Differential motivational properties of ethanol during early ontogeny as a function of dose and...

Alcohol 41 (2007) 41e55

Differential motivational properties of ethanol during earlyontogeny as a function of dose and postadministration time

Juan Carlos Molinaa,b, Ricardo Marcos Pautassia,*, Eric Truxella, Norman Speara

aCenter for Developmental Psychobiology, Binghamton University, Binghamton, NY 13902-6000, USAbInstituto de Investigacion Medica M. y M. Ferreyra (INIMEC e CONICET), Cordoba, C.P 5000, Argentina

Received 21 September 2006; received in revised form 29 January 2007; accepted 29 January 2007

Abstract

While appetitive reinforcement effects of ethanol are easily detected in rat neonates, such phenomena rarely have been observed in olderinfants. Recently, Molina et al. [Molina, J. C., Ponce L. F., Truxell, E., & Spear N. E. (2006). Infantile sensitivity to ethanol’s motivationaleffects: ethanol reinforcement during the third postnatal week. Alcohol Clin Exp Res 30, 1506e1519] reported such effects of ethanol in 14-day-olds using a second-order conditioning procedure. Infants also appear to be sensitive to biphasic reinforcement or general motivationaleffects of ethanol, with appetitive effects seeming to occur early in the state of intoxication and aversive effects predominant during latestages, but tests have been inconclusive. The present study examined the possibility of biphasic motivational effects of ethanol during in-fancy through the use of second-order conditioning procedures. Preweanling rats (14 days old) experienced intraoral water infusions (con-ditioned stimulus, CS) either 5e20 or 30e45 min after administration of 0.5 or 2.0 g/kg i.g. ethanol. Pups were then exposed to the CSwhile over a novel texture (second-order phase). Tests of tactile preference for that texture followed. Locomotive, thermal, hormonal (cor-ticosterone release), and pharmacokinetic patterns likely to underlie the acquisition of ethanol-mediated conditioning were also examined insubsequent experiments. Intraoral CSs paired with either early or late effects of low-dose ethanol (0.5 g/kg, blood ethanol concentration:40 mg%) became positive second-order reinforcers. Appetitive effects were also exhibited by pups exposed to the CS during commence-ment of the toxic episode induced by a 2.0 g/kg ethanol dose, 5e20 min after administration of ethanol, whereas aversions emerged whenCS presentation occurred 30e45 min postadministration time (blood ethanol concentrations: 157 and 200 mg%, respectively). Overall, theresults indicate that infants rapidly detect differential motivational properties of ethanol as a function of dose or drug postadministrationtime. Relatively neutral stimuli associated with these properties are later capable of acting as either positive or aversive reinforcers. Thermaland motor responses that accompany ethanol intoxication do not seem to be directly associated with differential hedonic properties of thedrug at this stage of development. � 2007 Elsevier Inc. All rights reserved.

Keywords: Ethanol; Motivational properties; Infant; Locomotion; Blood ethanol concentration; Thermoregulation

1. Introduction

Even though expressing different core views, most con-temporary theories of drug use and abuse agree that posi-tive, appetitive effects of psychotropics play a major rolein shaping drug seeking and consumption in human sub-jects (e.g., Deadwyler et al., 2004; Koob & Le Moal,2001; Robinson & Berridge, 2000). With regard to ethanol,this has encouraged behavioral researchers to develop ani-mal models whereby subjects demonstrate preference forthe drug in voluntary intake tests, exhibit changes in theprobability of execution of behaviors contingent with

* Corresponding author. Center for Developmental Psychobiology,

Binghamton University, Binghamton, NY 13902-6000, USA. Tel.: þ1-

607-237-2034; fax: þ1-607-777-2677.

E-mail address: [email protected] (R.M. Pautassi).

0741-8329/07/$ e see front matter � 2007 Elsevier Inc. All rights reserved.

doi: 10.1016/j.alcohol.2007.01.005

ethanol’s postabsorptive effects or approach discrete stimuli(conditioned stimuli, CSs) associated with administrationof the drug (Cunningham et al., 2000; Koob & Roberts,1997).

Appetitive properties of ethanol often have been re-ported when using genetically selected lines of rats andmice (Bechthold & Cunningham, 2005; Cicocciopo et al.,1999; Cunningham et al., 2002). However, the search forappetitive effects of ethanol in heterogeneous lines of etha-nol-naive rats has proven troublesome. Most often, aversivehedonic components of this drug have been found in adultand infant animals when ethanol doses equivalent to orabove 1 g/kg are paired with taste or tactile cues (Gauvin& Holloway, 1992; Hunt et al., 1991; Lee et al., 1998; Mo-lina et al., 1996; Pautassi et al., 2002, 2005b; Pueta et al.,2005; Sherman et al., 1983). During adulthood, long-term

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42 J.C. Molina et al. / Alcohol 41 (2007) 41e55

pre-exposure to the drug and/or concurrent use of otherdrug reinforcers has been necessary to allow the expressionof ethanol-mediated appetitive memories (Bienkowskiet al., 1995, 1996; Marglin et al., 1988). These experimentalmanipulations are not suitable to be used in preweanling(infant) animals. Specifically, the brevity of this ontogeneticperiod in the rat (0e21 days) as well as the likelihood ofteratological effects imposes serious limitations whenassessing ethanol’s motivational effects during earlyontogeny.

Nonetheless, increasing experimental evidence indicatesthat early infancy is a period of special sensitivity to etha-nol’s motivational properties, particularly when using ani-mals younger than 14 days of age. Exposure to ethanolduring late gestation induces an enhanced consumption ofethanol as well as an increased palatability toward the druglater in life (Arias & Chotro, 2005), a result likely to be ex-plained in terms of a conditioned preference acquired to thechemosensory properties of ethanol (Abate et al., 2002;Chotro et al., 2006). When an odor CS signals ethanol dur-ing late gestation neonates later show enhanced attachmentto a surrogate nipple scented with that olfactory stimulus(Abate et al., 2002). Using the surrogate nipple technique,Niznikov et al. (2006) have shown not only reinforcingproperties of ethanol in neonates but also heightened sensi-tivity to these effects as a function of prenatal ethanol expo-sure. Special sensitivity to sensory (Truxell & Spear, 2004)and behavioral and physiological (Hunt et al., 1991) conse-quences of ethanol in young rats may underlie the preced-ing results (for reviews see Bachmanov et al., 2003; Spear& Molina, 2005).

Evidence for nonaversive motivational effects of ethanol(i.e., appetitive and/or anxiolytic) in infants older than 14days postnatal (P14) is relatively scarce. Truxell and Spear(2004) have reported that 18-day-old pups will consumeethanol and reach pharmacologically relevant blood ethanolconcentrations (BECs) when having the possibility to drinkthe drug from a warmed floor, though to a lesser degreethan 12-day-old pups. It has also been reported that low-to-moderate doses of ethanol compete with or inhibit theexpression of associative learning mediated by variousaversive stimuli. Pautassi et al. (2005a) observed that pupsavoided a salient odor that had been paired with aversiveintraoral stimulation. The expression of this conditionedresponse was completely inhibited when pups were admin-istered with 0.25 g/kg ethanol during training. These resultssuggested that young animals are sensitive to nonaversiveeffects of the drug, including ethanol’s anxiolytic effects.Similar suggestions were derived from a subsequent study(Pautassi et al., 2006). Specifically, pups were exposed toan odor CS paired with an aversive unconditioned stimulus(US) (citric acid intraoral infusion). One day later, pupswere briefly re-exposed to the US while experiencing thepostabsortive effects of ethanol (0.5e1.25 g/kg) orvehicle. Ethanol exposure significantly reduced themagnitude of the conditioned odor aversion.

Molina et al. (2006) recently reported substantial ethanol-mediated appetitive learning in heterogeneous 14-day-oldanimals. In this study, no differential intake of water,quinine, or sucrose was found after pairing these stimuli(CSs) with early (postadministration time: 5e10 min) post-absorptive effects of ethanol (0.25, 0.50, or 2.0 g/kg). Acompletely different profile was observed when ethanol-mediated conditioning was analyzed in terms of the capa-bility of the taste CSs to act as second-order reinforcerswhen later associated with a neutral stimulus (sandpapertexture). Conditioned preference for the sandpaper surfacewas observed after pups were given that surface paired withan intraoral tastant, which itself had previously been pairedwith ethanol. Interestingly, none of the ethanol doses usedby Molina et al. (2006) resulted in behaviors indicative ofthe development of ethanol-mediated conditioned aver-sions. Also, the latter work made no systematic attemptto examine the mechanisms underlying the acquisition ofthe observed ethanol-mediated learning.

The procedure used by Molina et al. (2006) is referred toas second-order conditioning (SOC). The change in behav-ior derived from a CS1eUS experience is assessed notthrough direct exposure to the CS1 alone but rather throughthe capability of the original CS1 to transfer behavioral con-trol to a second, different neutral stimulus (CS2). If the orig-inal learning was successful, a substantial conditionedresponse should now be elicited by the CS2 (for a compre-hensive review, see Rescorla, 1980). This procedure hasbeen useful for detection of otherwise ‘‘silent’’ associa-tions, even in young organisms. Miller et al. (1990) foundthat although 8-day-old rats avoided an odor previously as-sociated with lithium chloride (LiCl), no signs of this con-ditioned aversion were observed in 4-day-old animals.Nevertheless, the younger pups were able to express the ac-quired odor-LiCl association when using a SOC procedure.Specifically, following initial training, pups were subjectedto a subsequent pairing of original odor (CS1) and a noveltactile stimulus (CS2). When later tested for their prefer-ence for this tactile cue, pups avoided the CS2 texture. Inother words, SOC allowed pups to express their acquiredaversion to the CS1 odor.

One possible reason for the capability of SOC to revealacquired associations is that this procedure minimizes ef-fects of conditioned responses that might otherwisecompete with the target conditioned response. For example,potentially interfering conditioned responses have beenobserved in tests of conditioned place preference with eth-anol as the US (Cunningham & Noble, 1992). Substantialbehavioral activation in young animals was also observedby Molina et al. (2006) in assessment of responsivenessto the tastants previously paired with ethanol. While no dif-ferences were observed in general locomotion, pups didmore wall climbing when exposed to a flavor previouslypaired with ethanol. This result suggests that conditionedmotor responses compete with the expression of ethanol’smotivational properties and might explain part of the

43J.C. Molina et al. / Alcohol 41 (2007) 41e55

failures observed in tests of ethanol-mediated appetitivelearning through more conventional first-order conditioningprocedures.

As observed with drugs such as cocaine (Ettenberg, 1999,2004) or amphetamine (Lett, 1988), ethanol seems to exertdifferential hedonic effects across the time course of the pro-cess of intoxication. In other words, the motivational value ofthe drug changes as a function of the development of theacute state of intoxication. In mice, ethanol exerts appetitiveeffects when BECs are rising (Risinger & Cunningham,1992). Aversive effects of ethanol, on the other hand, are eas-ily detected when pairing neutral taste stimuli with later post-absortive stages characterized by peak BECs (Chester &Cunningham, 1999). Cunningham and Prather (1992) alsoobserved that the longer the pairing between a distinctive lo-cation and postabsorptive effects of ethanol, the smaller themagnitude of the conditioned preference, a result suggestingthat the drug’s hedonic effects change from appetitive toaversive as a function of the temporal course of the intoxica-tion. In preweanling (infant) rats, we have observed that a tac-tile stimulus paired with the commencement of the toxic stateinhibits the expression of a conditioned taste aversion estab-lished through the association between a taste CS and peakblood ethanol levels (Pautassi et al., 2002). This result sug-gests that the tactile cue acquired nonaversive hedonic prop-erties (appetitive or anxiolytic) that counteract the aversiveconditioned taste response. Pautassi et al. (2006) also re-ported that ethanol’s capability to modify the magnitude ofan aversive memory changes not only as a function of dosebut also as a function of postadministration time intervals.While relatively low ethanol doses (0.5e1.25 g/kg, all BEC’s!70 mg%) inhibit the expression of a conditioned aversion,a higher dose (2.5 g/kg), particularly when resulting in highblood ethanol levels (175 mg%), exerts the opposite effectand inflates expression of the conditioned aversion.

The present study assessed whether infants are sensitiveto differential hedonic properties of ethanol as a function ofdose (0.5 or 2.0 g/kg) and time course of the acute state ofintoxication. The SOC procedure previously employed byMolina et al. (2006) was used. In the Molina et al. (2006)study, however, the original associative conditioning trialwas always conducted during the early stage of the etha-nol-induced toxic process (5e10 min postadministration).That is, there were no attempts to analyze whether motiva-tional properties of the drug change as a function of thetime course of the toxic process. As already discussed, pre-vious studies (Cunningham & Prather, 1992; Pautassi et al.,2006; Risinger & Cunningham, 1992; Wilson et al., 2004)suggest that early and delayed effects of ethanol differ intheir motivational value. Appetitive and/or anxiolytic prop-erties seem to prevail shortly after ethanol administrationwhile aversive effects are more prevalent during laterpostadministration times. Taking this into account, in thepresent study, we explicitly varied ethanol dose and postad-ministration time to examine possible changes in the moti-vational value of the drug during early ontogeny.

Specifically, pups were exposed to an intraoral stimulus(Water, CS1) during either an early or a late stage of thetoxic process induced by ethanol (postadministration times5e20 and 30e45 min, respectively). The water CS was thenpaired with a tactile cue (Sandpaper, CS2). Two-way loca-tion preference tests, meant to assess the preference for theCS2 relative to a novel cue, were later conducted. As can beobserved, this study not only aims to validate previousresearch suggesting early sensitivity to ethanol’s appetitiveeffects when assessed by an SOC procedure (Molina et al.,2006) but also intends to analyze changes in ethanol’s he-donic properties as a function of the temporal course of thestate of intoxication. Ethanol’s biphasic effects have longbeen recognized as important factors mediating ethanol useand abuse (for a classic review, see Pohorecky, 1977).

While the SOC procedure has been useful to analyzereinforcing properties of ethanol in early ontogeny, the be-havioral and/or physiological mechanisms associated withthe acquisition of these early memories still remain to beidentified. Hence, a second experiment analyzed severalvariables likely to underlie the acquisition, in young ani-mals, of ethanol-mediated second-order learning, includingacute and conditioned effects of ethanol upon locomotiveand thermal patterns. This set of variables was selectedon the basis of previous research indicating their involve-ment in ethanol-mediated reinforcement (Hunt et al.,1991; Masur et al., 1986).

Prior studies have also suggested a role for corticoste-rone in modulating reinforcing properties of severalsubstances of abuse, including ethanol. The hypothalamiceadrenalepituitary axis not only is activated by stressfulsituations (Aguilera et al., 1992) but also but psychotropicslike ethanol (Spencer & McEwen, 1997). This activation re-sults in augmented levels of corticosterone, a hormone thathas proven to exert reinforcing properties (Piazza et al.,1991). Removal of endogenous corticosterone decreasesethanol self-administration in rats, whereas long-term expo-sure to subcutaneous corticosterone increases consumptionof the drug (Fahlke et al., 1995). Moreover, it has beendemonstrated that levels of this hormone are modified notonly by acute ethanol but also by environmental cues asso-ciated with the drug (Seeley et al., 1996). Hence, in thepresent study, plasma corticosterone levels were measuredin infants immediately after test. Finally, BECs associatedwith the ethanol doses used in the behavioral experimentswere assessed.

2. Experiment 1

Preweanling rats express ethanol-mediated taste aver-sions following only one (Hunt & Spear, 1989) or two(Hunt et al., 1990) conditioning trials. Infants are also ableto rapidly acquire and transfer information between neutraland biologically relevant stimuli when using high-order


conditioning procedures (e.g., sensory preconditioning:Chen et al., 1993; SOC: Cheslock et al., 2003; Milleret al., 1990; revaluation of the representation of the US:Kraemer et al., 1992; Molina et al., 1996). The use of a pro-cedure akin to SOC has recently revealed that ethanol ex-erts positive reinforcing effects quickly after the onset ofthe toxic process when using doses between 0.25 and2.0 g/kg (Molina et al., 2006). As stated, previous studiessuggest that the hedonic value of the drug is likely tochange during later stages of the intoxication, particularlywhen relatively higher doses are used. For instance, Pautas-si et al. (2006) observed in 15-day old rats that ethanol’s ca-pability (2.5 g/kg) to inhibit a previously acquired aversivememory changes with development of the toxic episode.

In this experiment, SOC procedures were used toscrutinize the possibility that ethanol exerts differentialmotivational properties depending on dose and postadmi-nistration time. If the drug exerts appetitive and aversiveeffects associated with early and late postadministrationtimes, respectively, the behavioral control acquired by anintraoral CS experienced during a given stage should varyaccordingly. Hence, ethanol-mediated learning was nottested directly in terms of responsiveness to the intraoralCS, but rather as a function of its capability to endow a tac-tile CS with reinforcing properties. In other words, the aimof this experiment was to assess whether a relatively neutralstimulus would be likely to later act as an appetitive oraversive second-order reinforcer as a function of a precedingassociation with different ethanol doses, or within eachdose with varying postadministration time intervals.

2.1. Material and methods

2.1.1. AnimalsSeventy-one preweanling SpragueeDawley pups (14

days old at the start of the experiment; body weight range:29e42 g), representative of eight litters were used. All pupswere born and reared at the Vivarium of the Center for De-velopmental Psychobiology (Binghamton University, NY,USA). Births were examined daily and the day of parturi-tion was considered as Postnatal Day 0 (PD 0). Newbornswere always kept with their biological mothers in standardmaternity cages (47� 25� 20 cm) partially filled withwood shavings. All animals were housed in a temperature-controlled (22�C) vivarium maintained under a 14-h light/10-h dark cycle (lights on at 0700 h) with ad libitum accessto food (Purina Mills, St. Louis, MO) and water. At P1,litters were culled to 10 pups (five males and fivefemales whenever possible) and were left undisturbed untilcommencement of the experiment. Maintenance andexperimental procedures were in accordance with the Guidefor Care and Use of Laboratory Animals (Institute ofLaboratory Animal Resources, 1996) and the guidelinesindicated by the Binghamton University animal handlingreview committee.

2.1.2. Experimental designThe experimental approach was defined by a factorial de-

sign with two orthogonal between-group factors: ethanoldose (0.5 or 2.0 g/kg) and conditioning procedure (the condi-tioned stimulus was presented either during an early[5e20 min; early pairing, EP] or a later [30e45 min; latepairing, LP] ethanol postadministration time, or was explic-itly unpaired with ethanol’s postabsorptive effects, UP). Sixgroups were thus formed. Each group had 11 or 12 animals.To avoid overrepresentation of litters within each specificgroup, no more than two animals per litter (one male andone female) were assigned to each particular treatment. Sexwas also considered a factor in the present and following ex-periments. Yet, because this variable failed to exert signifi-cant main effects or interact with the remaining factors inall experiments, data were collapsed across this variable.

2.1.3. ProceduresOn PD 14, pups were removed from the maternal cage

and placed in pairs in a holding chamber. These chamberswere maintained at 35�C by means of a heating pad placedunderneath. A polyethylene cannula was then implanted inthe pup’s cheek, as described elsewhere (i.e., Abate et al.,2000; Pautassi et al., 2002). This procedure has been provento be minimally stressful to rat pups (Spear et al., 1989).After the cannulation procedure, pups were left undisturbedfor 2 h until commencement of the conditioning session.

Conditioning procedures followed those used by Molinaet al. (2006) closely. Pups on P14 were individually placedin a trapezoid-shaped chamber (wall lengths: front 5 29 cm,back 5 18 cm, sides 5 11.5 cm, height 5 12.5 cm). Theback wall and sidewalls of this cage were made of mirroredPlexiglas while the front wall and the floor were lined withclear Plexiglas. The floor of the chamber was covered withcotton. A 10-min habituation phase was initially conductedto familiarize pups with the chambers as well as to facilitatea better discrimination of the forthcoming CS. Immediatelyfollowing habituation, paired pups were weighed to thenearest 0.01 g (Sartorius, Gottingen, Germany) and intra-gastrically administered with 0.5 or 2.0 g/kg ethanol. Theseethanol doses were achieved by administering 0.015 ml/kgof a 4.2 or 16.8% vol/vol ethanol solution, respectively(190-proof Ethanol, Pharmaco, Brookfield). Pups were thenreturned to the holding chambers where they remained untilpostadministration times 5 (paired early group) or 30 min(paired late group). Conditioning took place after pups werereturned to the trapezoid chambers. In these chambers, pre-weanlings received intraoral pulses of distilled water (con-ditioned stimulus, CS1). Fifteen 5-s pulses (5 ml per pulse)were delivered throughout the conditioning trial (interstim-ulus interval: 55 s). To control fluid delivery, the cannulaeof the pups were attached to polyethylene tubing (PE50,Clay Adams) connected to an infusion pump (AutoPump,Kashinsky 5/2000, Binghamton, NY). Paired pups werethen returned to the holding chambers until completinga 2-h period that began with administration of the


corresponding ethanol dose. Pups assigned to the unpairedcontrol groups received the same intraoral water and etha-nol as paired subjects, but in this case the water CS was ex-perienced 2 h before intragastric ethanol administration.Immature rats consistently fail to exhibit conditioning withlong-delay intervals between CSs and biologically relevantstimuli (e.g., Miller et al., 1990; Pepino et al., 1998; Rudy& Cheatle, 1979), and this is in any case a conservative un-paired control. Unpaired pups were returned to the motherafter CS exposure. Following intragastric ethanol adminis-tration they were deprived from maternal care for 2 h.These manipulations allowed maternal deprivation to beequated across paired and unpaired groups.

At PD 15, the intraoral cannulae were positioned in thecheek opposite to that used during PD 14. After 1 h pupswere placed in the trapezoid chambers. A sandpaper liningwas now covering the floor of these cages (second order-conditioning stimulus; CS2, coarse: 50, Gatorgrit, USA).While in contact with sandpaper, four pulses of the originalwater CS were delivered through the intraoral cannula (vol-ume per pulse: 5 ml, duration: 5 s, interstimulus interval:55 s). The sandpaper sheets were changed after each ani-mal. A 5 min two-way texture preference test followed30 min later. This test took place in a clear Plexiglas rect-angular chamber (28� 13� 15.5 cm). Half of the floorwas lined with the sandpaper tactile stimulus (CS2) thathad been previously paired with the water CS1. The remain-ing floor surface was covered with the smooth backside ofa similar piece of sandpaper. New, clean textures were usedfor each individual pup. Texture preference assessmentsstarted by placing the animal in the middle section of theapparatus. Time spent over each particular texture of the ap-paratus was recorded. A subject was considered to be ina particular texture when three paws and the head were overthat section. Time spent over each section was recordedduring each minute of the test session. Testing was con-ducted by experimenters blind with regard to treatmentconditions.

2.1.4. Data analysisThe dependent variable under analysis was absolute time

spent per minute on each tactile surface. This variable wasanalyzed by means of a four-way mixed analysis of vari-ance (ANOVA). The between-group factors under consider-ation were ethanol dose (0.5 and 2.0 g/kg) and conditioningprocedures (EP, LP, and UP) while the within-subject fac-tors were texture (sandpaper vs. smooth) and evaluationbin (1, 2, 3, 4, and 5 min). In this, as well as in the follow-ing experiments, the loci of significant main effects or inter-actions were further examined through follow-up ANOVAsand by means of pair-wise post hoc comparisons (Fisher’sLeast Mean Significant tests, with an alpha level setat 0.05).

2.1.5. ResultsAs stated, absolute time spent over each texture during

the second-order test was subjected to a four-way mixedANOVA (dose� conditioning procedure� texture� bin).The ANOVA yielded significant main effects of textureand bin, F(1, 65) 5 8.37, P ! .05; F(4, 260) 5 2.63,P ! .05. The interactions between dose and texture, condi-tioning and texture and texture and bin also reached signifi-cance, F(1, 65) 5 5.52, P ! .05; F(2, 65) 5 7.07, P ! .005;F(4, 260) 5 3.38, P ! .05. More importantly, the four-wayinteraction between the factors was also significant, F(8,260) 5 2.21; P ! .05. Absolute time spent over each tex-ture at test is depicted in Fig. 1. To better understand thesignificant interactions shown by the overall analysis, fol-low-up ANOVAs (texture� bin) were performed for eachof the six groups derived from the original experimentaldesign (UP/0.5, UP/2.0, EP/0.5, EP/2.0, LP/0.5, LP/2.0,letters indicate conditioning procedures while numbers al-lude to ethanol dosage). In the case of Unpaired animalsno significant effects were observed. When consideringPaired pups that experienced the water CS paired with theearly postabsorptive effects of 0.5 g/kg ethanol (EP/0.5)the ANOVA indicated a significant main effect of texture,F(1, 10) 5 7.99, P ! .025. These pups exhibited height-ened preference to the tactile cue (sandpaper) paired withthe water CS that was originally associated with this lowethanol dose. Similar heightened time spent over sandpaperwas observed in groups LP/0.5 and EP/2.0; F(1,11) 5 6.75, P ! 0.025 and F(1, 11) 5 15.19, P ! .0025,respectively. Yet for pups given water infusions as a first-order CS paired with late postabsorptive effects of 2.0 g/kgethanol and subsequently exposed to the same water infu-sions paired with sandpaper (group LP/2.0), the resultswere markedly different. In this group, there were indica-tions of an acquired aversion to sandpaper. The ANOVArevealed an interaction between texture and bin, F(1,44) 5 2.97, P ! .05. Post hoc Fisher tests showed that dur-ing the first 3 min of the test, these animals avoided sand-paper relative to the alternative smooth surface. Thisexpression of a second-order conditioned aversion was nolonger observed during the last 2 min of evaluation.

In summary, when infants were intraorally stimulatedwith water under a sober state (Groups UP), later pairingsbetween this liquid and a distinctive texture failed to induceeither preference or aversions toward the tactile CS. Inother words, regardless of the ethanol dose administeredduring the first phase of the conditioning, animals assignedto unpaired control conditions spent roughly 50% of thetime over the target CS. In sharp contrast, intraoral infusionof water served in the present experiment as an effectivesecond-order reinforcer provided it had been initially pairedwith either early (5e20 min) or late (30e45 min) post-absorptive consequences of low-dose ethanol (0.5 g/kg).These animals exhibited a robust preference toward thesandpaper texture, which appeared to persist during the en-tire testing procedure. Differential hedonic effects of the


Fig. 1. Time spent over sandpaper and the novel surface as a function of intraoral water paired with early (5e20 min postadministration) or late (30e45 min

postadministration) ethanol dose (0.5 or 2.0 g/kg) and time at test (1e5 min). Unpaired groups, which do not exhibit significant differences between them,

have been collapsed across drug treatment and postadministration time. In this study, intraoral water was subsequently paired with sandpaper. Vertical lines

illustrate standard error of the means.

drug were evident when a higher ethanol dose (2.0 g/kg) wasused. Positive reinforcing effects of ethanol were observedwhen the original CS was associated with commencementof the intoxication, whereas a second-order conditioned aver-sion was evident when this pairing was delayed until a laterpostadministration time period, a period characterized bymuch higher BECs (200 mg%, see Experiment 3). This aver-sion was particularly evident during the first minutes of thetest; apparently, extinction took place during the later phaseof this behavioral assessment.

3. Experiment 2

The previous experiment indicates that infant rats encodedifferential hedonic effects of ethanol as a function of doseand postadministration time. Specifically, intraoral CSspaired with either early or late effects of low-dose ethanol(0.5 g/kg) became effective positive second-order rein-forcers. Appetitive effects were also exhibited by pups ex-posed to the CS during commencement of the toxic episodeinduced by a higher ethanol dose (2.0 g/kg), whereas a condi-tioned aversion was developed when the first-order CS waspresented during a later postadministration time.

The purpose of the present experiment was to analyze pos-sible mechanisms associated with ethanol-mediated SOC. Tothis end, a substantial part of the procedures conducted inExperiment 1 was replicated while recording acute andconditioned effects of the drug upon thermal, motor, andhormonal responses. Specifically, first and SOC phases sim-ilar to those used in Experiment 1 were conducted. In thisexperiment, vehicle-treated infants rather than unpairedcontrols were used to adequately determine unconditioned

effects of each ethanol dose at each postadministration time.On PD 14, preweanling rats given vehicle (0.0 g/kg ethanol)or ethanol (0.5 or 2.0 g/kg) intragastrically were intraorallystimulated with water (CS) during postadministration times5e20 or 30e45 min. After 24 h, pups were exposed to thewater CS while placed over a distinctive texture (sandpaper).Locomotive patterns as well as rectal body temperatureswere recorded during both phases of the experiment. Cortico-sterone levels were also measured on PD 15 after pups wereexposed to the water CS, which, as mentioned, had been pre-viously paired with ethanol. As will be discussed (see Section5) the behavioral, physiological, and hormonal variables un-der consideration seem to be associated with ethanol’smotivational properties (Cunningham et al., 1991, 1993;Fahlke et al., 1995).


3.1.1. AnimalsSixty-six SpragueeDawley derived pups representative

of 10 litters born and reared at the Center for Developmen-tal Psychobiology were used. Genetic, housing, and breed-ing conditions of these animals replicated those describedin Experiment 1. Animals (14-days old at the beginningof the experiment) had a mean body weight of 33.8 g.

3.1.2. Experimental designA randomized, factorial, experimental design was used.

Animals were assigned to one of six groups defined asa function of ethanol dose administered during conditioning(0.0, 0.5, or 2.0 g/kg of the drug) and postadministrationtime at which intraoral infusions of the water CS took place(EP: 5e20 min or LP: 30e45 min). Each of the groups had


a minimum of 10 and a maximum of 12 pups. Precautionswere taken to avoid overrepresentation of litter and sexacross groups. No more than one male and one female cor-responding to each particular litter were assigned to a spe-cific treatment. Due to blood sampling problems, eight datapoints corresponding to corticosterone assessments wereunavailable. No more than two data samples for a givengroup were lost.

3.1.3. ProceduresAs in Experiment 1, pups were implanted with intraoral

cannulae and left undisturbed for 2 h in heated holdingchambers (34e35�C). Throughout the experiment (condi-tioning and testing procedures) ambient room temperaturewas maintained at 22�C.

The first-order conditioning phase started with a 10 minhabituation trial. Pups were individually introduced in clearPlexiglas activity chambers (10� 10� 12 cm) lined withcotton. These chambers were equipped with six infraredphoto emitters and six infrared photoreceptors. The photobeams crossed the chamber generating a matrix of ninecells. Each activity chamber was in turn connected to a com-puter. Custom-made software served to analyze the numberof beams crossed by each subject every 10th of a second.Data were subsequently grouped using 1-min timeintervals.

At the end of the habituation phase animals wereweighed to the nearest 0.01 g (Sartorius, Gottingen, Ger-many) and their temperature was recorded using a Physi-temp Temperature Monitor (TH8 Model, Clifton, NJ)equipped with a rectal probe (RET-3, tip diameter: 0.065in.). Specifically, the probe was lubricated with mineraloil kept at room temperature and was then inserted2.5 cm in the rectum. Temperature recordings were ob-tained 20 s following insertion of the probe. Immediatelyafter temperature recording, animals received an intragas-tric administration of ethanol (0.0, 0.5, or 2.0 g/kg, vehicle:tap water) and returned to the holding chambers. Ethanoldoses were achieved by administering 0.015 ml of a 0.0,4.2, or 16.8% vol/vol ethanol solution per gram of bodyweight.

Pups remained in the heated holding chambers until 5 or30 min postadministration. Pups were then gently placed inthe activity chambers and intraorally stimulated with waterfor 15 min, using the parameters already described in Ex-perimental 1 (15 pulses, 5 ml per pulse, duration: 5 s, inter-pulse interval 55 s). Throughout the conditioning trial,locomotive patterns were recorded by means of the activitydevices previously described. The PE 50 tubing connectingeach subject’s cannula to the infusion pump exited from theactivity chamber through a hole located in its upper section.This prevented accidental activity counts due to beamsbreaks caused by the tubing. Following the infusion proce-dure, rectal temperatures were again recorded to estimateethanol-induced thermoregulatory changes. After comple-tion of water stimulation procedures, oral cannulae were

removed and pups were returned to their holding chambers.After 2 h following vehicle or ethanol administration proce-dures, pups were returned to their respective maternitycages.

On PD 15, oral cannulae were again implanted in thecheeks of the animals. After 1 h, they were weighed andrectal temperatures were recorded (baseline measurement).Pups were then immediately placed in the activity cham-bers that were now lined with sandpaper. While over therough surface pups received four intraoral pulses of water(5 ml, pulse duration: 5 s, interpulse interval: 55 s) in eachof two consecutive 5-min trials. The intertrial intervalwas also 5 min. Activity levels were recorded during eachtrial while rectal temperatures were registered at the endof each trial. It should be noted that, relative to the proce-dures of Experiment 1, a second 5-min conditioning trialcomprising pairings between water pulses (CS1) and sand-paper (CS2) was added. The rationale for this was to ana-lyze whether conditioned effects of ethanol likely tomediate transfer of information during the SOC procedurewould change as a function of successive presentations ofCS1. It is known that while short SOC trials allow transferof information to the CS2, additional trials can result in ex-tinction of the original memory (Rescorla, 1980). Immedi-ately after the second temperature recording, animals weretransferred to the sacrifice room. Following decapitation,blood trunk samples were collected. Samples were centri-fuged at 6,000 rpm to create a plasma phase. The vials con-taining the plasma phase were stored at �15�C for lateranalysis. Corticosterone levels were obtained by means ofradioimmunoassay of the plasma samples. 3H Kits reagents(MP Biomedicals, Orangeburg, NY) were used and thevalues obtained were expressed as ng/ml.

3.1.4. Data analysisRectal temperatures (�C), locomotion scores (total num-

ber of beams crossed) and plasma corticosterone levels(ng/ml) were considered as dependent variables. Due to dif-ferent procedural manipulations inherent to each particularday (PD 14, first-order conditioning; PD 15, presentation offirst-order CS) and different time points at which data werecollected, separate analyses were conducted for each spe-cific day. Rectal temperatures on PD 14 were analyzed us-ing a three-way mixed ANOVA. Ethanol dose (0.0, 0.5, or2.0 g/kg) and postadministration time (5e20 min or30e45 min, EP or LP, respectively) served as between fac-tors. Time of rectal temperature assessment (baseline andpostadministration) was included as a within-measure fac-tor. Ethanol dose and postadministration time also servedas between factors in the ANOVA used to analyze thermalrecordings during the SOC phase (PD 15). In this particularinstance, the within-time factor comprised three repeatedmeasures (baseline and immediately after the first or secondtrial).

Overall, locomotion during baseline and during the first-order conditioning trial (PD 14) were analyzed by means of


a 3 (Ethanol dose: 0, 0.5 or 2.0 g/kg)� 2 (Postadministra-tion time, Early or Late)� 2 (Recording time: Baseline orduring conditioning). Motor scores at PD 15 were scruti-nized by means of a 3 (Ethanol dose)� 2 (Postadministra-tion Time)� 2 (Infusion trial: 1 or 2 as a within-measurefactor) ANOVA. Finally, a 3 (ethanol dose)� 2 (Postadmi-nistration Time) ANOVA served to analyze corticosteroneplasma levels at PD 15.

3.1.5. Results

3.1.5.1. Temperature responsiveness. The analysis on rec-tal temperatures during PD 14 indicated a significant maineffect of time of assessment, F(1, 60) 5 70.55, P ! .0001.The interaction between this factor and ethanol dose alsoachieved significance, F(2, 60) 5 11.60, P ! 0.0001. Posthoc tests revealed that all groups exhibited similar rectaltemperatures during baseline. In turn, all groups exhibitedwithin-differences between baseline and postadministrationrecordings. Temperatures decreased following vehicle orethanol administrations. Nevertheless, the highest ethanoldose (2.0 g/kg) resulted in significantly lower rectal temper-atures when compared to the values attained in vehicle-treated pups or those exposed to the lowest ethanol dose(0.5 g/kg). This dose effect was similar in both postadmi-nistration times (Early or Late). At PD 15, time of assess-ment exerted a significant main effect, F(2, 120) 5 3.85,P ! .05, that was tempered by a significant interaction withethanol dose, F(4, 120) 5 3.48, P ! .05. The locus of thisinteraction was analyzed by subsequent post hoc compari-sons. No differences were observed among groups during

baseline. As was the case during first-order conditioning,pups originally treated with vehicle showed a significantdecrement in rectal temperature relative to baseline valuesduring subsequent recordings. Interestingly, this was notthe case in groups that during conditioning experiencedthe water CS under the effects of ethanol. Further posthoc comparisons demonstrated that the difference betweenthermal responsiveness following CS presentations onlyachieved significance when comparing pups previouslytreated with vehicle and those administered with the highestethanol dose (2.0 g/kg). These results are depicted in Fig. 2.Since postadministration time at which intraoral infusionsof the water CS took place did not exert a significant maineffect upon thermal responsiveness nor interacted with theremaining factors, data in Fig. 2 have been collapsed acrossthis factor to facilitate visualization of the results.

In summary, thermoregulation was disrupted during con-ditioning when animals were intragastrically administeredwith ethanol, particularly when using a 2.0 g/kg dose,which resulted in a clear hypothermic response. Lesser de-grees of hypothermia were also encountered in vehicle pupsand those treated with 0.5 g/kg ethanol. In these groups, it islikely that the temperature of the solution or the fact of be-ing isolated during habituation and conditioning affectedthermoregulatory homeostasis. Mild hypothermia wasagain observed during PD 15 when vehicle pups were sim-ply exposed to the water CS. Interestingly, and despite thefact that heightened hypothermia was observed during PD14 when using 2.0 g/kg ethanol, these pups appeared tobe as resistant to thermoregulatory disruptions as vehiclecontrols during the second phase of the experiment. These

Fig. 2. Rectal temperatures (�C) during postnatal day (PD) 14 and 15 (first- and second-order conditioning phases, respectively) as a function of ethanol dose

(0.5, 2.0, or 0.0 g/kg, water) and time point of measurement (PD 14: Baseline and after conditioned stimulus exposure; PD 15: Baseline and after the first and

second conditioned stimulus exposure). To facilitate visualization, data have been collapsed across postadministration time (early or late). This factor failed to

significantly affect the pattern of results during both PD 14 and 15 and did not significantly interacted with the remaining factors. Vertical lines illustrate

standard error of the means.


particular effects of drug treatment upon unconditioned andpossible conditioned responses comprising thermoregula-tion will be analyzed in detail in Section 5.

3.1.5.2. Locomotive patterns. As illustrated in Fig. 3, itseems clear that at PD 14 motor activity was similar acrossgroups during the habituation phase. This was not the casewhen activity was scored following drug administration.The highest ethanol dose (2.0 g/kg) resulted in motor activ-ity decrements. These observations were supported by thecorresponding ANOVA which indicated a significant maineffect of time of assessment and a significant interactioncomprising this factor and ethanol dose; F(1, 60) 5 41.19and F(2, 60) 5 25.67, both P’s ! .001. As indicated bypost hoc analysis, animals treated with 2.0 g/kg ethanolshowed a significant decrease in locomotive behaviorrelative to both water-treated controls and pups that hadreceived the lowest ethanol dose (0.5 g/kg). The lattergroup also showed a lessened pattern of locomotion whencompared to vehicle controls (P 5 .08).

At PD 15, the ANOVA indicated a significant main ef-fect of infusion trial, F(1, 60) 5 15.62; P ! .0005. Specifi-cally, a decrease in motor activity was observed during thesecond infusion trial relative to the previous one, an effectprobably attributable to progressive habituation to theenvironment or to the water infusions. According to theANOVA, neither dose of ethanol received during the first-order conditioning phase (PD 14) nor postadministrationtime of the original associative process (early or late) ex-erted significant main effects upon motor activity scoresregistered on PD 15. Also, no significant interaction be-tween these factors was observed. These results have been

Fig. 3. Locomotor activity during postnatal days (PD) 14 and 15 as a func-

tion of ethanol dose (0.5, 2.0, or 0.0 g/kg, water) and time point of mea-

surement (PD 14: Baseline and First-order conditioning phase; PD 15:

Infusion Trial 1 and 2). To facilitate visualization, data have been collapsed

across postadministration time (early or late). This factor did not have a sig-

nificantly effect neither during the habituation and conditioning phases

conducted on PD 14 nor on the second-order conditioning phase (PD

15). Also, postadministration time did not significantly interacted with

the remaining factors. Vertical lines illustrate standard error of the means.

depicted in Fig. 3. Results in this figure are presented afterhaving collapsed data across postadministration time (earlyor late). As stated, this factor did not exert a significantmain effect upon locomotor patterns nor interacted withthe remaining factors.

3.1.5.3. Corticosterone levels. With regard to corticoste-rone levels after exposure to the water CS during PD 15,the ANOVA indicated no significant main effects or interac-tions between the factors under consideration. Mean levelof corticosterone across groups was 148 6 8 ng/ml,mean 6 S.E.M.

In summary, the higher dose of ethanol (2.0 g/kg) ex-erted significant depressant effects upon locomotor activitywhen acutely administered on PD 14, regardless of postad-ministration time. The lower ethanol dose also induceda somewhat depressed pattern of locomotor activity. Specif-ically, animals administered with 0.5 g/kg ethanol tended tomove less than vehicle controls. When pups were re-exposed to the water CS on PD 15, locomotive patternsand corticosterone levels were similar across conditioningtreatments.

A substantial decrease in rectal temperature was ob-served after administering a 2.0 g/kg ethanol dose. Thishypothermic response was observed at both postadministra-tion time periods. In terms of thermoregulatory process dur-ing the second phase of the experiment, pups with no priorethanol experience showed decrements in rectal tempera-ture while isolated, a response very similar to the one at-tained during first-order conditioning. Interestingly, pupsthat exhibited marked hypothermia as an unconditionedresponse to ethanol during conditioning were not observedto exhibit significant changes in thermoregulation when iso-lated and exposed to the water CS during the second phaseof the experiment. Possible explanations relative to this ef-fect will be presented in the Section 5. The thermoregula-tory effects observed in this experiment failed to varyacross trials, a result that suggests that the amount of expo-sure to CS1 was not sufficient to result in significant levelsof extinction of the memory under analysis.

4. Experiment 3

Pharmacological effects of ethanol vary as a function ofdose and postadministration time, both variables being reg-ulated by the particular genetic and housing conditions ofthe organisms under analysis (Bau et al., 2005; Cunning-ham et al., 1993). In the present experiment, we examinedBECs resulting from the ethanol doses and postadministra-tion times used in Experiments 1 and 2.


4.1.1. AnimalsFifty SpragueeDawley derived rats were used. These

animals were representative of six litters, born and reared


at the vivarium of the Center for Developmental Psychobi-ology. Housing conditions were similar to those describedin Experiments 1 and 2. Animals had 14-days of age atthe start of the experiment and weighed 27e34 g.

4.1.2. Experimental design and proceduresThe experimental design comprised two independent

factors: ethanol dose and postadministration time. Each ofthe four groups resulting from this design was composedof 12e13 pups. As was the case in prior experiments, pre-cautions were taken to avoid overrepresentation of littersand sex within each specific group. Animals received either0.5 or 2.0 g/kg of ethanol. BECs were registered at 12.5 or37.5 min after the corresponding administration, with inde-pendent groups of animals being used at each time point.These time periods were chosen since they are temporallyequidistant to the onset and offset of the early and late con-ditioning phases used in Experiments 1 and 2. Ethanoldoses were achieved by intragastrically administering0.015 ml of a 4.2 or 16.8% vol/vol ethanol solution pergram of body weight, respectively. On PD 14, infant ratswere placed in pairs in holding cages maintained at 35�Cthrough the use of heating pads placed beneath them. Pupsremained in these cages 120 min and were then randomlyassigned to one of four conditions defined by ethanol doseadministered and postadministration time when blood col-lection took place. Trunk blood was obtained through de-capitation. Samples were collected using a heparinizedcapillary tube. They were immediately centrifuged at highspeed (6,000 rpm; Micro-Haematocrit Centrifuge, Hawks-ley & Sons LTD, Sussex, England) and stored at �70�Cfor later analysis. An AM1 Alcohol Analyzer (Analox In-struments, Lunenburg, MA) was used to process the sam-ples. Calculation of BECs was made by oxidating ethanolto acetaldehyde in the presence of ethanol oxidase. The ap-paratus measures the rate of oxygen required by this pro-cess, which is proportional to ethanol concentration, andproduces a printout 20e30 s after the plasma is injected.All BECs values were expressed as milligrams of ethanolper deciliter of body fluid (mg/dl 5 mg%).

4.1.3. ResultsBECs were analyzed by means of a two-way ANOVA

(ethanol dose [0.5 or 2.0 g/kg]� postadministration time[12.5 or 37.5 min]). Significant effects of both main factorswere detected, F(1, 46) 5 636.33; F(1, 46) 5 15.19; bothP’s ! .0001. The interaction between the factors alsoachieved significance, F(1, 46) 5 15.5, P ! .0005. Ascould be expected, post hoc tests indicated that BECs de-rived from 2.0 g/kg ethanol were significantly higher thanthose obtained with 0.5 g/kg ethanol. While BECs associ-ated with the 0.5 g/kg dose remained constant across post-administration times, BECs derived from the 2.0 g/kg doseat the latter time period were significantly higher than theones corresponding to the early phase of the toxic state.These results have been depicted in Fig. 4.

5. Discussion

The main experimental finding of the present study is thatpreweanlings (PD 14e15) rats appear to be sensitive to bi-phasic motivational properties of ethanol. Specifically, sec-ond-order conditioned preferences and aversions wereestablished as a function of ethanol dose and postadministra-tion time. More in detail, after intraoral infusion of water(CS) was paired with postabsorptive effects of a relativelylow ethanol dose (0.5 g/kg), this CS acted as a second-orderappetitive reinforcer when subsequently paired with sandpa-per. This resulted in preference for sandpaper. Control ani-mals exposed to water intraoral infusions (CS) explicitlyunpaired with ethanol’s postabsorptive effects spent equiva-lent time over sandpaper and the alternative tactile cue. Thelatter results are in agreement with those originally foundby Molina et al. (2006). Interestingly, a novel finding of thisstudy is that conditioned tactile preferences were also foundin pups originally exposed to the CS while intoxicated witha higher ethanol dose (2.0 g/kg), but only if the CS was pre-sented during the initial phase of the toxic state. In contrast,second-order conditioned aversions emerged during the ini-tial intervals of testing whenever the water CS was originallyexperienced at a later stage of the intoxication induced by the2.0 g/kg dose. That is, biphasic motivational effects of thedrug are observed as a function of dose and temporal courseof the ethanol intoxication. When taking into account theblood ethanol levels associated with dose and postadminis-tration time (Experiment 3; see Fig. 4), some preliminaryconclusions can be drawn. The motivational value of low-dose ethanol remains constant across postadministrationtimes characterized by similar amounts of circulating ethanol(40 mg%). Appetitive hedonic effects seem to be also presentafter a relatively high dose of ethanol (2.0 g/kg) during the as-cending limb with BECs about 160 mg% (postadministrationtime: 12.5 min). When BECs reached approximately210 mg% (postadministration time: 37.5 min) aversive

Fig. 4. Blood ethanol concentrations (mg%) as a function of ethanol dose

(0.50 or 2.0 g/kg) and postadministration time (12.5 or 37.5 min). Vertical

lines illustrate standard errors of the means.


rather than appetitive motivational properties of ethanolseem to prevail.

Is it possible that pups in group PL/2.0 spent less timeover sandpaper not because of ethanol’s aversive effects,but rather due to sedative effects of ethanol that competewith sensory processing of the CS? This possibility seemshighly unlikely in light of prior literature. Several studiesindicate that, while experiencing the toxic effects inducedby ethanol doses equivalent to 2.0 g/kg or above, pups arecapable of processing tactile and chemosensory CSs andto associate them with different USs, including uncondi-tioned effects of ethanol (e.g., Molina et al., 1996; Pautassiet al., 2002, 2005b). In this respect, Pautassi et al. (2006)trained pups to avoid an odor CS by pairing it with an in-traoral infusion of citric acid (US). After 24 h, animals werere-exposed to the CS under the delayed toxic effects ofa 2.5 g/kg dose (postadministration interval 25e30 min).Interestingly, when later tested for their odor preferencethese animals exhibited an enhanced avoidance response.That is, delayed effects of 2.5 g/kg ethanol inflated the aver-sive value of citric acid. Obviously, this result implies thatethanol intoxication did not preclude perception of the tasteUS. Reliable odor aversive conditioning promoted by foot-shock is also detected in rat pups under the effects ofa 2.0 g/kg ethanol dose (Lopez et al., 1996).

The results obtained in this study replicate the previousfinding of Molina et al. (2006) showing positive reinforcingproperties of ethanol assessed by means of higher-order con-ditioning. The motivational profile induced by ethanol is alsoin accordance with previous experiments showing that thedrug, specifically during the early ontogeny of the rat, exertsbiphasic hedonic effects when using relatively high doses(Pautassi et al., 2002, 2006). This work also adds to a growingbody of experimental evidences indicating that neonates, in-fants, and periadolescent rats rarely develop conditionedaversions when treated with relatively low ethanol dosesand, on the contrary, seem to be highly sensitive to either ap-petitive or negative (antianxiety) reinforcing effects of etha-nol (Cheslock et al., 2001; Fernandez-Vidal et al., 2003;Molina et al., 2006; Niznikov et al., 2006; Pautassi et al.,2005a, 2005b). These effects are observed using proceduresthat require minimal experience with the drug, a phenomenonrarely encountered in heterogeneous adult animals.

Drug-induced locomotive activity has long been consid-ered as an index of reinforcing properties of substances ofabuse such as cocaine and amphetamines (Orsini et al.,2004; Wise & Bozarth, 1987). In mice, increased levelsof ethanol-induced activity have been associated with rein-forcing properties of this drug (Cunningham et al., 1993,but also see Risinger et al., 1992). In Experiment 2, sub-stantial acute motor depressant effects of ethanol wereobserved in pups administered with 2.0 g/kg ethanol,independently of postadministration time. Even thoughnot significant in statistical terms, pups treated with 0.5 g/kg ethanol also exhibited a somewhat depressed motorunconditioned response. Despite this motor profile, both

ethanol doses induced substantial appetitive learning as as-sessed through SOC (Experiment 1). Hence, these resultsindicate dissociation between ethanol-induced changes inmotor activity and reinforcing capabilities of the drug inearly infancy. These findings also fit well with previoussuggestions that although low doses of ethanol stimulatelocomotive activity in mice (Masur et al., 1986), ratstypically show a dose-dependent suppression of motoractivity (Chuck et al., 2006).

Conditioned motor responses to discrete or contextualcues are likely to be acquired during ethanol intoxication(Cunningham & Noble, 1992) and apparently can mask ex-pression of ethanol-induced reinforcement with first-orderconditioning procedures. When focusing on activity pat-terns elicited by the water CS during the second-orderphase (PD 15, dependent variable: locomotion operational-ized through number of beams crossed), there were noindications of ethanol-mediated conditioned motor re-sponses (Experiment 2). Although Molina et al. (2006)described motor conditioned responses after conditioningprocedures similar to that of the present study, the motorbehavior that proved sensitive in that study was wall climb-ing. The results of Molina et al. (2006) agreed with thepresent study in the absence of conditioned motor respond-ing in terms of overall locomotion.

Previous studies indicate that ethanol-induced hypother-mia may underlie the capability of the drug to supportlearned aversions. Drug-mediated taste aversions are dis-rupted when ethanol-induced hypothermia is attenuatedby exposing rats to high ambient temperatures (Cunning-ham et al., 1988, 1992). This experimental manipulationalso interferes with the acquisition of ethanol-induced placeaversion in rats (Cunningham & Niehus, 1988). In younganimals, the effectiveness of ethanol as an aversive USseems to be associated with the capability of the drug to de-crease body temperature (Hunt et al., 1991). Cunningham(1994) also observed that a stimulus paired with self-administered ethanol acquires the ability to evoke aconditioned increase in body temperature. Interestingly,animals subsequently self-administered more ethanol whenre-exposed to the original CS than when this stimulus wasabsent. This result suggests that the hyperthermic responsehelped counteract aversive consequences of the drug de-rived from its hypothermic consequences. In the presentstudy, high-dose ethanol (2.0 g/kg) induced substantial hy-pothermia soon after its intragastric administration. Ther-moregulatory disruptions of pups administered with 0.5 g/kg ethanol were very similar to those encountered in vehi-cle control animals. A similar doseeresponse effect in pre-weanling rats has been previously reported by Hunt et al.(1991). In the present study, a decrease in rectal tempera-ture following baseline recordings was observed in vehicleand pups administered with 0.5 g/kg ethanol, even thoughthe magnitude of this decrement was significantly lowerthan encountered with the highest ethanol dose (Experi-ment 2). This drop in temperature in vehicle controls is


probably due to the administration of water at room temper-ature (PD 14) and/or to removal of animals from theirheated holding cages and placement into chambers keptat room temperature where they remained for a relativelylong period of time (PD 14 and PD 15). Similar and evenhigher temperature decrements have been previously re-ported in vehicle-treated young animals (Hunt et al., 1991).

On PD 15, when exposed to intraoral water, control an-imals again showed a decrease in body temperature relativeto initial baseline recordings. This was not the case inethanol-treated pups, which showed relatively stable rectaltemperatures throughout the entire experimental session.This difference in thermal responsiveness after CS deliveryonly attained significance, however, when contrasting pupspreviously treated with vehicle and those given the highestethanol dose (2.0 g/kg). What mechanisms might helpexplain this differential thermoregulatory response asa function of prior drug treatment? A possibility is thatethanol-treated pups developed conditioned tolerance todisruptive thermoregulatory effects of ethanol. Duringconditioning, the water CS was presented while pups expe-rienced procedural manipulations and drug effects leadingto hypothermia. Under the assumption of conditioned toler-ance, subsequent exposure to the CS could elicit opponentthermoregulatory processes in an effort to mitigate thepredicted homeostatic disturbance. This hypothesis issupported by several reports indicating that conditionedtolerance develops to several effects of ethanol. For exam-ple, ethanol-induced ataxia is significantly attenuated whenadult rats suffer the state of intoxication under environmen-tal conditions originally associated with the drug’s motor-impairing effects (White et al., 2002). This also seems tobe the case for ethanol-induced hypothermia. Le et al.(1979) observed that chronic tolerance to ethanol-inducedhypothermia was disrupted when subjects were moved toa novel environment prior to drug administration. In thisstudy, it was also noted that the context paired withethanol-induced hypothermia subsequently elicited compen-satory conditioned responses (hyperthermia) when animals re-ceived vehicle rather than ethanol. Similar results wereobserved in later studies that incorporated more adequatecontrol conditions to assess Pavlovian associative learningmechanisms (Mansfield & Cunningham, 1980; Melchior,1990; Tirelli et al., 1992). While in these studies theputative CS was the general environmental context,ethanol-induced hyperthermic conditioned responses havealso been observed when using more discrete CSs (Cunning-ham, 1994). In these studies, repeated administrations of eth-anol were performed before assessing conditioned tolerance(e.g., Le et al., 1979), while in the present study, this phenom-enon was detected after a single ethanol administration. It isnecessary to observe that the above stated studies were con-ducted in adult rodents, organisms that possess fully devel-oped thermoregulatory capabilities. In infants, self-controlof body temperature is still developing (Leon et al., 1986).Hence, we cannot discard the possibility that rates of

development of ethanol-mediated thermoregulatory re-sponses vary as a function of maturational processes.

One possible objection to the proposed learned tolerancehypothesis is that the temporal duration of the ethanol-mediated hypothermia was too short to warrant the acquisi-tion of this learned response. This possibility is notsupported by the data obtained in Experiment 2 or by priorliterature. Specifically, the 2.0 g/kg ethanol caused a signif-icant drop in temperature not only shortly after the onset ofthe intoxication but also in those pups tested during the latepostadministration interval. Temperatures measurements inthese animals were conducted 45 min after ethanol admin-istration. Hypothermic effects of ethanol (1.6 g/kg) havebeen observed in 16-day old pups even 120 min after ad-ministration of the drug (Hunt et al., 1991). The results ofHunt et al. (1991) were obtained while using the same ratstrain and similar test conditions as those used in Experi-ment 2. These findings support the possibility that pupstested in Experiment 2 experienced ethanol-mediated hypo-thermia for a considerable amount of time.

Yet, there is an alternative nonlearning explanation re-lated with the enhanced thermal responsiveness observedduring PD 15. Specifically, it could be the case that ethanoladministration (2.0 g/kg, PD 14) induced an unconditionedchange in thermoregulatory capabilities that caused animalsto exhibit hyperthermia when tested on PD 15. This possi-bility cannot be dismissed since Experiment 2 did not in-clude unpaired control conditions. Unconditioned thermalrebound effects as a function of prior ethanol exposure havebeen previously reported in 2-week old rats (Spiers & Fus-co, 1992). It should be noted that the latter study useda much higher ethanol dose (4 g/kg, i.p.) than the one usedhere. In addition, the unconditioned rebound effect reportedby Spiers and Fusco (1992) was only observed when pupswere severely challenged in terms of thermoregulatory ca-pabilities (i.e., test was conducted at 27.5�C). This wasnot the case in the present study.

Is there a relationship between the observed thermoreg-ulatory effects of the drug and the establishment of ethanol-mediated appetitive or aversive effects? Results reported byHunt et al. (1991) suggest that drug-induced hypothermia isa necessary condition for the acquisition of ethanol-mediated aversions in preweanling rats. In the study ofHunt et al. (1991), ethanol-mediated conditioned aversionswere only observed when treated pups showed reliableethanol-induced hypothermia. In the present study, infantstreated with 2.0 g/kg during first-order conditioning didshow marked hypothermia during early and later phasesof the process of intoxication. But second-order condi-tioned aversions were only observed in those subjectsinitially exposed to the CS during the later phase of thetoxic process, a stage characterized by higher BECs. Ac-cording to these observations in conjunction with those re-ported by Hunt et al. (1991), ethanol-induced hypothermiacan represent a necessary but not a sufficient factor to pro-mote aversive learning in the developing rat. This statement

53hol 41 (2007) 41e55

receives further support when considering that pups under-going early postabsorptive effects of the 2.0 g/kg ethanoldose expressed conditioned preferences rather than aver-sions despite the fact that ethanol-induced hypothermiawas also evident in these subjects.

Ethanol administration is associated with activation of thehypothalamiceadrenalepituitary axis, which results inheightened levels of corticosterone (Spencer & McEwen,1997). The role of this hormone in ethanol reinforcement,however, is far from clear. For instance, antalarmin, a com-pound that antagonizes the type 1 corticotropin-releasingfactor receptor, has been observed to reduce oral intake ofethanol in rodents, a result not related to possible antianxietyeffects of the drug (Lodge & Lawrence, 2003). Type 1 corti-cotropin-releasing factor antagonists are also capable of miti-gating the enhanced anxiety-like behaviors associated withrepeated ethanol withdrawal episodes (Overstreet et al.,2004). On the other hand, neither acquisition nor expressionof ethanol-induced conditioned place preference in mice isaltered as a function of the administration of aminoglutethi-mide, an inhibitor of corticosterone synthesis (Chester &Cunningham, 1998). More recently, it has been observed thatknockout mice for the type 2 corticotropin-releasing factorreceptor do not show differences in a wide array of ethanol-related behaviors, including drug intake, ethanol-inducedconditioned taste aversions and hypothermia (Sharpe et al.,2005). The results obtained in Experiment 2 seem to agreewith this latter set of studies indicating that corticosterone re-sponsiveness is not strongly related with the motivational ef-fects of ethanol. Specifically, it was observed that neitherethanol dose nor postadministration time affected later corti-costerone responses to the water CS.

In summary, the results obtained in this study argue in fa-vor of infantile sensitivity to differential hedonic propertiesof ethanol. Both appetitive and aversive effects of the drugare evident as a function of dose and the time course of theacute state of intoxication. Results also indicate that pulsat-ing water infusions can acquire either appetitive or aversivevalue as a function of associative learning processes medi-ated by ethanol. The SOC procedure seems to be a valuabletool allowing detection of differential motivational proper-ties of ethanol. Further studies are needed to dissect themechanisms underlying the capability of this second-orderpreparation to detect ethanol-mediated learning. Indepen-dent from these considerations, and in agreement witha growing body of literature (for a review on this matter seeSpear & Molina, 2005), the present results indicate that evenbrief experiences with ethanol during early development aresufficient to affect subsequent responsiveness to the drugitself or to stimuli that predict its postabsorptive effects.

Acknowledgments

This work was supported by supported by grants fromthe NIAAA (AA11960, AA013098) and the NIMH

J.C. Molina et al. / Alco

(MH035219) to NES and the Agencia Nacional de Promo-cion Cientifica y Tecnologica (PICT 05-14024) to JCM.The authors wish to express their gratitude to ShwanaBawer, Naveed Sheik, and Judy Sharp for their technical as-sistance, as well as to Carlos Arias for helpful suggestionsmade during the writing of this manuscript.

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