ggroupwerehigherat eachsampling pointthanthoseof the 0.8-ggroup.In bothgroups, peakconcentrationsoccurred approximately 30 min afterthe ethanoliniection. The mean(•sD) bloodethanolconcentrations of the 2.4-gand0.8-g groupsat the 10-,30-, 60- and 120-minsampling pointswere,respectively, 160ñ 36, 185ñ 37, 175ñ 39 and150ñ 32 mg per 100 ml and 59 ñ 28, 68 + 27, 45 ñ 23 and 27 +- 12 mg per 100 mi. A 2 X 4 (ethanoldoseX sampling periods)analysis of variance established that the over-alldifference betweenthe groups wasreliable(F = 217.84,1/22 dr,p < .001) andthat the changein blood ethanollevelsacross sampling periodswasreliable(F = 4.44,3/66 dr, p
