Alcohol 30 (2003) 45–60

Adolescent rats discriminate a mild state of ethanolintoxication likely to act as an appetitive unconditioned stimulus

Juan M. Fernandez-Vidala,*, Norman E. Spearb, Juan Carlos Molinaa,b,1

aInstituto de Investigacion Medica M. y M. Ferreyra, Cordoba, C.P. 5000, ArgentinabCenter for Developmental Psychobiology, Binghamton University, Binghamton, NY 13902-6000, USA

Received 29 June 2002; received in revised form 7 March 2003; accepted 22 March 2003

Abstract

Practically no information is available in relation to the capability of the adolescent animal in terms of discriminating postabsorptiveeffects of ethanol. Three experiments were conducted to analyze whether young, genetically heterogeneous rats discriminate different stagesof the process of intoxication exerted by a low dose (0.5 g/kg) of ethanol. An ethanol pharmacokinetic profile was first examined to selecttwo stages within the process of ethanol intoxication that, as a function of the corresponding blood ethanol concentrations (BECs), couldrepresent two potentially discriminable drug states. In a second experiment, sucrose was available when the BECs of rats peaked or wereof a lesser magnitude (5 and 30 min postadministration time, respectively). When animals were tested under similar or different drug statesrelative to the training procedure, no behavioral evidence indicative of differential sucrose expectancy was obtained. In Experiment3, rats discriminated each of the previously defined ethanol states from a non-drug state. Unexpectedly, it was also found that thepharmacological effects of the 0.5-g/kg dose of ethanol are likely to support appetitive associative learning that involves the taste ofsucrose as a conditioned stimulus. The apparent positive affective components of the state of ethanol intoxication have rarely been observedin genetically heterogeneous rats with rather brief experiences with the drug’s effects. � 2003 Elsevier Inc. All rights reserved.

Keywords: Ethanol; State discrimination; Appetitive conditioning; Nose-poking; Sucrose; Adolescent rat

1. Introduction

Interoceptive effects originated by drugs of abuse repre-sent an integral stimulus that has a significant impact onsubsequent seeking behavior and subsequent use of thesesubstances (Duka et al., 1998). Drugs, such as amphet-amines, barbiturates, and ethanol, have the capability toact as an interoceptive context that, when present during theacquisition and retrieval phases of a given learning situation,optimizes the expression of specific memories. That is, im-provements in performance tend to occur when the interocep-tive context is similar between training and test (Bruins Slotet al., 1999; Lowe, 1986), and a decrement in retentionoften occurs when the context of testing differs from that oftraining (Riccio et al., 1984; Spear & Riccio, 1994). Thisindicates that animals acquire information about not only

* Corresponding author. Instituto Ferreyra, Casilla de Correo 389,CP: 5000 Cordoba, Argentina. Tel.: �54-351-468-1465; fax: �54-351-469-5163.

E-mail address: molina3@immf.uncor.edu (J.M. Fernandez-Vidal).1 J.C. Molina is also working at Facultad de Psicologıa, Universidad

Nacional de Cordoba, CP: 5000 Cordoba, Argentina.Editor: T.R. Jerrells

0741-8329/03/$ – see front matter � 2003 Elsevier Inc. All rights reserved.doi: 10.1016/S0741-8329(03)00093-4

the reinforcing properties of a drug, but also the internalcontext provided by other effects of the drug. This phenome-non has been referred to frequently as state-dependent learn-ing (SDL) (Bruins Slot et al., 1999; Deutsch & Roll, 1973;Lowe, 1986; Spear & Riccio, 1994) and has been verifiedeven in early development (Hunt et al., 1990; McKinzieet al., 1994).

The capability to encode the effects of a drug as aninteroceptive state has also been tested frequently throughthe use of operant training procedures (Bruins Slot et al.,1999; Krimmer, 1992; Overton, 1979). In drug-discriminationprocedures, animals learn to execute an instrumental re-sponse to obtain a given appetitive reinforcer or to avoid anaversive stimulus on the basis of the action of a drug or anon-drug state as a discriminative stimulus. In these par-adigms, training procedures are long. Drug-discriminationcapabilities can be tested only after behavioral shaping tech-niques take place (Colpaert & Koek, 1995); after strict per-formance criteria, in terms of operant responding, are met;and after specific training procedures, with drug-related in-teroceptive effects that act as discriminative stimuli, areconducted. The temporal brevity of some ontogenetic stagesof development requires procedures that require correspond-ingly brief periods. In rats, for example, it is difficult to achieve

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consistent operant performance for drug-discriminationeffects during infancy or adolescence because of the brief-ness of these developmental stages. Perhaps for this reason,very little is known regarding how young animals processdiscriminative properties of drugs such as ethanol. Duringearly ontogeny, ethanol has been used frequently as a condi-tioned stimulus (CS) because of its particular chemosensoryattributes [see Molina et al. (1985, 1986, 1999)] or as anunconditioned stimulus (US) capable of supporting associa-tive learning [see Berman and Cannon (1974), Domınguezet al. (1994), and Hunt et al. (1991)]. Very rarely has this drugbeen used as an interoceptive context or as a discriminativestimulus that aids in the retrieval of early memories relatedto its postabsorptive effects.

Numerous findings obtained from studies have demon-strated SDL generated by ethanol when focusing on adultanimals (Bruins Slot et al., 1999; Holloway, 1972;Nakagawa & Iwasaki, 1995). In general in these studies,drug versus non-drug conditions as discriminative stimulihave been compared. Furthermore, drug-discrimination pro-cedures can be defined through the use of differential doses ofthe same drug or effects derived from different psychotropicagents (Appel et al., 1999; Jarbe & Swedberg, 1998) or fromthe combination of these agents (Mariathasan et al., 1999;Stolerman et al., 1999).

The literature concerning discrimination of differentstages within the development of the toxic process inducedby a given ethanol dose is scarce [see, for example, Schechter(1989) and Shippenberg and Altshuler (1985)]. Differentialeffects across the state of intoxication, with the use of anethanol dose, have been analyzed mainly through condi-tioning studies in which the postabsorptive effects of eachparticular stage of the toxic process are used as differentialunconditioned stimuli (Cunningham & Prather, 1992;Krimmer, 1992; Risinger & Cunningham, 1992). It has beenhypothesized that ethanol has positive-reinforcing qualitiesrelatively soon after exposure, during the rising limb ofthe curve of blood ethanol concentrations (BECs), andaversive effects during the falling limb of the curve of BECs(Reid et al., 1985). This hypothesis has been supportedmainly in terms of heightened ethanol self-administrationpatterns by mice or genetically selected rats. Heterogeneousstrains of rats generally do not express appetitive contents ofmemories comprising postabsorptive effects of ethanol. Onthe contrary, these animals tend to encode primarily theaversive consequences of ethanol. Conditioned place andtaste aversions are easily established with ethanol dosesequal to or higher than 1 g of ethanol per kilogram of bodyweight (1 g/kg) (Cordoba et al., 1990; Cunningham et al.,1993; Gauvin & Holloway, 1992; Schechter & Krimmer,1992). Lower doses rarely exert significant aversive or appe-titive conditioning (Stewart & Grupp, 1985; van der Kooyet al., 1983). To attain ethanol-mediated conditioned prefer-ences in heterogeneous rats, extensive training procedures(Bozarth, 1990), long-term preexposure to the interoceptive

effects of the drug (Reid et al., 1985), or concurrent presenta-tion of other non-ethanol reinforcers (Marglin et al., 1988)—or a combination of these conditions—has been needed.

In the current study, we tested whether adolescent ratscan use the effects of a relatively low dose (0.5 g/kg) ofethanol as a discriminative state and whether they perceivedifferences in the effects of such a dose throughout the processof intoxication. An additional goal, but critical in terms ofbeing able to analyze the above-stated possibilities, was togenerate a technique that would enable us to verify drug-discrimination capabilities after minimal behavioral train-ing procedures. Obviously, this is a prerequisite to train andevaluate rats within the adolescent stage of development. Inthe rat, this stage lasts no more than 15 days [postnatal days(PNDs) 28–42] (Spear, 2000; Spear & Brake, 1983).

In the current study, mildly dehydrated adolescent ratshad easy access to a sucrose solution when sober or whenintoxicated with ethanol. They were later tested in terms ofsucrose-seeking behavior when in the state under which theywere trained or in a different state. Although the study wasfocused on discriminative properties of a low dose of ethanolduring adolescence, it was found that the training procedureallowed the expression of apparently appetitive conse-quences of a 0.5-g/kg dose of ethanol in the adolescent rat.In other words, beyond the original experimental intention,a brief, simple, and economical training procedure involvingexplicit pairings between a sweet solution and the interocep-tive effects of ethanol resulted in the expression of behaviorsthat support the suggestion of appetitive contents of ethanol-related memories.

The original goals of the current study required that wefirst determine, in Experiment 1, pharmacokinetic profilesof ethanol in adolescents administered a low dose (0.5 g/kg) of the drug. This experiment allowed identification, oper-ationally, of distinctive states of intoxication as indicated byBECs. For the second and third experiments, we used thisinformation to determine whether adolescents can discrimi-nate such states of intoxication and/or an ethanol versus anon-ethanol state.

2. Experiment 1

Toward assessing the capacity of adolescents to discrimi-nate acute states of ethanol intoxication, a pharmacokineticanalysis of BECs resulting from exposure to a low dose (0.5g/kg) of ethanol was performed. The goal was to selectpostadministration intervals that yielded significantly differ-ent BECs. The dose was selected in accordance with prelimi-nary results, supporting the suggestion that adolescent ratsare capable of discriminating the interoceptive context gener-ated by this dose from the state of sobriety (Godoy et al.,1999). More explicitly, the intention was to choose onepostadministration interval in which BECs peaked and asecond one with significantly lower BECs, still detectableas different from a non-drug state. This experiment was

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–60 47

considered necessary for a subsequent behavioral study thatrequired two potentially different drug states.

2.1. Materials and methods

2.1.1. SubjectsFifteen Wistar-derived adolescent male rats (each

weighing between 60 and 80 g) were used. The animals wereborn and reared in the animal colony at the Instituto Ferreyra.Rats were housed in standard opaque cages filled with pineshavings and maintained on a 14-h light/10-h dark cycle (lightonset at 0700) and temperature-controlled (22ºC–24ºC) con-ditions. Unless specified, subjects had free access to rat chow(Cargill; Cordoba, Argentina) and water delivered throughautomatic dispenser valves. The day of birth was consideredas PND 0. After delivery, on PND 1, pups were culled toeight per litter (four males and four females whenever possi-ble) and weaned on PND 21. After weaning, animals fromeach litter were kept together in an environment similarto the original maternity cage. Only males were used forexperimental purposes. In accordance with the precedingliterature (Spear, 2000; Spear & Brake, 1983), adolescencein the rat takes place between 28 and 42 days of age. Takingthis, as well as maturational parameters corresponding to theabove-mentioned strain of rats, into account we ensuredthat all animals used in the current experiment were 34days of age at the start of the experiment. Animal care andprocedures were in accordance with the Guide for the Careand Use of Laboratory Animals (Institute of LaboratoryAnimal Resources, Commission on Life Sciences, NationalResearch Council, 1996).

2.1.2. ProceduresTwenty-four hours before blood samples were obtained

for analysis of BECs, rats were subjected to a surgical proce-dure aimed at placing a catheter in the right jugular vein.This surgery was necessary to allow serial blood samplingduring the course of the ethanol toxicity process. The animalswere anesthetized by means of an intraperitoneal injectionof ketamine hydrochloride (80 mg/kg) (Vetanarcol, Konig;Buenos Aires, Argentina) supplemented with xylazine hy-drochloride (13.5 mg/kg) (Kensol, Konig; Buenos Aires,Argentina). An incision was then made in the ventral portionof the neck, and a catheter (filled with heparin dilutedin physiological saline) was inserted into the jugular veinuntil reaching the cardiac atrium. The catheter was keptin this position by means of a suture procedure to maintain itsattachment to the sternocleidomastoid muscle. The free endof the catheter was inserted subcutaneously until it reached thedorsal side of the neck. A small incision was made to expressthe free end of the cannula and attach it, with one suturestitch, to the skin. To diminish the possibility of postsurgicalnociception, buprenorphine hydrochloride (0.03 mg/kg)(Temgesic, Schering-Plough; Buenos Aires, Argentina) wasadministered subcutaneously and used as an analgesic agent.

All subjects were water deprived for 22 h during fourconsecutive days, the deprivation procedure to be used in

subsequent behavioral experiments to test discriminationbetween states of intoxication (see Experiments 2 and 3).Four hours before intragastric administration of a 0.5-g/kgdose of ethanol (PND 34), the rats were also deprived offood. This ensured relatively equivalent stomach contentsof food to minimize individual differences in absorption anddistribution of the drug. To attain the 0.5-g/kg dose of etha-nol, rats were subjected to intragastric administration of0.015 milliliters per gram of body weight of a 4.2% [volume/volume (vol./vol.)] ethanol solution. Blood samples (100 µl)were collected at each of five postadministration times (5,15, 30, 60, and 90 min) after intubation of ethanol. Adminis-tration and sampling procedures took place between 1000and 1200.

Blood samples were subjected to head-space gas chroma-tography (Hachemberg & Schmidt, 1985; Molina et al.,1992). Samples were placed in microvials (total volumecapacity: 700 µl) equipped with a rubber stopper. Each vialwas placed on crushed ice to prevent ethanol vaporization.For assessment of BECs, samples were kept in a water bathat 60ºC for 30 min. Gas-tight syringes (Hamilton; Reno,Nevada; 10 µl) were used to collect the volatile componentof the samples and to inject them into the gas chromatograph(Hewlett-Packard, Model 5890, Palo Alto, CA) column (Car-bowax 20M; 10 m × 0.53 mm × 1.33 mm film thickness).Oven, injector, and detector temperatures were 60ºC, 150ºC,and 250ºC, respectively. Nitrogen served as the carrier gas(flow rate: 15 ml/min). Blood ethanol concentrations werecomputed by using linear regression analysis of known stan-dards. Twenty microliters of butanol (52 mg/dl) was addedto each blood sample to provide an internal standard control.Blood ethanol concentrations were expressed as milligramsof ethanol per deciliter of body fluid (mg/dl � mg%).

2.2. Results and discussion

Blood ethanol concentrations resulting from the admin-istration of a 0.5-g/kg dose of ethanol in adolescent ratscan be seen in Fig. 1. The data were processed by using a one-way analysis of variance (ANOVA), with postadministrationtime as the factor under analysis. This ANOVA yieldeda significant main effect [F(4,56) � 23.44, P � .001]. TheBECs increased rapidly after administration of the drug andpeaked [40.98 � 5.35 mg% (mean � S.E.M.)] 5 min afteradministration of the 0.5-g/kg dose of ethanol. These levelswere similar to those encountered at the 15-min interval.Indeed, post hoc comparisons (Fisher least significant differ-ence test, with an alpha level of .05) revealed that BECs at5 and 15 min did not differ. Post hoc tests also indicatedthat BECs at 30 min were significantly lower than thoseregistered in previous postabsorptive time intervals and alsosignificantly higher than those obtained 60 and 90 min afterethanol administration. The scores in these two last intervalswere not different from a 0 mg% theoretical value.

Rate of ethanol elimination was estimated from individ-ual slopes of the linear regression of ethanol concentrations

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–6048

Fig. 1. Blood ethanol concentrations (mg%) registered in Experiment 1 as a function of postadministration time resulting from intragastric administrationof a 0.5-g/kg dose of ethanol in periadolescent male rats. Vertical lines represent standard error of the mean (S.E.M.).

across 5-, 15-, 30-, and 60-min sampling points. The analy-sis was not conducted for the 90-min sampling period be-cause all animals showed null levels of ethanol in blood.The elimination rate was equivalent to 46.13 � 5.9 mg%/h(mean � S.E.M). The rate of metabolism is relativelysmaller when compared with the one reported by Silveri andSpear (2000) during periadolescence (PNDs 26 and 36).These investigators used Sprague–Dawley rats subjected tointraperitoneal administrations of higher ethanol doses (1.5and 4.5 g/kg) relative to the dose used in the current experi-ment. The route of administration as well as genetic factorscould be responsible for the differences observed across stud-ies. Kelly et al. (1987) reported lower ethanol eliminationrates in 30-day-old, male, Sprague–Dawley rats relative tothe ones observed in the current experiment. In their study, a2.5-g/kg dose of ethanol was administered intragastricallywith the use of milk as a vehicle. This factor is probably re-sponsible for lower rates of absorption and distribution ofthe drug, which will obviously have an impact on ethanolrate of elimination.

3. Experiment 2

Experiment 2 was conducted to assess whether adoles-cents can discriminate two temporally separate states ofethanol intoxication, operationally defined by significantlydifferent amounts of ethanol in blood. In accordance withthe results of Experiment 1, we decided to use the followingpostadministration intervals for Experiment 2: 5–15 versus30–40 min. To assess discrimination between these drugstates, we used a behavioral technique that allows completion

of training and testing within the adolescent stage of develop-ment. A very simple and naturally occurring behavior (nosepoking) sustained by a sweet reinforcer (sucrose) wasselected. Nose-poking behavior was easily completed bythese adolescents to gain access to sucrose solution, duringeither peak or intermediate BECs derived from the samesubnarcoleptic dose (0.5 g/kg) of ethanol used in Experiment1. While still adolescents, animals were evaluated in termsof behavioral reactivity (nose poking) within the environ-ment in which the sucrose solution was originallyencountered. This reactivity was tested when the animalswere under a similar or different drug state than that origi-nally experienced during access to sucrose.

3.1. Materials and methods

3.1.1. SubjectsGenetic as well as housing conditions of the animals

replicated those described in Experiment 1. A total of 47adolescent male rats were used. These animals were repre-sentative of 12 litters. At the beginning of the experiment,all subjects were 30 days of age.

3.1.2. Experimental designOn the basis of results of Experiment 1, two ethanol post-

absorptive states were defined: state A (5–15 min postadmin-istration time) and state B (30–40 min postadministrationtime). Animals were quasirandomly assigned to one of twotraining groups: A�B� (during state A, rats had accessto sucrose, whereas during state B, rats did not have access tosucrose) and A�B� (state A was experienced while sucrosewas absent, and state B was experienced while sucrose was

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–60 49

present). The quasirandom distribution was executed toensure equivalent litter representation and body weightsacross groups. Half the animals in each group were laterevaluated when experiencing the same drug state experi-enced while having access to sucrose (evaluation groupsame; included training groups A�B� tested in A andA�B� tested in B). The remaining rats were evaluated underthe state in which they did not have access to sucrose(evaluation group different; included training groups A�B�tested in B and A�B� tested in A).

3.1.3. Apparatus and proceduresBlack Plexiglas chambers (25 × 25 × 25 cm), which had

a hole (diameter: 4 cm) in one of their lateral walls, wereused. The center of this hole was 4.5 cm above the floorand equidistant from the adjacent walls. A transparent Plexi-glas cup (volume capacity: 16 ml; diameter: 3.4 cm) waspositioned in the external side of each chamber. The rim ofthe cup was in contact with the inferior border of the hole.This cup was removable. One of these devices was used topresent a 10% [weight/volume (wt./vol.)] sucrose solutionand a second one was always presented empty. Nose pokingallowed the animals to have access to the empty or thesucrose-filled cup.

The experiment had a total duration of 5 days (training:days 1, 2, 3, and 4, which correspond to PNDs 30, 31, 32,and 33, respectively; evaluation: day 5 or PND 34). Beforethe training and testing sessions, all subjects were waterdeprived during 22 consecutive hours. A pair of subjectswas first weighed (�0.1 g) and subjected to intragastricadministration of a 0.5-g/kg dose of ethanol. Five minuteslater (state A), animals were placed in individual cham-bers: one with an empty cup (group A�B�) and the otherone with a cup containing sucrose (group A�B�). Subjectsremained in these chambers for 10 min. Immediately after,they were weighed and placed in individual holding cageswhere they stayed until postadministration time 30 min (stateB). Under state B, animals were placed in the Plexiglaschambers where they remained 10 additional minutes. Ani-mals in group A�B� had access to the cup filled withsucrose, whereas animals in group A�B� were exposed toan empty cup. Once again, body weights were registeredbefore and after subjects were placed in the correspondingchambers. Two hours after ethanol administration, rats hadfree access to water for 100 consecutive minutes. They werethen water deprived until commencement of the followingtraining session. Fig. 2 summarizes the training and test-ing procedures that were used.

Before evaluation procedures (day 5, PND 34), animalscorresponding to each training group were subdivided intotwo evaluation groups: different and same. Different refersto the groups of rats that were tested under a different drugstate than that previously associated with sucrose availabil-ity; that is, group A�B� evaluated under A (5–15 minpostadministration time, n � 12) and group A�B� evalu-ated under B (30–40 min postadministration time, n � 12).

Same refers to the groups of rats that were tested under thesame drug state that previously signaled sucrose availability;that is, group A�B� evaluated under B (30–40 min postad-ministration time, n � 11) and group A�B� evaluatedunder A (5–15 min postadministration time, n � 12). Testswere conducted in a black Plexiglas box similar to the oneused during training. All test sessions were conducted inabsence of sucrose to evaluate sucrose-seeking behavior asoperationalized through nose poking (see following sectionfor details on this matter). During tests, animals were fluiddeprived as when trained.

Immediately after termination of the test, blood sampleswere collected from each adolescent after induction of etheranesthesia. Blood ethanol concentrations were determinedby using the same procedures as those described in Experi-ment 1. The blood was collected through cardiac puncturewith the use of a 271/2-gauge needle attached to a dispos-able syringe.

3.1.4. Collection of behavioral dataAll subjects were videotaped (Samsung SCA20) during

the evaluation procedure. A real-time computer-based pro-gram served to determine parameters related to nose-pokingbehavior (duration, frequency, and latency to perform thefirst nose poke). The experimenter who used this computerprogram was blind relative to training and test conditionsof the animals. Preliminary examination of the results of thecurrent study (Experiments 2 and 3) and those correspondingto prior pilot experiments indicated that, very rapidlyduring the test session, animals decreased their nose-pokerate, probably because of the fact that they readily sensedthe absence of sucrose. When considering both behavioralexperiments of the current study (Experiments 2 and 3,overall n � 143 rats) we observed that, in terms of relativefrequency of nose-poke behavior, 70.1% occurred duringthe first 4 min of the test. Hence, in the current and subse-quent experiments, data subjected to inferential statisticalanalysis were mean nose-poke duration in the first 4 min ofthe test session. Latency to perform the first nose pokewas also subjected to inferential analysis. These behavioralscores seem to represent the most sensitive indexes ofsucrose-seeking behavior.

3.2. Results and discussion

3.2.1. Sucrose intake during training sessionsBody weights at the start of the experiment did not

differ between groups [A�B�: 61.96 � 1.33 g and A�B�:65.58 � 2.27 g (mean � S.E.M.)]. Despite water-deprivationprocedures, all animals gained weight across training days,and this increase was similar across groups [overall weightgain between days 1 and 4: 8.30 � 0.39 g (mean � S.E.M.)].A two-way ANOVA showed significant increases in bodyweights across training days [F(3,135)� 309.28, P � .001].Post hoc Fisher tests indicated that body weights for each

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–6050

Fig. 2. Diagram of training and test procedures for groups A�B� and A�B� in Experiment 2. Vertical arrows signal the time corresponding to theintragastric administration of ethanol during training. When training procedures are considered, “A” or “B” stands for ethanol postadministration intervalsof 5–15 or 30–40 min, respectively. Signs “�” and “�” represent presence or absence of sucrose in a novel context (black chamber), respectively. Atconditioning, black bars indicate postadministration intervals in which the rat was placed in the black Plexiglas box having access, or no access, to sucrose.Before test, all rats were administered ethanol. They were placed in the black box without having access to sucrose either during similar (group same) oralternative (group different) ethanol postadministration intervals relative to those used at training.

specific day were always significantly higher relative to theones recorded during the preceding day (data not shown).

Percent body weight gains (%BWG) were calculated foreach training session in which animals had explicit accessto sucrose. This intake index was calculated by using thefollowing formula:

100 × (postsession body weight � presession body weight)/presession body weight

This dependent variable was processed by using a twotraining group (A�B� and A�B�) × 4 days of trainingtwo-way mixed ANOVA. This analysis indicated only asignificant main effect of days [F(3,135) � 9.46, P � .001].Post hoc Fisher least significant difference tests (P � .05)showed that sucrose intake was significantly lower duringthe first day in relation to the next three training days. Theseresults have been depicted in Fig. 3.

3.2.2. Behavioral responsiveness during testLatency to perform the first nose poke when ado-

lescents were evaluated by using an empty cup was analyzedthrough a two-way ANOVA. The factors under considerationwere training (A�B� or A�B�) and evaluation (same ordifferent). Latency was observed to vary significantly onlyas a function of training [F(1,43) � 6.17, P � .01]. Animalsassigned to group A�B� showed significantly lower laten-cies than did subjects assigned to group A�B�.

Mean duration of nose poking was analyzed through anANOVA that incorporated the training and evaluation groupsas independent factors. Neither the main effects nor theinteractions between them were statistically significant.

As stated, BECs were determined after completion of thetest. A two training group (A�B� and A�B�) × two statesat test (A, B) ANOVA revealed that animals tested understate A had significantly higher BECs than did animals testedunder state B [F(1,43) � 43.7, P � .001]. This differencewas not affected by prior training procedures, nor by the in-teractions of the factors under consideration. The differencebetween BECs of animals previously tested during state Aor B was very similar to that observed in Experiment 1 whencontrasting BECs at 5 and 30 min postadministration time.

Latency to perform the first nose poke, mean duration ofnose poke, and BECs at termination of test for the varioustreatment conditions are shown in Table 1.

Clearly, the results of the current experiment failed toshow any obvious behavioral sign indicative of discrimina-tion between two drug states characterized by significantlydifferent BECs. Performance during the test did not depend onthe drug state present during the test. Behavior seemedessentially the same when the drug state was the same fortraining and when it was different. The only factor that hada significant impact on a specific dependent variable (latencyto exhibit the first nose-poke behavior) was the nature ofthe training group. As stated, animals in group A�B� morerapidly approached the cup previously associated withpresence or absence of sucrose than did animals in groupA�B� (Table 1). Could this difference be due to differentialunconditioned properties of a particular intoxication statethat might favor appetitive or aversive conditioning? Undercertain experimental conditions, ethanol has been found toexert biphasic hedonic effects during the course of the state

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Fig. 3. Percent body weight gain (%BWG) across training days in Experiment 2, as a function of the training procedure (group A�B� or group A�B�).Values represent relative body weight gains of periadolescent rats after consuming a 10% (weight/volume) sucrose solution during state “A” (5–15 minpostadministration time) or state “B” (30–40 min postadministration time). Signs “�” and “�” represent presence or absence of sucrose, respectively.Vertical lines represent standard error of the mean (S.E.M.).

of intoxication (Cunningham & Prather, 1992; Krimmer,1992; Risinger & Cunningham, 1992) or to act either as anappetitive or an aversive US (Cordoba et al., 1990; Stewartet al., 1988). If any of these possibilities was responsiblefor the observed latency differences in the test situation,probably some differences should also have been observedin sucrose-intake patterns as a function of the accumulationof training trials. This speculation is based on the possibilitythat sucrose sensory properties can act as a CS likely to beassociated with ethanol’s toxic consequences (Berman &Cannon, 1974; Bormann & Cunningham, 1998; Cordobaet al., 1990; Hunt et al., 1990, 1991). Yet, sucrose-intakepatterns, as a function of training procedures, did not differbetween groups across the entire training procedure.

An alternative explanation related to the observation thatgroup A�B� rats approached the cup faster than did group

Table 1Behavioral scores registered in Experiment 2 during evaluation phaseand the corresponding blood ethanol concentrations (BECs)

Training State Nose-poke Nose-poke mean BECs aftergroup at test latency (s) duration (s) test (mg%)

A�B� A 4.23 � 0.90 1.82 � 0.19 41.15 � 1.74B 3.53 � 0.66 1.63 � 0.15 26.58 � 2.35

A�B� A 7.74 � 1.80 1.80 � 0.18 38.07 � 2.75B 6.21 � 1.27 2.28 � 0.22 22.72 � 2.03

All values are expressed as mean � S.E.M.A � First of two ethanol postabsorptive states (on the basis of results

of Experiment1)defined as5–15 min postadministration time;B � secondoftwo ethanol postabsorptive states defined as 30–40 postadministration time;A�B� � group that during state A had access (�) to sucrose but duringstate B did not have access (�) to sucrose; A�B� � group that during stateA did not have access to sucrose but during state B had access to sucrose.

A�B� rats during test can be related to the sequence ofsucrose presentation during the training phase of the experi-ment. It is likely that subjects that received sucrose the firsttime that they were placed in the training apparatus (A�B�)would exhibit shorter latencies in terms of seeking the sweetstimulus than those that received sucrose the second timethat they were placed in the black Plexiglas chamber duringtraining (A�B�). Animals encode not only exteroceptiveand interoceptive information related to presence or ab-sence of a particular salient stimulus, in this case a sucrosesolution, but also information related to the sequence ofaccess to such a stimulus. In other words, rats learned thatsucrose was available during the first, but not the second,trial of a day, and the test was, in effect, the first trialof that day. Such learning has been appreciated for manyyears, primarily because of the work of E. J. Capaldi [see,for example, Capaldi et al. (1986) and La Fiette et al. 1994)].

4. Experiment 3

The intention of Experiment 3 was to analyze whetherrats can discriminate either of the ethanol-induced statesused in Experiment 2 with respect to a non-drug state (state ofsobriety, S). In Experiment 2, adolescents did not indicatediscrimination between an ethanol postabsorptive periodcharacterized by peak BECs (state A) and a later period inwhich the contents of the drug were significantly lower (stateB). In the current experiment, adolescent rats had access tosucrose while nonintoxicated (state S) or while experiencingpeak BECs (state A) resulting from a 0.5-g/kg dose of ethanol(training groups S�A� or S�A�, respectively). Two

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other additional groups were incorporated. In these groups,sucrose availability was associated with either the non-drug state or with levels of ethanol that characterize a timesegment of the falling limb of the ethanol pharmacokineticcurve (training groups S�B� or S�B�, respectively). Un-expectedly, and as will be described later in detail, therewere clear indications supporting the suggestion that ethanolhad a strong impact on sucrose-intake patterns after the firsttraining session.

4.1. Materials and methods

4.1.1. SubjectsGenetic and housing conditions of the animals replicated

those described in Experiments 1 and 2. A total of 96 adoles-cent male rats representative of 24 litters were used. Theage of animals ranged between 30 and 31 days at com-mencement of the experiment.

4.1.2. Experimental designTwo factors defined the training conditions. The first

factor comprised the two combinations of states that definedthe training procedures. Animals were trained to discriminateeither a non-drug state (state S) from that of a period ofpeak BECs (state A) or a non-drug state (state S) from thatof a period during the falling limb of the pharmacokineticcurve (state B). Within each training procedure, the secondindependent factor was the state in which animals had access,or no access, to sucrose. Hence, the two independent factors inthis study resulted in four different training groups: S�A�,S�A�, S�B�, and S�B�. During the test, each groupwas subdivided into two new groups (evaluation groups).Animals were evaluated under either the state in which theyoriginally had access to sucrose (group same) or the stateassociated with the absence of sucrose (group different).Each of the eight independent groups included 11–13 adoles-cents. The animals were distributed across groups in a qua-sirandom manner. The constraints were to attain equivalentrepresentation of litters and body weights across groups.

4.1.3. Apparatus and proceduresThe experiment included the same apparatus, schedule

of deprivation, and drug administration procedure as in Ex-periment 2. The sole difference between these experimentswas in the drug states to be discriminated. These includedthe A and B states, as defined in Experiment 2, and the S(sobriety) state. For the S state, animals were weighed andsubsequently intubated intragastrically, but no solution wasdelivered into their stomachs (sham administration). Fiveminutes later, these animals were placed in the black Plexi-glas chamber with access, or no access, to a 10% (wt./vol.) sucrose solution. Under this non-drug state, animalsremained in the chambers for 10 min. Intragastric fluid ad-ministration was not used to avoid an overload of the stom-ach, because all subjects were later subjected to intragastricadministration of a 0.5-g/kg dose of ethanol. A second expo-sure to the black training chambers occurred 30 min after

completion of the initial experience within these chambers.Subjects assigned to group S�A� or group S�A� re-ceived the intragastric administration of ethanol 5 min beforebeing exposed for the second time to the training chambers,whereas those assigned to group S�B� or group S�B� wereintubated with ethanol 30 min before the second exposureto the training chambers. These administration parametersallowed training the animals under drug states homologous tostates A and B, respectively, in Experiment 1. In summary,for some animals sucrose was available when they werenot intoxicated with ethanol (groups S�A� and S�B�),whereas in the remaining groups availability of sucrose wasassociated with peak BECs (group S�A�)or with significantly lower BECs (group S�B�).

As in Experiment 2, tests had a total duration of 10 min andwere executed by using similar chambers and deprivationschedules as those used during training. As specified in theExperimental Design section, rats were evaluated undereither a similar or a different state in which they originallyhad access to sucrose. Once again, sucrose was not availablein the evaluation phase. Latency to perform the first nosepoke and mean duration of nose-poking behavior servedas dependent variables. Fig. 4 provides a summary of thetraining and testing procedures that were used.

4.2. Results and discussion

Body weights across groups were similar at commence-ment of the experiment [S�A�: 62.12 � 1.73 g; S�A�:57.22 � 1.54 g; S�B�: 61.25 � 1.00 g; and S�B�:59.51 � 1.11 g (mean � S.E.M.)]. As in Experiment 2, bodyweights increased significantly as a function of progressionof days [F(3,276) � 850.59, P � .001]. This effect failed tointeract significantly with the nature of the training proce-dures [overall body weight gain between training days 1 and4: 7.41 � 0.21 g (mean � S.E.M.)].

Percent body weight gains (%BWG), as a function ofsucrose intake during training, are shown in Fig. 5. A com-pletely unexpected result was obtained. Adolescent rats ex-posed to sucrose under either peak (state A) or lower (stateB) BECs drank significantly more sucrose than did thoseexposed to this stimulus under a non-drug state (state S).Apparently this difference did not exist during the first dayof training. A 4 × 4 mixed ANOVA confirmed these observa-tions. The independent factor under consideration was thetraining procedure (group S�A�, S�A�, S�B�, orS�B�). The repeated measures were derived from the daysof training. This analysis showed significant main effects oftraining [F(3,92) � 14.89, P � .001] and of days of training[F(3,276) � 26.85, P � .001]. The interaction betweenthese factors also achieved significance [F(9,276) � 4.30,P � .001]. The locus of the two-way interaction was anal-yzed with the use of post hoc Fisher least significant differ-ence tests. All groups failed to differ in terms of sucroseintake during the first day of training. During the next 3days, the intake scores of groups S�A� and S�B� were

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–60 53

Fig. 4. Diagram of training and test procedures for groups S�A�, S�A�, S�B�, and S�B� in Experiment 3. Vertical arrows indicate ethanol or shamintragastric administration procedures during training. In accordance with the conditioning procedures, “S” represents an ethanol-free state (sobriety), whereas“A” or “B” indicates ethanol postadministration intervals of 5–15 or 30–40 min, respectively. Signs “�” and “�” represent presence or absence of sucrosein a salient environmental context (black chamber), respectively. During training, horizontal black bars indicate time intervals in which the animal waspositioned in the black box while having access, or no access, to a sucrose solution. Before test, some rats were only sham-intubated or administered withethanol. Animals were then placed in the black compartment with no sucrose available. The state of the animal (S, A, or B) coincided or not (same ordifferent) with the state in which they had access to sucrose while being trained.

significantly higher than those corresponding scores ofgroups S�A� and S�B�. Furthermore, the intake scoresof groups S�A� and S�B� during the second, third, andfourth training days were significantly higher than thoseshown by these same groups during the first day. This wasnot the case in groups S�A� and group S�B� animals; their

intake scores did not differ between groups, nor across train-ing days.

Latency to perform the first nose poke during test wasanalyzed by a two-way ANOVA defined by the followingindependent factors: training group and evaluation group(same or different, relative to the state paired with sucrose

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–6054

Fig. 5. Percent body weight gain (%BWG) across training days in Experiment 3, as a function of the training procedure (S�A�, S�A�, S�B�, orS�B�). Values represent relative body weight gains of periadolescent rats after consuming a 10% (weight/volume) sucrose solution during state “A”(5–15 min postadministration time) or state “B” (30–40 min postadministration time). “S” represents an ethanol-free state (sobriety). Signs “�” and “�”represent presence or absence of sucrose, respectively. Vertical lines represent standard error of the mean (S.E.M.).

during training). This ANOVA indicated only a significantmain effect of evaluation [F(1,88) � 4.01, P � .05]. Whenanimals were evaluated under the same state in which theyoriginally had access to sucrose, latency to seek this stimuluswas significantly shorter than when rats were evaluated undera different state (overall mean � S.E.M. collapsed acrosstraining groups: same � 3.51 � 0.36 s; different � 6.70 �

1.59 s). Latency scores of the different groups have beenillustrated in Fig. 6.

Mean nose-poke duration during test was analyzed withthe same form of ANOVA used for latencies. Only the trainingfactor exerted a significant effect on nose-poke mean duration[F(3,88) � 3.09, P � .05]. Additional post hoc tests indi-cated that nose-poke duration was significantly higher in

Fig. 6. Latencies (seconds) to perform the first nose-poke behavior during test in periadolescent male rats as a function of training (S�A�, S�A�, S�B�,or S�B�) and evaluation conditions (same or different) in Experiment 3. “A” or “B” indicates ethanol postadministration intervals of 5–15 or 30–40 min,respectively. “S” represents a non-drug state. Signs “�” and “�” represent presence or absence of sucrose, respectively. Vertical lines represent standarderror of the mean (S.E.M.).

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–60 55

groups S�A� and S�B� in comparison with findingsfor groups S�A� and S�B�, respectively. No differenceswere encountered when contrasting the values for groupsS�A� and S�B� or for groups S�A� and S�B�. Thesedata have been illustrated in Fig. 7.

As could be expected, differences in BECs correspondingto state A or B after test were consistent with the resultsobtained in Experiments 1 and 2. Those subjects that weretested 5 min after ethanol administration had significantlyhigher BECs than did adolescents tested at postadministra-tion time 30 min (Student t test for independent groups:t � 9.56, df � 46, P � .001). Latency scores, mean nose-poke duration, and BECs for all independent groups havebeen incorporated in Table 2.

In the current experiment, there was empirical evidenceto support the possibility that adolescents are able to discrim-inate a moderate ethanol toxic state from a non-drug state.When the state associated with sucrose during training wasreplicated during the test, animals exhibited shorter nose-poke latencies than when the state varied between thesephases of the experiment.

Unexpectedly, those animals trained with access to su-crose while intoxicated with ethanol (groups S�A� andS�B�) not only increased sucrose intake after the firstassociation between these stimuli, but also exhibited agreater nose-poke mean duration than the other groupsduring test. The possibility that ethanol intoxication duringtraining acts as an appetitive US seems to aid in the under-standing of these results. This hypothesis obviously impliesthat the postabsorptive effects of a low dose of ethanol are

Table 2Behavioral scores registered in Experiment 3 during evaluationprocedures and the corresponding blood ethanol concentrations (BECs)

Training State Nose-poke Nose-poke mean BECs aftergroup at test latency (s) duration (s) test (mg%)

S�A� A 5.70 � 3.06 2.18 � 0.27 42.68 � 3.43S 2.82 � 0.60 2.02 � 0.23 0.00

S�A� A 3.66 � 0.87 2.36 � 0.29 44.17 � 2.15S 9.31 � 3.56 3.06 � 0.35 0.00

S�B� B 3.58 � 0.96 1.96 � 0.23 22.32 � 1.49S 3.80 � 0.61 2.05 � 0.23 0.00

S�B� B 3.74 � 0.78 2.88 � 0.41 21.95 � 1.20S 8.45 � 4.36 2.41 � 0.28 0.00

All values are expressed as mean � S.E.M.A � State in which BECs peaked (corresponding to 5–15 postadminis-

tration time); B � state occuring during the falling limb of the pharmacoki-netic curve for BECs (corresponding to 30–40 min postadministration time);S � non-drug state (sobriety); “�” � access to sucrose; “�” � no accessto sucrose.

an effective reinforcer, capable of supporting conditioningto sucrose’s sensory cues.

5. General discussion

The original goal of the current study was to examinewhether young rats are able to discriminate interoceptive ef-fects of a low dose (0.5 g/kg) of ethanol after having rela-tively little experience with the drug. The first step wasto select two postadministration periods, characterized bysignificantly different BECs, as distinctive states of intoxica-tion (Experiment 1). These temporal parameters were usedin a relatively simple behavioral study in which availability

Fig. 7. Nose-poke mean duration (seconds) in group S�A�, S�A�, S�B�, or S�B� during the test session corresponding to Experiment 3. Data havebeen collapsed across testing conditions (same or different) corresponding to each particular training treatment. “A” or “B” indicates ethanol postadministrationintervals of 5–15 or 30–40 min, respectively. “S” represents a non-drug state. Signs “�” and “�” represent presence or absence of sucrose, respectively.Vertical lines represent standard error of the mean (S.E.M.).

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–6056

of a palatable sucrose solution through nose poking wasassociated with one state but not with the other. Tests ofdifferential nose poking in the different ethanol-inducedstates associated with sucrose or no sucrose, however, pro-vided no evidence that the adolescent rats discriminatedthese states (Experiment 2). When animals experiencedeither an ethanol-induced state associated with sucrose and anon-drug state associated with absence of this reinforcer orvice versa, however, behavioral evidence of state discrimina-tion was obtained (Experiment 3). In Experiment 3, adoles-cents exhibited shorter nose-poke latencies when in the samestate in which sucrose had been available. The results ofExperiment 3 not only indicated discrimination between drugversus non-drug states, but also supported the suggestionthat, at this age, the low dose (0.5 g/kg) of ethanol can supportappetitive conditioning to the taste of sucrose.

In the current study, a specific drug state was associatedwith sucrose availability, whereas an alternative state was not.This training procedure is analogous to the procedures usedby Winter (1975) and York (1978), in which, ideally, animalslearn to respond or to refrain from responding as a functionof a given drug state that was or was not previously associ-ated with delivery of an appetitive stimulus. The procedurewas selected to rapidly train and test subjects within the onto-genetic stage of adolescence, and it was effective in thecurrent study for discriminating either of two states of etha-nol intoxication from a sober state.

As mentioned, results of different studies, with the useof a wide range of training procedures, endorse the capabilityof adult rats to exhibit ethanol-mediated SDL (Bruins Slotet al., 1999; Holloway, 1972; Lowe, 1986; Nakagawa &Iwasaki, 1995). Nevertheless, Bruins Slot et al. (1999) foundethanol-induced SDL in adult rats only with a higher dose(1.25 g/kg, i.p.) relative to the one used in the current study.There were no indications of adult learning when ethanoldoses equivalent to 0.32 or 0.64 g/kg were used. The findingsof the current study indicate that periadolescents rapidlyencode discriminative properties of the state of intoxicationeven when a 0.5-g/kg dose of ethanol is used. Also, Hunt etal. (1990) reported ethanol-mediated SDL in preweanlingrats when a mild dose (0.4 g/kg, i.g.) of ethanol was used.Despite the need to consider different factors across studies(e.g., training procedures, modes of expression, and routes ofadministration), it seems that early in ontogeny the rat ishighly responsive to discriminative properties of the acutestate of intoxication induced by relatively low doses ofethanol.

In terms of explicit comparisons (see Fig. 6), it seemsthat young rats that always experienced sucrose under eitherof the states of ethanol intoxication (groups S�A� andS�B�) took longer to exhibit the first nose poke whentested under a sober state than when tested under the stateassociated with sucrose availability. A weaker effect of thiskind occurred for rats that originally had access to sucrosewhile not intoxicated (groups S�A� and S�B�). It ispossible that state discrimination is more likely to occur for

subjects in which the toxic state was associated with sucrosethan when a non-drug state was paired with this appeti-tive stimulus, similar to a common feature of discriminationlearning, termed feature-positive effect (Sainsbury, 1971).Hence, the possibility of asymmetrical state discriminationshould not be ruled out. During training, the contingencybetween ethanol intoxication and sucrose presence seemsto be optimal in comparison with the contingency betweennon-drug state and sucrose availability. In other words, sobri-ety represents the animal’s most common state, not expe-rienced only when the organism is placed in a particularenvironment in which an appetitive stimulus such as sucroseis available. It may also be significant that for animals ingroups S�A� and S�B�, state was confounded by tempo-ral order in that the sober state was associated with sucroseand also with being placed in the training chamber for the firsttime during each conditioning session. As previously dis-cussed, temporal cues (sequence) can be encoded readilyas relevant signals that will affect the expression of a particu-lar memory (Capaldi et al., 1986; La Fiette et al., 1994). Inthe test situation, all animals were placed in the apparatusonly once, hence the “first” occasion in that session and solikely to yield maximal responsiveness in terms of sucrose-seeking behavior. Therefore, both drug state and temporalsequence might have determined test performance. This hy-pothesis also seems to apply to the results of Experiment 2,in which animals were trained only while intoxicated andthere was no evidence for discrimination of alternativeethanol-induced states. In this experiment, rats that experi-enced sucrose when first placed in the conditioning chamberduring training (group A�B�) showed shorter latencies inthe test situation than did animals that had access to thisappetitive stimulus during the second phase of each trainingsession (group A�B�).

Ethanol has been described as a drug with unspecificpharmacological actions relative to other drugs, such as ben-zodiazepines and barbiturates. Results of drug-discriminationstudies, in which animals have been trained to exhibit agiven response under ethanol intoxication, have shown thatthis response also occurs when the animals are tested underseveral other drug states (e.g., benzodiazepines) but not viceversa (Barry, 1974; Barry & Krimmer, 1977; Rees et al.,1987). This result supports the suggestion that, as a discrimi-native stimulus, ethanol has diverse interoceptive attributes(Barry, 1991). Likewise, the pharmacological effects of etha-nol have been defined as a stimulus complex with a redun-dant nature. In drug-discrimination procedures, this stimulusredundancy can determine overlapping qualities of ethanolacross doses, postadministration intervals, or both (Grant,1999). In other words, one interoceptive state derived froma given ethanol dose or postadministration interval couldhave an ample spectrum of effects that includes the discrimi-native effects of other ethanol-generated interoceptive states.This overlapping could impair the discrimination betweensuch drug states. This phenomenon could help explain thediscrimination failure observed for the two ethanol-induced

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–60 57

states in Experiment 2. Furthermore, it is operationally im-possible to avoid the effects of peak BECs (state A in Experi-ment 2) when the experimental design includes the need topresent sucrose during the effects of lower BECs registeredwithin the falling limb of the ethanol elimination curve (stateB). Under these circumstances, the possibility exists rela-tive to close temporal proximity between the first toxic state(A) and sucrose presentation during the following toxicstate (B). This contiguity factor may be sufficient to supportthe association between the first state and the appetitive tasteand hence impede the expression of discrimination acrossthese hypothetically separable states.

Results of Experiment 3 indicated that adolescents wereable to discriminate an ethanol-induced state of intoxicationfrom a non-drug state. Results also showed that ethanolwas probably acting not only as a relatively redundantstimulus, but also as an appetitive US. Sucrose, which wasused to establish the process of drug state discrimination,has a distinct flavor likely to become associated withunconditioned stimuli, such as lithium chloride (Eylamet al., 2000; Fouquet et al., 2001), morphine withdrawal(McDonald et al., 1997), and apomorphine (Wang et al.,1997). Both infant (Hunt et al., 1990) and adult (Cordobaet al., 1990) rats readily avoid this flavor when previouslypaired with postabsorptive consequences of ethanol doses(higher than the dose in the current study). In geneticallyheterogeneous rat strains, ethanol has generally been foundto act as an aversive stimulus when associated with eitherchemosensory (Abate et al., 2001; Deutsch & Eisner, 1977;Eckardt, 1975) or tactile and visual (Cunningham et al.,1993; Gauvin & Holloway, 1992; Schechter & Krimmer,1992) cues. Extensive training procedures (Bozarth, 1990),long-term preexposure to the interoceptive effects of thedrug (Reid et al., 1985), concurrent presentation of otherreinforcers [for example, food (Stewart & Grupp, 1985)or morphine (Marglin et al., 1988)], or stressful events (Mat-suzawa et al., 2000) have been necessary to attain ethanol-mediated conditioned preferences in heterogeneous rats. Itis interesting to note that, in Experiment 3, the possibilityof a relatively mild stressor could have favored heightenedsucrose-intake patterns in groups S�A� and S�B�. Thesham intragastric administration procedure can imply stress-related effects that could be alleviated through the anxiolyticeffects of ethanol (Pohorecky, 1981) and, hence, facilitatesucrose intake. Under this perspective, negative-reinforcingor appetitive-reinforcing effects (or both types of effects) ofethanol could have determined sucrose-preference patternsin these groups relative to pertinent controls and modulatedsubsequent seeking behavior of the taste originally pairedwith the drug.

It was recently suggested that characteristics of the ado-lescent brain predispose the adolescent to be highly respon-sive to appetitive properties of different drugs of abuse,including ethanol (Spear, 2000). The age factor, in conjunc-tion with different procedural characteristics of the currentexperiments, might help explain the unexpected outcome in

relation to the US properties of ethanol. The dose (0.5 g/kg) of ethanol used in the current study is lower than thedoses reported to support aversive conditioning (normally,equal to or higher than 1 g/kg). Also, the strength of theassociation between sucrose and ethanol was probably fa-vored by at least two factors. First, when appetitive condi-tioning became evident (Experiment 3), animals had beentrained in a distinct environment under a non-drug condi-tion in which sucrose was explicitly absent and in the sameenvironment under the effects of ethanol paired with thistastant. Preexposure to the exteroceptive cues in whichsucrose–ethanol pairings took place probably diminishesthe possibility that other cues will interfere with the estab-lishment of the associative memory between sucrose andthe ethanol intoxication (Fernandez-Vidal & Molina, 2001).Second, the lack of interference effects on the acquisitionof this memory is probably also favored by a rather shortprocess of intoxication. One hour after administration of the0.5-g/kg dose of ethanol, the drug was practically undetect-able in blood (Experiment 1). Hence, because of the lowethanol dose and its pharmacokinetic profile, the possibil-ity that other cues will become associated with the toxiceffects of ethanol seems minimized. With regard to this lastissue, it is also important to note that, after training, adoles-cents were placed in a holding chamber that was very similarto the home environment. This implies a lack of salientcues that might retroactively compete with the signalingproperties of sucrose relative to the effects of ethanol.

It could be argued that the results of Experiment 3 donot reflect the reinforcing properties of ethanol but, rather,indicate the development of ethanol-mediated conditionedtaste aversions (Cappell et al., 1973; Cunningham, 1979;Hunt et al., 1990). According to this perspective, groupsS�A� and S�B� could be drinking less sucrose relativeto the amount consumed by groups S�A� and S�B�because in animals in the former group the sweet solutionsensed when first exposed to the black box becomes associ-ated with later aversive effects derived from the state ofethanol intoxication. The following considerations argueagainst this possibility. Results of different studies have dem-onstrated that low doses (lower than 0.8 g/kg) of ethanolpaired with flavors or alternative environmental cues failto establish conditioned aversions (Cappell et al., 1973;Cunningham et al., 1993; Lester et al., 1970; Sherman etal., 1983; Stewart & Grupp, 1985; van der Kooy et al.,1983). Furthermore, results of a recently conducted study(unpublished observations, J. M. Fernandez-Vidal, N. E.Spear, & J. C. Molina, 2003) also seem to argue againstthe possibility of conditioned taste aversions. We examinedwhether the heightened sucrose intake observed in S�A�rats is more likely to occur when alternative training proce-dures are used. The complete design included groups S�A�and S�A�, as well as S� and A� alone groups. The S�rats received sucrose in the black box, followed 5 h laterby intragastric administration of ethanol (0.5 g/kg) in the

J.M. Fernandez-Vidal et al. / Alcohol 30 (2003) 45–6058

home cage. The A� rats received sucrose paired with intoxi-cation in the black box, followed 5 h later by a sham intragas-tric intubation in the home cage. These procedures allowedus to keep ethanol-related experiences and intubations con-stant across groups. The S� group implies a longer delaybetween the CS and US than the one used in group S�A�.There were no differences in sucrose intake between groupsduring the first training day. In subsequent days, S�A�subjects drank significantly more than did subjects in anyremaining conditions. If conditioned taste aversions are de-rived from forward pairings between sucrose and ethanol,it would be logical to expect lesser consumption in S�A�rats when compared with findings for the S� alone groupbecause of notable differences in delays between the CS andUS. These groups failed to differ between them and bothdrank significantly less than did S�A� periadolescents.Interestingly, A� rats also failed to exhibit enhanced su-crose-consumption patterns. This latter result argues againstnonspecific activating or dipsogenic effects of ethanolleading to increases in intake behavior and supports thesuggestion that discriminative procedures could facilitate ac-quisition of ethanol-mediated sucrose-acceptance patterns.

In Experiment 2, sucrose was presented when BECs eitherwere at peak or during the falling limb of the pharmacoki-netic curve. The design of this experiment does not allowfocusing on the possibility of ethanol as an appetitive rein-forcer. All groups (A�B� and A�B�) tasted sucrose whileexperiencing the postabsorptive effects of ethanol. Interest-ingly, in this experiment, sucrose consumption increasedafter the first day of training. Nevertheless, this increaseseems to be of a lesser magnitude than the one observed inExperiment 3 in group S�A� or group S�B� (for explicitcomparisons, see Figs. 2 and 3). In these last groups (S�A�and S�B�), the contingency between sucrose and the phar-macological effects of ethanol in a distinctive training envi-ronment seems optimal. Under a non-drug state, sucrose wasabsent, whereas under the state of intoxication, sucrosewas present. In Experiment 2, comparable contingenciesare less optimal: Although these rats also were placed intothe training environment on two occasions, on both of thesethey were under the effects of ethanol and only once wassucrose available.

These results indicate that, within relatively brief periods,the adolescent rat is capable of learning about specific char-acteristics of the acute state of intoxication. Under appro-priate experimental circumstances, these young organismsseem highly sensitive to positive hedonic consequences ofthe state of intoxication. This ontogenetic considerationshould be tempered by the fact that appetitive effects ofethanol have been described in food-deprived adult rats asa consequence of the caloric properties of ethanol (Shermanet al., 1983). It cannot be completely dismissed that a similarmechanism was responsible for the effects reported in thisarticle, despite the fact that the regimen of fluid deprivationand, hence, possible partial food deprivation is markedlyminor when compared with the one used by Sherman et al.

(1983). Yet, it is necessary to note that periadolescence ischaracterized by a unique rate of growth associated with thegreatest caloric intake relative to body weight of any time inthe life span (Nance, 1983; Spear, 2000). It remains to bedetermined whether the age factor, the present discriminationprocedures, or both facilitated the emergence of phenomenareported in this article that have been rarely observed inheterogeneous strains of rats.

Acknowledgments

This work was supported by grant PICT 5-7053 fromAgencia Nacional de Promocion Cientıfica y Tecnologica,grant Ramon Carrillo and Arturo Onativia from Ministeriode Salud, Argentina (J.C.M.), and grant RO1AA10223 andgrant RO1AA11960 from NIAAA (N.E.S.), as well as byfellowship from Fundacion Interior Argentina and fellow-ship from Consejo Nacional de Investigaciones Cientıficasy Tecnicas awarded to J.M.F.V. We wish to express ourgratitude to Beatriz Haymal and Teri Tanenhaus for theirtechnical assistance.

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