Acute alcohol intoxication paired with appetitive reinforcement: Effects upon ethanol intake in infant rats

BEHAVIORAL AND NEURAL BIOLOGY

51, 326-345 (1989)

Acute Alcohol Intoxication Paired with Appetitive Reinforcement: Effects upon Ethanol Intake in Infant Rats JUAN CARLOS MOLINA AND MARIA GABRIELA CHOTRO 1

Instituto de lnvestigacion Medica Mercedes y Martin Ferreyra, Casilla de Correo 389, 5000--Cordoba, Argentina A recent study suggested that infant rats process alcohol odor and/or taste during acute ethanol intoxication probably dlie to ethanol elimination via respiration and salivation. The present set of experiments was meant to analyze the possibility that this orosensory processing may act as a conditioned stimulus when an appetitive reinforcer is paired with the state of intoxication. In the first experiment it was observed that intragastric administration of a mildly intoxicating ethanol dose (1.5 g/kg), paired during postabsorptive time intervals with oral infusion of sucrose, was sufficient to promote a significant preference to ethanol. In Experiment 2 different doses of ethanol were either paired or explicitly unpaired with sucrose administration. The result,reported in Experiment 1 was replicated and it was observed that a higher dose (3.0 g/kg) unpaired with the reinforcer resulted in alcohol aversions in terms of alcohol consumption patterns. However, when the reinforcer was paired with this dose, the aversion was inhibited. Finally, in the third experiment results indicated that preexposure to alcohol odor eliminates sucrose-conditioned alcohol preferences, These results indicate that, in physiologically immature rats, alcohol preference can be regulated by prior associative experiences involving the state of intoxication and consequences internal and/or inherent to this state. © 1989AcademicPress, Inc.

A series of recent studies have consistently demonstrated that preweanling rats show a remarkable capacity to associate ethanol (EtOH) odor with different reinforcers (Molina, Hoffman, & Spear, 1986a; Molina, Serwatka, & Spear, 1984, 1986b; Molina, Serwatka, Spear, & Spear, 1985; Serwatka, Molina, & Spear, 1986). Depending on the type of reinforcer paired with this sensory cue, the infant will subsequently accept i This research was supported by Grants P.I.D. 681/86 and 890/87 from Cordoba's State Council of Research (CONICOR) awarded to Juan Carlos Molina. The authors express their gratitude to Arcor Co. which provided the odorants employed in this research and to Irma Orshinger for her English assistance. Requests for reprints should be addressed to Juan Carlos Molina, Instituto Ferreyra, Casilla de Correo 389, 5000--Cordoba, Argentina. 326 0163-1047/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

ETHANOL INTOXICATION IN INFANT RATS

327

or reject ethanol solutions presented singly or in tests providing simultaneous access to alcohol and nonalcohol flavored solutions (Molina et al., 1985; Molina et al., 1986a). In one of these studies EtOH-conditioned aversions were potentiated when the organism suffered acute alcohol intoxication resulting from forced alcohol consumption (Molina et al., 1984). Furthermore, while intoxicated, the animal seems to discriminate EtOH and nonethanol odors (Molina et al., 1984; Molina, Serwatka, Enters, Spear, & Spear, 1987). On the basis of these results the question arose as to possible alcohol orosensory processing during a state of ethanol intoxication. Such a possibility is supported by the fact that about 10-12% of EtOH in rats is not metabolized but is excreted unchanged through respiratory and other nonalveolar elimination routes (salivation, perspiration, and urine) (Goldstein 1983; Hollstedt 1981). It has been previously suggested that this fraction is even higher in infant organisms due to (a) immaturity of the enzymatic systems involved in ethanol metabolism and (b) the high respiratory rate of the infant rat (Abel, 1984; Hollstedt & Rydberg, 1985; Kelly, Bonhius, & West, 1987). If t h e odor and/or taste o f alcohol are sensed during postabsorptive periods, then after being intoxicated, the rat will subsequently recognize these sensory cues. Recently this speculation received experimental support in a series of studies performed with preweanling rats. In these studies, it was noted that after suffering a highly intoxicating intragastric ethanol dose, rat pups consumed less alcohol and avoided EtOH odor. Moreover, when a lower dose, insufficient to promote such aversive responses, was paired with nociceptive electrical stimulation, a similar pattern of ethanol acceptance performance was also displayed (Chotro, 1987; Molina, Chotro, & Spear, in press). Apparently, sensorial cues arising from EtOH's direct elimination processes were paired with the electrical stimulation present during the postabsorptive state. If the suggested associative process takes place during acute alcohol intoxication, appetitive reinforcers present at postadministration times should promote ethanol acceptance rather than ethanol rejection behaviors. In other words, it could be assumed that direct elimination represents a conditioned sensorial stimulus capable of being associated with positive reinforcement. In turn, this type of pairing should increase subsequent ingestion of the drug. This hypothesis was investigated in a series of experiments in which rat pups were administered EtOH intragastrically and 30-60 min later received oral infusion of a sweet solution. Sucrose was employed as a reinforcer because even at very early ages the rat pup exhibits a high preference toward this flavor (Johanson & Shapiro, 1983; Rozin & Zellner, 1985; Spear & Kucharski, 1984). In addition, young pups display odor-conditioned responses when a flavored solution is employed as an unconditioned stimulus (Molina & Spear, in preparation).

328

MOLINA AND CHOTRO EXPERIMENT 1

The aim of the present experiment was to study ethanol acceptance patterns in I 1-day-old pups as a function of a prior episode in which alcohol intragastric administration was followed by intraoral infusion of a sucrose solution. EtOH was delivered using intragastric intubation in order to avoid explicit exposure to the taste and/or odor of the drug (Waller, McBride, Gatto, Lumeng, & Li, 1984). A 1.5 g/kg EtOH dose was used since this dose fails to alter subsequent preference toward the orosensory cues of EtOH. Nevertheless, and as previously demonstrated, if 30-60 min after drug treatment a shock is delivered, the animal later expresses aversion toward alcohol (Chotro, 1987; Molina et al., in press). Therefore, it appears that even if the dose does not change EtOH preference, it represents a dose sufficient to provide EtOH-related sensorial cues which can be associated with aversive shock. This dose yields blood alcohol levels which peak (120-140 mg%) 30 min after administration and remain stable for 60-90 min (Molina et al., 1987, in press). Similar findings have been reported for pups of a different strain receiving a slightly lower dose (1.25 g/kg) (Hollstedt, Olsson, & Rydberg, 1980). On the basis of these ethanol elimination parameters, it was decided to apply the positive reinforcer during a postadministration interval characterized by peak and stable blood alcohol concentrations since there exists a positive correlation between this index and respiratory elimination of the drug (Goldstein, 1983). Following the associative procedure, EtOH ingestion and EtOH odor preference were assessed.

Methods Subjects. Forty-five 11-day-old Wistar-derived pups were used in this experiment. These infants were representative of 7 litters originally composed by 8-10 pups/litter. All animals were born and reared at the Instituto Ferreyra. At birth (postnatal Day 1) pups were housed with their biological parents in standard opaque maternity cages partially filled with wood shavings and equipped with automatic water dispenser valves. Parents had free access to rat chow (ADIABIC). All animals were maintained on a 14-h light/10-h dark cycle where light onset occurred at 0700 h. Temperature in the colony room ranged between 22 and 25°C. Taking into account normal weight development of the pups, an infant was only included in the experiment if by postnatal Day 11 its overall body weight was over 15 g. Apparatus and procedures. At l l days of age the pups were randomly assigned to one of four groups defined by Drug Treatment (EtOH, 1.5 g/kg or Vehicle, 0.0 g/kg) and Sucrose Pairing Procedure (Paired, P or Unpaired, UP). The number of pups assigned to each group was as

ETHANOL INTOXICATION IN INFANT RATS

329

follows: EtOH-Paired, n = 11; EtOH-Unpaired, n = 12; Vehicle-Paired, n = 10; and Vehicle-Unpaired, n = 12. Animals were then placed in standard maternity cages in accordance with treatment condition without the presence of biological parents. The temperature in these holding devices was kept at 32-34°C through the use of adjustable heating pads. Following this procedure, pups were orally cannulated using a method similar to that previously described (Chotro, 1987; Gemberling & Domjan, 1981; Gemberling, Domjan, & Amsel, 1980; Hall & Rosenblatt, 1977; Molina et al., in press; Vogt & Rudy, 1984). Cannulated pups were returned to their respective holding environments where they remained deprived of maternal care for 4 h. These oral cannulae were then used for sucrose delivery procedures which took place in individual Plexiglas chambers (15 x 7 x 15 cm) equipped with grid floors. Unpaired pups received sucrose infusion 1 h prior to EtOH or vehicle administration. Paired pups received the infusion 30-60 min after intragastric administration of alcohol or vehicle. Sucrose infusion was performed via a 10-channel peristaltic infusion pump (Manostat Cassette Pump, standard model). Each channel was equipped with polyethylene tubing (internal diameter 0.5 mm). This tubing was attached to the oral cannulae of the pups. A sucrose solution (10% w/v, vehicle: tap water) at room temperature was delivered through the cannulae using pulsating administration for 30 consecutive min. Each pulse had a duration of 3 s (interval between pulses: 10 s). The pump was set to deliver the solution at a rate of 0.096 ml/min. Intragastric intubation was performed using polyethylene tubing (ClayAdams PE 10) carefully placed in the mouth of the infant and gently pushed until the free end of the cannula was inserted approximately 3.0 cm beyond the infant's tongue and into its stomach. This procedure normally took less than 20 s. Prior to administration, and in order to avoid explicit taste or odor stimulation during the intragastric procedure, the external portion of the cannula was paper towel dried. The EtOH dose (1.5 g/kg) was achieved by administering 0.015 ml/g body weight of a 12.6% (v/v) alcohol solution. Vehicle (tap water)-treated pups received similar volumes of administration as did EtOH-treated rats. Once pups had been administered vehicle or alcohol as well as sucrose, they were returned to their holding cages where they remained for 24 h without maternal care. At 12 days of age an ethanol intake test was performed. Pups were individually placed in Plexiglas chambers (15 x 7 x 15 cm) equipped with grid floors. The oral cannulae were attached to a polyethylene tubing exiting from the peristaltic infusion pump. A 6.0% EtOH solution was delivered during three trials (trial duration, 150 s; interval between trials, 60 s). The infusion rate was 0.096 ml/min. The total amount of the infused EtOH solution represented approximately 5.5% of the preinfusion body weights of the pups. Percentage body weight

330

MOLINA AND CHOTRO

gain [100 × (postinfusion weight - preinfusion weight/preinfusion weight)] during the infusion test represented the dependent variable under consideration. All body weights were registered with an error of 0.01 g. Prior to testing, the preweanling's bladders were voided and defecation was stimulated by stroking the anogenital region with cotton swabs. After performing the intake test, oral cannulae were removed and pups were returned to their parents. Twertty four hours later, all pups received an olfactory preference test. This test was conducted in a clear Plexiglas chamber (33 x 14 × 15 cm.) equipped with a stainless-steel grid floor. This apparatus was divided into two equal sections. Cotton scented with 5.0 cc of alcohol (190 proof EtOH) or 5.0 cc of a 0.1% (v/v) nonethanol lemon extract solution (Arcor Co.) was placed 2.0 cm beneath the grid floor and proximal to either end section of the chamber. Testing began when the infant was individually placed in the center of the grid. Time spent over each section of the experimental environment was recorded during 180 s. A rat was considered to be over the lemon- or alcoholscented section whenever its nose and front paws were above it. Untreated 11-day-old rats exhibit similar preference for both odorants under the above-described experimental setting (Molina, unpublished results). Total time spent over the EtOH odor was recorded for statistical analysis. Results and Discussion Ethanol infusion test. All groups exhibited similar loss in body weight between conditioning and testing day. This null effect was confirmed by a two-way (Drug Treatment x Pairing Procedure) analysis of variance (ANOVA) which failed to reveal significant differences attributable either to the main factors under consideration or to the interaction between such factors (all p's > . 1). Overall percentage body weight loss between 11 and 12 days of age was 8.27 + 0.25%; mean _+ standard error. Percentage body weight gain during the EtOH consumption test has been illustrated in Fig. 1. As can be observed, animals from groups Vehicle-Paired and -Unpaired and EtOH-Unpaired exhibited similar increases in body weight as a result of the infusion. The figure also shows that animals assigned to group EtOH-Paired evidenced higher intake scores. These statiscally descriptive observations were confirmed by a two-way ANOVA (Drug x Pairing) as well as by post hoc comparisons (Fisher's least significance difference test, p < .05) (Keppel, 1982). The ANOVA revealed a significant effect of drug treatment and pairing procedure, F(1, 41) = 14.51, p < .001 and F(1, 41) = 9.64, p < .005. In turn, the main effects were tempered by a significant interaction between the factors under study, F(1, 41) = 10.66, p < .005. Subsequent post hoc comparisons showed that EtOH-Paired pups exhibited a significantly higher intake score when compared to EtOH-Unpaired rats. In turn,

ETHANOL INTOXICATION IN INFANT RATS

331

haired • Poired

o~

z (D I-I kl.I >D 0 m

1

0 0.0

1.5 Et OH DOSE (g/Kg)

Fio. 1. Ethanol ingestion (percentage body weight gain) as a function of ethanol dose (0.0 or 1.5 g/kg) and treatment (paired or unpaired with sucrose delivery). Vertical bars represent standard errors of the mean. group EtOH-Paired also differed significantly from Vehicle-Paired and Unpaired groups. Furthermore, no significant differences were recorded when comparing scores of Vehicle-Paired, Vehicle-Unpaired, and EtOHUnpaired infants. According to these results, temporal parameters involving alcohol and sucrose delivery were responsible for the significant effects observed during the alcohol intake test. Specifically, if the reinforcer (sucrose) was presented during a post-EtOH administration interval, the pup later exhibited higher alcohol consumption values than those observed when the drug was administered following sucrose delivery. Furthermore, and in accordance with prior results (Chotro, 1987; Molina et al., in press), it is clear that alcohol administration of a dose equivalent to 1.5 g/kg fails to affect later alcohol intake behavior. Indeed, animals treated with this dose, which was unpaired with sucrose delivery, exhibited similar body weight gain as did pups treated with vehicle. Thus, it appears that the critical circumstance that promoted changes in a forced alcohol intake test was the temporal arrangement existing between alcohol administration and delivery of an appetitive reinforcer. EtOH odor preference test. Time spent over EtOH odor is shown in Fig. 2. Results closely paralleled those reported in the intake test. Once again, infants assigned to group EtOH-Paired exhibited higher scores in terms of EtOH acceptance as compared with the remaining groups. A two-way ANOVA and Fisher's post hoc comparisons confirmed this interpretation. The type of pairing procedure exerted a significant effect while drug treatment approached significance, F(1, 41) = 5.14, p < .05

332

MOLINA AND CHOTRO

~ 150

[] Unpeired • Poired

120 ne" 0 Z

W I--

0.0

1.5 Et OH DOSE (g/Kg)

FI~. 2. T i m e spent over ethanol odor (s) as a function of ethanol dose (0.0 or 1.5 g/kg) and t r e a t m e n t (paired or unpaired with s u c r o s e delivery). Vertical bars represent standard errors of the m e a n .

and F(1, 41) = 3.76, .10 < p < .05, respectively. The ANOVA also revealed a significant effect of the interaction under analysis, F(1, 41) = 5.75, p < .025. Fisher tests (p < .05) demonstrated that pups treated with alcohol paired with sucrose spent more time over alcohol odor than pups which received alcohol unpaired with sucrose or pups that received vehicle administration paired or unpaired with sucrose delivery. No significant differences were found among these last three groups. These results suggest that a mildly intoxicating alcohol dose (1.5 g/kg) fails to modify subsequent ethanol odor preference. Nevertheless, when administration of this dose is followed by sucrose infusion, the infant later exhibits a marked preference for this sensory cue. This effect is contrary to the one previously reported when, following alcohol administration, nociceptive stimulation was delivered to pups of a similar age (Chotro, 1987; Molina et al., in press). It is necessary to observe that sucrose consumption during conditioning day was not assessed. Therefore, no empirical data allow rejecting the possibility that differential alcohol preference across groups simply obeyed to previous differential consumption of the reinforcer. In the following experiment sucrose intake was recorded during conditioning trials. EXPERIMENT 2

In order to clarify different aspects regarding subsequent changes in alcohol consumption, different doses of EtOH, including those employed in Experiment 1, were either paired or unpaired with sucrose delivery.

ETHANOL INTOXICATION IN INFANT RATS

333

Independently from replication purposes and dose-response considerations, the present experiment also pursued the goal of assessing sucrose consumption for the different treatment conditions. A 3.0 g/kg EtOH dose has been reported to act as an aversive unconditioned stimulus in taste aversion paradigms (Berman & Cannon, 1974; Chotro, 1987; Lester, Nachman, & Le Magnen, 1970). This dose was included in the present study in order to analyze the interaction among the seemingly aversive nature of such a dose with the administration of an appetitive reinforcer. It was previously suggested that EtOH smell and taste avoidance responses resulting from intragastric administration of 3.0 g/kg were probably due to an association between sensory cues arising from EtOH direct elimination and interoceptive aversive effects of such dose (Chotro, 1987). Under this assumption, it could be expected that during postabsorptive intervals, the presence of an appetitive reinforcer could compete with the aversive effects of the drug. Two other doses, 0.375 and 0.75 g/kg, were also used in order to determine whether the results previously reported (Experiment 1) are limited by the amount of alcohol administered.

Methods Subjects. The subjects were 79 male and female 11-day-old Wistarderived rats. Infants were representative of 11 litters (litter size = 811 pups). Housing and rearing conditions for these subjects were similar to those described in the previous experiment. Apparatus and procedures. At 11 days of age pups were randomly distributed in 10 groups defined by the following factors: EtOH dose (0.00, 0.375, 0.75, 1.50, or 3.00 g/kg) and Pairing Procedure (Dose Paired with Sucrose, P; Dose Unpaired with Sucrose, UP). The number of subjects assigned to each particular group ranged from 7 to 9 pups. Cannulation, sucrose infusion, and intragastric intubation procedures during conditioning day replicated those described in Experiment 1 with the sole exception that body weight gain attributable to sucrose infusion was registered [100 x (sucrose preinfusion weight - sucrose postinfusion weight)/sucrose preinfusion weight]. EtOH doses were achieved by administering 0.015 ml/g of the following alcohol solutions in tap water: 3.15% (v/v) (0.375 g/kg); 6.30% (v/v) (0.75 g/kg); 12.6% (v/v) (1.50 g/kg); and 25.2% (v/v) (3.0 g/kg). Animals treated with 0.0 g/kg received similar volumes of tap water. As in the first experiment, paired subjects experienced sucrose infusion 30-60 min after intragastric administration. Unpaired animals were exposed to sucrose infusion 60 min before receiving the corresponding drug treatment. EtOH intake was assessed using similar devices and procedures to those previously described. Percentage body weight gain as a function of alcohol infusion served as the dependent variable under analysis. In

334

MOLINA AND CHOTRO TABLE 1 Sucrose Intake (Percentage Body Weight Gain) Pairing procedure

EtOH dose (g/kg)

Unpaired

0.00 0.37 0.75 1.50 3.00

3.19 ± 0.33" 3.37 --+ 0.56 2.93 -+ 0.60 3.61 - 0.26 3.32 ± 0.43

Paired 3.13 3.75 3.26 2.87 1.88

± 0.69 ± 0.56 -+ 0.44 - 0.57 ± 0.68

Values representmean percentages _+ standard error.

this experiment odor preference tests were not performed. This test was avoided since the odorants normally employed were found to be degraded probably due to expiration dates. In studies performed independently from the present, preferences varied as time elapsed (Molina, unpublished observations). Further technical advice from the company that supplied the chemical cues confirmed our impressions. Results and Discussion

A two-way ANOVA (EtOH Dose x Pairing Procedure) indicated that relative body weight gain did not differ as a function of sucrose delivery. It should be noted that 3.0-P pups appeared to be highly intoxicated while receiving sucrose and exhibited a considerable lower relative increase in body weight when compared with any of the remaining groups. When sucrose was delivered into their mouths, these pups, although behaviorally characterized by an observable degree of ataxia, exhibited similar tongue and lip movements to those observed in the remaining treatment conditions. Sucrose consumption expressed through relative changes in body weight for the different groups is shown in Table 1. Relative body weight loss between 11 and 12 days of age was similar for all treatment conditions. The corresponding ANOVA failed to indicate significant differences as a consequence of dosage, pairing procedure, or the interaction between these factors. Overall percentage weight loss was equivalent to 8.33 + 0.21%, X +- SE. Changes in body weight due to EtOH infusion at postnatal Day 12 were significantly affected by the interaction between dose and pairing procedure (two-way ANOVA: F(4, 69) = 3.28, p < .025). Post hoc comparisons (Fisher's least significance difference test with a probability Type I error set at 0.05) were performed in order to analyze the locus of such interaction. These tests revealed that pups receiving the 1.5 or

ETHANOL INTOXICATION IN INFANT RATS

A

o~ v

o--o Unpaired Paired

/ ~

335

I

Z w.-i <( D I.--

"3£.9 >£3

O rn L

0U00

i

037

I

~

1.50 EtOH DOSE (g/Kg) (175

i

3.00

Fro. 3. Ethanol ingestion (percentage body weight gain) as a function of ethanol dose (0.00, 0.37, 0.75, 1.50, or 3.0 g/kg) and treatment (paired or unpaired with sucrose delivery). Vertical bars represent standard errors of the mean.

the 3.0 g/kg dose paired with sucrose delivery consumed significantly more alcohol than their respective controls (1.5-UP and 3.0-UP). Animals assigned to groups 0.0-P, 0.37-P, and 0.75-P failed to differ from their respective control groups. Furthermore, group 1.5-P exhibited significantly higher values than those observed in animals receiving lower doses (0.0P, 0.37-P, and 0.75-P). On the other hand, animals assigned to the abovementioned group (1.5-P) exhibited similar consumption patterns to those recorded in group 3.0-P. In turn, pups which experienced the highest dose unpaired with sucrose infusion (3.0-UP) were found to exhibit significantly lower body weight gains than pups assigned to groups 0.0-UP, 0.37-UP, and 0.75-UP. Results registered during the EtOH infusion test have been illustrated in Fig. 3. The present results replicate those reported in Experiment 1. Once again when the 1.5 g/kg EtOH dose was paired with an appetitive reinforcer, subjects exhibited higher intake scores than those receiving similar dose unpaired with the reinforcer or vehicle paired or unpaired with sucrose delivery. Additionally, when considering sucrose ingestion at 11 days of age, it appears that higher EtOH intake is not attributable to differential consumption of the reinforcer which might in turn affect later intake patterns during EtOH oral administration. As previously described, with the sole exception of group 3.0-P, all animals consumed similar quantities of sucrose. It is also interesting to observe that subjects exposed to highly intoxicating doses (3.0 g/kg) unpaired with the reinforcer evidenced lower intake values than those reported for groups treated with vehicle, 0.37 and 0.75

336

MOLINA AND CHOTRO

g/kg EtOH doses. In a previous study it was observed that the administration of this high dose (3.0 g/kg) was sufficient to promote aversive responses toward alcohol odor and taste (Chotro, 1987). In turn, according to the relative high intake exhibited by group 3.0-P, it appears that the presence of a positi~,e reinforcer during postabsorptive intervals is sufficient to partially reverse the aversive consequence inherent to this dose. The guiding hypothesis for this experiment and the former one is supported by results obstained with doses higher than or equal to 1.5 g/kg. Apparently the organism is capable of establishing associations between elements present during acute alcohol intoxication. As previously suggested, EtOH orosensory processing can occur during postabsorptive periods due to direct elimination of the drug, via for example respiration and salivation. If during such processing an appetitive stimulus is present, the organism later expresses a preference toward a solution containing the drug. If the dose administered is sufficient to promote aversive conditioned responses in different learning paradigms (Cappell & LeBlanc, 1977; Cappell, LeBlanc, & Endrenyl, 1973; Chotro, 1987; Gamzu, Vincent, & Boff, 1985; Riley & Tuck, 1985; Sherman, Hickis, Rice, Rusiniak, & Garcia, 1983) the infant will later reject an EtOH solution. This rejection is blocked if the dose has been accompanied by delivery of a sweet reinforcer. Taken as a whole the results seem to validate associative processes occurring during postabsortive intervals. Such processes seem to involve EtOH's inherent effects as well as other stimuli (for example sucrose) present at the time of intoxication. If the proposed associative process occurs during intoxication, then it should be expected that prior experience with the sensory cues of EtOH (odor and/or taste) that apparently act as conditioned stimuli will affect the strength of the conditioned response under consideration. This possibility guided the design of the following experiment. EXPERIMENT 3

It has been repeatedly demonstrated that preexposure to a neutral or conditioned stimulus attenuates subsequent conditioning toward it (Lubow, 1973). The CS preexposure effect has been observed in studies related with learned associations in which an olfactory and/or gustatory cue is employed as a conditioned stimulus (Domjan, 1980, 1983; Kalat & Rozin, 1973; Misanim, Guanowsky, & Riccio, 1983; Nachman, Rauschemberger, & Asher, 1977). This effect has been reported not only in adult organisms but also in pups as young as 2 and 8 days of age (Rudy & Cheatle, 1977, 1979, 1983; Vogt & Rudy, 1984). In the present experiment pups were preexposed to alcohol odor. Subsequently, these organisms experienced alcohol intragastric administration followed by sucrose delivery. The experimental purpose of this associative arrangement was to examine whether unreinforced exposure

ETHANOL INTOXICATION IN INFANT RATS

337

to alcohol odor could affect the high consumption that results from paired experiences involving alcohol administration and delivery of an appetitive reinforcer (Experiments 1 and 2). As previously suggested and on the basis of the drug's direct elimination process, the orosensory cues of alcohol were suspected to act as a conditioned stimulus in an associative process where sucrose represented the: reinforcer. The 1.5 g/kg EtOH dose was selected on the basis of the high intake scores achieved by subjects that received paired administration of sucrose. Alcohol odor was used as the preexposed stimulus since prior studies demonstrated that EtOH odor and taste share similar sensorial and/or functional importance for the physiologically immature rat (Molina et al., 1986a). Furthermore, acute ambient odor stimulation was used in order to avoid other forms of ethanol sensory stimulation, such as direct taste perception through oral infusion techniques, where alcohol intoxication may result from drug intake.

Methods Subjects. Subjects were 53 11-day-old male and female Wistar-derived rats representative from 7 litters (litter size = 8-11 pups). Housing and rearing conditions for these subjects were similar to those previously reported. Apparatus and procedures. Pups were randomly distributed in four groups defined by Drug Treatment (Vehicle, 0.0 g/kg or EtOH, 1.5 g/kg) and Odor Preexposure Procedure (Preexposed, P; Nonpreexposed, NP). The number of infants assigned to each particular group was as follows: 0.0-NP, n = 12; 0.0-P, n = 13; 1.5-NP, n = 14; and 1.5-P, n = 14. As in the previous experiments, pups were orally cannulated and placed in holding chambers in accordance with treatment condition. Preexposure treatments were performed 30 min prior to intragastric administration. Such treatments took place in individual Plexiglas chambers (15 x 7 x 15 cm) placed over stainless-steel grid floors. Unpreexposed subjects (0.0-NP and 1.5-NP) were placed in these chambers where they remained for 30 min. Cotton unscented with odorants was placed 2-3 cm beneath the grid floors. Preexposed pups were treated in a similar manner except that cotton was scented with EtOH (1.5 cc of 190 proof alcohol). Immediately after preexposure treatment, pups were removed from the chambers and administered vehicle or alcohol. Thirty minutes later sucrose was delivered into their mouths. Intragastric intubation and sucrose infusion procedures were performed using similar devices and parameters as those described in Experiment 1 and groups 0.0-P and 1.5-P of Experiment 2. Body weight gain due to sucrose consumption was registered. Twenty four hours later, an EtOH intake test, similar to those described in the previous experiments, was performed. Percentage body weight gain was individually recorded.

338

MOLINA AND CHOTRO

xp0sed .~d z

[ ,

I--

-I-

I

UJ

>O

o

i

m

!

I 0.0

1.5 Et OH DOSE (g/Kg)

FIG. 4. Ethanol ingestion (percentage body weight gain) as a function of ethanol dose (0.0 or 1.5 g/kg) and treatment (preexposed or nonpreexposed to ethanol odor). Vertical bars represent standard errors of the mean.

Results and Discussion

A two-way ANOVA failed to indicate differences in sucrose intake as a function of the main factors under study or the interaction between them (all p's > .1). Similar null effects were obtained when processing percentage body weight loss between 11 and 12 days of age. Of major importance for the present experiment were the results attained in the alcohol drinking test which are illustrated in Fig. 4. As can be noted, group 1.5-NP exhibited a higher percentage body weight gain when compared to the remaining treatments. Interestingly, alcohol odor preexposure per se exerted an effect upon subsequent EtOH consumption. Subjects which experienced alcohol odor ingested somewhat higher amounts of EtOH solution than pups which were unexposed to this cue and treated with vehicle. Yet, in terms of percentage body weight gain, odor preexposure effects were lower than those resulting from pairing EtOH intoxication and sucrose delivery. Furthermore, when comparing the scores of groups 1.5-NP and 1.5-P, it seems that odor preexposure tends to inhibit partially the effect induced following EtOH-sucrose associations. These assumptions were confirmed by the corresponding statistical analysis. A two-way ANOVA revealed significant dose effects tempered by an interaction with preexposure procedures, F(1, 49) = 5.56, p < .025 and F(1, 49) = 8.23, p < .01. Fisher's post hoc comparisons (p < .05) showed that 1.5-NP preweanlings gained significantly higher weights than unpreexposed pups treated with vehicle (0.0-NP). Treated pups (1.5-

ETHANOL INTOXICATION IN INFANT RATS

339

NP) also exhibited significantly higher intake scores than those observed in animals treated with a similar dose but previously exposed to EtOH ambient odor stimulation (1.5@). In turn, preexposed pups treated with vehicle (0.0-P) exhibited significantly higher intake scores than those recorded in nonpreexposed pups receiving similar drug treatment (0.0NP). Finally, no statistical difference was obtained when comparing relative body weight gains of groups 0.0-P and 1.5-P. As was the case with the first two experiments, pups which experienced the alcohol dose paired with the reinforcer exhibited higher intake rates than their counterparts. This preference appears to be partially inhibited when the organism previously experiences unreinforced presentation of alcohol odor. This effect occurs even when odor preexposure per se represents a factor which promotes higher intake values. Such odor preexposure outcome has been previously reported for older preweanling rats (Molina et al., 1984). Furthermore, recent research has also demonstrated olfactory preexposure effects ifi terms of heightened preference toward the experienced sensory cue using different types of assessment paradigms (Fillion & Blass, 1986; Galef, 1981; Leon, 1987; Schwarze & Muller, 1971), ontogenetic stages (Caza & Spear, 1984; Galef & Kaner, 1980; Goldblatt, 1978; Leon, Galef, & Behse, 1972; Rudy & Cheatle, 1979), or different rodent species (Brunjes & Alberts, 1979; Carter & Marr, 1970; Cornwell, 1970; Cooper & Hathorn, 1977).

GENERAL DISCUSSION High intake and/or EtOH odor preference resulting from intragastric administration of a 1.5 g/kg EtOH dose followed by sucrose delivery represents a common denominator in the present experiments. As observed in Experiment 1 and replicated in the next two experiments, when sucrose was delivered 30-60 min after administration of the above-mentioned EtOH dose, animals subsequently exhibited heightened EtOH intake scores. Similar heightened scores were also recorded in tests based on alcohol odor locational preference (Experiment 1). Evidence was also obtained suggesting that this dose represents a threshold or suprathreshold value in the establishment of heightened EtOH intake. Lower doses (0.37 and 0.75 g/kg), although paired with the appetitive reinforcer, failed to induce changes in EtOH's intake patterns. Results also suggest that a 3.0 g/kg dose is sufficient to induce changes in alcohol intake tests. If this dose is unpaired with sucrose, pups display aversions when assessing alcohol consumption. This dose-dependent aversive behavior appears to be inhibited when sucrose delivery follows drug administration (Experiment 2). The results of the third experiment also indicate that acute unreinforced experience with alcohol odor partially blocks heightened alcohol intake scores attributable to associative processes involving a 1.5 g/kg dose and sucrose administration.

340

MOLINA AND CHOTRO

The above-described results appear unrelated to differential sucrose consumption during conditioning day. Pups treated with different EtOH doses and different pairing procedures failed to differ in terms of sucrose intake (Experiments 2 and 3). In only one experimental circumstance, depressed sucrose intake scores were observed. Specifically, when sucrose was infused after administration of a 3.0 g/kg EtOH dose, organisms gain less weight than pups treated with lower alcohol doses. These organisms, even when exhibiting clear behavioral signs of alcohol intoxication, also seemed to react to the sweet taste of sucrose during infusion procedures. Subsequently, such infants expressed enhanced EtOH intake when compared with pups receiving a similar EtOH dose unpaired with sucrose. What is more, alcohol intake scores of these animals were statistically similar to those obtained in groups which consistently exhibited heightened alcohol acceptance due to pairings between a 1.5 g/kg dose and a sucrose infusion. Therefore, sucrose reinforcing properties seem related to its taste rather than to liquid intake caused by prior fluid deprivation. Body weight loss between conditioning and testing days was found to be very similar across treatments. In the three experiments, all groups displayed similar weight loss resulting from maternal deprivation. Thus, this variable does not seem to play a significant role in terms of treatment differences in alcohol intake tests. This interpretation is also supported by results attained in an EtOH odor preference test performed with pups which were maternally undeprived prior to such assessment (Experiment 1). This circumstance probably attenuated motivational factors related to liquid and/or nutritional deprivation. Results suggest that associative processes may occur while the organism is under the effects of alcohol. Cues derived from alcohol administration seem to act as conditioned stimuli which are paired with hedonically positive attributes of sucrose. These cues could possibly be related with alcohol elimination processes. EtOH present in exhaled air or saliva could represent orosensory stimulation for the organism under study. In turn, these cues can be associated with other events which occur during postadministration intervals. In these studies, sucrose presentation prior to EtOH administration failed to affect later alcohol ingestion or alcohol odor preference. If this reinforcer was administered at a postadministration interval characterized by high and stable blood alcohol concentration, animals later expressed preferences in both behavioral assessments. It is interesting to observe that blood alcohol concentrations correlate positively with the amount of EtOH directly excreted by the intoxicated subject. The plausible associative phenomenon is also supported by prior results showing that when the intoxicated pup is exposed to peripheral nociceptive stimulation, it will later avoid alcohol olfactory cues and EtOH consumption

ETHANOL INTOXICATION IN INFANT RATS

341

(Chotro, 1987; Molina et al., in press). A similar outcome has been observed after rats suffer highly intoxicating doses (Chotro, 1987; Molina et al., 1984). Such was the case of group 3.0-NP during Experiment 2. Under this circumstance, and accepting the associative hypothesis, postabsorptive consequences seem to provide both conditioned and unconditioned stimuli. Relative to EtOH's unconditioned property, it has been consistently demonstrated that flavors are avoided after being paired with highly intoxicating EtOH doses (Cappell & LeBlanc, 1977; Riley & Tuck, 1985). Even when accepting such an associative phenomenon, the nature of the conditioned stimulus might still be questioned. Pharmacologically defined contextual learning studies indicate that alcohol intoxication can act as a discriminative cue regulating expression of memories (Cunningham, 1979; Holloway, 1972; Ryback, 1969). From this view, it might be argued that the internal context defined by alcohol becomes associated with reinforcers present at postadministration intervals. Recreation of this context through subsequent intake of the drug might then promote behavioral changes in order to sustain or avoid such context, depending on the hedonic nature of the original association. Nevertheless, this interpretation is weakened when taking into account conditioned preferences observed in odor preference tests (Experiment 1). Recreation of a toxic context is difficult to conceive due to the brief duration of such assessments and its noningestional nature. As previously stated, EtOH olfactory preexposure was sufficient to inhibit heightened alcohol intake which resulted from intragastric alcohol administration paired with sucrose (Experiment 3). This result appears to partially validate the proposed orosensory nature of the conditioned stimulus paired with sucrose. If the organism perceives alcohol odor and/or taste during intoxication, then it follows that prior exposure to such cues would affect the strength of the conditioning procedure. Such preexposure effect has been consistently observed in both infants and adults using nonethanol stimuli (Domjan, 1973; Kalat & Rozin, 1973; Logue, 1979; Misanim et al., 1983; Rescorla, 1980; Rudy & Cheatle, 1979, 1983). In Experiment 3 it was also noted that alcohol odor preexposure per se increased later alcohol intake. This effect has also been reported for older rats when using different testing procedures (Molina et al., 1984, 1986b). If preexposure exerts such an effect, then orosensory perception of alcohol during EtOH's postadministration intervals should also promote heightened acceptance patterns. In this study, such effect was not observed under circumstances in which the effects of the drug were unpaired with the reinforcer. Accepting that alcohol direct elimination provides odor and/or taste cues, the absence of a preexposure effect could be related with parametric properties of the perceived stimulus (intensity, duration,

342

MOLINA AND CHOTRO

etc.). From this viewpoint it is difficult to compare effects resulting from expficit manipulation of alcohol odor versus those resulting from perception of a similar cue related with direct elimination of EtOH. Furthermore, during postabsorptive intervals the organism is probably exposed to other nonsensory effects of alcohol that may define a complex internal stimulus. These findings suggest that alcohol preference can be regulated by the pairing of intoxication and external stimuli. It is necessary to point out that these results have been recorded in infant animals. It is difficult to predict what would happen in older subjects under similar circumstances. Such difficulty arises from ontogenetic changes in EtOH pharmacokinetics (Abel, 1984; Hollstedt, 1981; Kelly et al., 1987), learning and retention capacities (Campbell & Spear, 1979; Spear & Kucharski, 1984; Spear & Smith, 1978), functional importance and transfer of olfactory and gustatory information (Spear & Molina, 1987), as well as central nervous sensitivity to alcohol (Hollstedt et al., 1980; Hollstedt & Rydberg, 1985). REFERENCES Abel, E. L. (1984). Fetal alcohol syndrome and fetal alcohol effects. New York: Plenum. Berman, R., & Cannon, D. (1974). The effect of prior ethanol experience on ethanolinduced saccharin aversions. Physiology and Behavior, 12, 1041-1044. Bronstein, P., & Crokett, D. (1976). Exposure to the odor of food determines the eating preferences of rat pups. Behavioral Biology, 18, 387-392. Brunjes, P., & Alberts, J. (1979). Olfactory stimulation induces filial preferences for huddling in rat pups. Journal of Comparative and Physiological Psychology, 93, 548-555. Campbell, B., & Spear, N. E. (Eds.). (1979). Ontogeny of learning and memory. Hillsdale, NJ: Erlbaum. Cappell, H., & LeBlanc, A. (1977). Gustatory avoidance conditioning by drugs of abuse. N. Milgram, L. Krames, & T. Alloway (Eds.), Food aversion learning (pp. 133,167). New York: Plenum. Cappell, H., LeBlanc, A., & Endrenyi, L. (1973). Aversive conditioning by psychoactive drugs: Effects of morphine, alcohol and chlordiazepoxide. Psychopharmaeologia, 29, 239-246. Carter, C., & Marr, J. (1970). Olfactory imprinting and age variables in the guinea pig. Animal Behavior, 18, 238-244. Caza, P., & Spear, N. E. (1984). Short term exposure to an odor increases its subsequent preference in preweanling rats: A descriptive profile of the phenomenon. Developmental Psychobiology, 17, 407-422. Chotro, M. G. (1987). Intoxicacion aguda con etanol en crias de rata: Subsiguiente disminucion de la preferencia hacia el alcohol. Unpublished master's thesis, Faculty of Exact, Physical and Natural Sciences, State University of Cordoba, Argentina. Cooper, A., & Hathorn, S. (1977). Modification of flavor preference by olfactory preexposure in normal and zinc sulfate treated mice. Bulletin of the Psychonomic Society, 10, 369-370. Cornwell, C. (1970). Golden hamster pups adapt to complex rearing odors. Behavioral Biology, 14, 175-188. Cunningham, C. (1979). Alcohol as a cue for extinction: State dependency produced by conditioned inhibition. Animal Learning & Behavior, 7, 87-95. Domjan, M. 0980). lngestional aversion learning: Unique and general processes. In R.

ETHANOL INTOXICATION IN INFANT RATS

343

Rosenblatt, C. Hinde, C. Beer, & M. Busnel (Eds.), Advances of the study of behavior Vol. 2, pp. 275-336). New York: Academic Press. Domjan, M. (1983), Biological constraints on instrumental and classical conditioning: Implications for general processes theory. In G. Bower (Ed.), The psychology of learning and motivation (pp. 215-277). New York: Academic Press. Fillion, T., & Blass, E. (1986). Infantile experience with suckling odors determines adult sexual behavior in male rats. Science, 231, 729-731. Galef, B. (1981). Development of flavor preference in man and animals. R. Aslin, J. Alberts, & M. Petersen (Eds.), Development of perception (Vol. 1, pp. 411-431). New York: Academic Press. Galef, B., & Kaner, H. (1980). Establishment and maintenance of preference for natural and artificial olfactory stimuli in juvenile rats. Journal of Comparative and Physiological Psychology, 94, 588-596. Gamzu, E., Vincent, G., & Boff, E. (1985). A pharmacological perspective of drug use in establishing conditioned food aversions. Experimental assessments and clinical applications of conditioned food aversions. In N. Braveman, & P. Bronstein (Eds.), Annals of the New York Academy of Sciences (Vol. 443, pp. 231-249). New York: New York Academy of Sciences. Gemberling, G., & Domjan, M. (1981). Selective associations in one-day-old rats: Tastetoxicosis and texture-shock aversion learning. Journal of Comparative and Physiological Psychology, 96, 105-113. Gemberling, G., Domjan, M., & Amsel, A. (1980). Aversion learning in 5-day-old rats: Taste-toxicosis and texture-shock associations. Journal of Comparative and Physiological Psychology, 94, 734-745. Goldblatt, A. (1978). Effects of early olfactory experience on tilt table odor preference. Behavioral Biology, 22, 269-273. Goldstein, D. (1983). Pharmacology of alcohol. London/New York: Oxford Univ. Press. Hall, W., & Rosenblatt, G. (1977). Suckling behavior and intake control in the developing rat pup. Journal of Comparative and Physiological Psychology, 91, 1232-1247. Hollstedt, C. (1981). Alcohol and the developing organism: Experimental and clinical studies. Stockholm: Opuscula Medica. Hollstedt, C., Olsson, O., & Rydberg, U. (1980). Effects of ethanol on the developing rat. II. Coordination as measured by the tilting plane test. Medical Biology, 58, 164-168. Hollstedt, C,, & Rydberg, U. (1985). Postnatal effects of alcohol on the developing rat. In U. Rydberg (Ed.), Alcohol and the developing rat (pp. 69-82), New York: Academic Press. Holloway, F. (1972). A state dependent effect of ethanol on active and passive avoidance learning. Psychopharmacologia, 25, 238-261. Johanson, I., & Shapiro, E. (1983). Ontogeny of aversive responses to taste stimuli in rats. Paper presented at International Society of Developmental Psychobiology, Hyannis, MA. Kalat, J., & Rozin, P. (1973). "Learned safety" as a mechanism in long delay learning in rats. Journal of Comparative and Physiological Psychology, 83, 198-207. Kelly, S., Bonhius, D., & West, J. (1987). Developmental changes in alcohol pharmacokinetics in rats. Alcoholism: Clinical and Experimental Research, 11, 281-286. Keppel, G. (1982). Design and analysis: A researcher's handbook. Hillsdale, NJ: Erlbaum. Leon, M. (1987). Plasticity of olfactory output circuits related to early olfactory learning. Trends in Neurosciences, 10, 434-438. Leon, M., Galef, B., & Behse, J. (1972). Establishment of pheromonal bonds and diet choice in young rats by odor preexposure. Physiology and Behavior, 18, 387-391. Lester, D., Nachman, M., & LeMagnen, J. (1970). Aversive conditioning by ethanol in the rat. Quarterly Journal of Studies of Alcohol, 31, 578-586.

344

MOLINA AND.CHOTRO

Logue, A. (1979). Taste aversion and the generality of the laws of learning. Psychological Bulletin, 86, 276-2%. Lubow, R. (1973). Latent inhibition. Psychological Bulletin, 79, 398-407. Misanim, J., Guanowsky, V., & Riccio, D. (1983). The effect of CS preexposure on conditioned taste aversion in young and adult rats. Physiology and Behavior, 30, 859862. Molina, J. C., Chotro, M. G., & Spear, N. E. (in press). Early recognition of alcohol's orosensory cues resulting from acute ethanol intoxication. Behavioral and Neural Biology. Molina, J. C., Serwatka, J., Enters, K., Spear, L. P., & Spear, N. E. (1987). Acute alcohol intoxication disrupts brightness but not olfactory conditioning in preweanling rats. Behavioral Neuroscience, 101, 846-853. Molina, J. C., Serwatka, J., & Spear, N. E. (1984). Changes in alcohol intake resulting from prior experience with alcohol odor in young rats. Pharmacology, Biochemistry and Behavior, 21, 387-391. Molina, J. C., Hoffmann, H., & Spear, N. E. (1986a). Conditioning of aversion to alcohol orosensory cues in 5 and 10 day rats: Subsequent reduction in alcohol ingestion. Developmental Psychobiology, 19, 175-183. Molina, J. C., Serwatka, J., & Spear, N. E. (1986b). Alcohol drinking patterns of young adult rats as a function of infantile aversive experiences with alcohol odor. Behavioral and Neural Biology, 46, 257-271. Molina, J. C., Serwatka, J., Spear, L. P., & Spear, N. E. (1985). Differential ethanol olfactory experiences affect ethanol ingestion in preweanling but not in older rats. Behavioral and Neural Biology, 44, 90-100. Nachman, M., Rauschemberger, J., & Ashe, J. (1977). Stimulus characteristics in food aversion learning. In N. Milgram, L. Crames, & T. Alloway (Eds.), Food Aversion Learning (pp. 105-131). New York: Plenum. Rescorla, R. A. (1980). Pavlovian second order conditioning: Studies in associative learning. Hillsdale, N J: Erlbaum. Riley, A., & Tuck, D. (1985). Conditioned taste aversion: A behavioral index of toxicity. Experimental assessments and clinical applications of conditioned food aversions. In N. raveman, & P. Bronstein (Eds.), Annals of the New York Academy of Sciences, (Vol. 443, pp. 273-292). New York: New York Academy of Sciences. Rozin, P., & Zellner, D. (1985). The role of Pavlovian conditioning in the acquisition of food likes and dislikes. Experimental assessments and clinical applications of conditioned food aversions. In N. Braveman, & P. Bronstein (Eds.), Annals of the New York Academy of Sciences, (Vol. 443, pp. 189-202). New York: New York Academy of Sciences. Rudy, J. W., & Cheatle, M. D. (1983). Odor aversion learning by rats following LiCI exposure: Ontogenetic influences. Developmental Psychobiology, 16, 13-22. Rudy, J. W., & Cheatle, M. D. (1977). Odor aversion learning in neonatal rats. Science, 198, 845-846. Rudy, J. W., & Cheatle, M. D. (1979). Ontogeny of associative learning: Acquisition of odor aversion in neonatal rats. In B. Campbell, & N. E. Spear (Eds.), Ontogeny of learning and memory (pp. 157-188). Hillsdale, NJ: Erlbaum. Ryback, R. (1969). State dependent or dissociated learning with alcohol in the goldfish. Quarterly Journal of Alcohol Studies, 30, 598-608. Schwarze, H., & Muller, S. (1971). Olfactory imprinting in a precocial mammal. Nature (London), 229, 55-56. Serwatka, J., Molina, J. C., & Spear, N. E. (1986). Weanlings' transfer of conditioned ethanol aversion from olfaction to ingestion depends on the unconditioned stimulus. Behavioral and Neural Biology, 45, 57-70.

ETHANOL INTOXICATION IN INFANT RATS

345

Sherman, J., Hickis, A., Rice, K., Rusiniak, K., & Garcia, J. (1983). Preferences and aversions for stimuli paired with ethanol in hungry rats. Animal Learning & Behavior, 11, 101-106. Spear, N. E., & Kucharski, D. (1984). Ontogenetic differences in stimulus selection during conditioning. In R. Kail, & N. E. Spear (Eds.), Perspectives in development of memory (pp. 227-252). Hillsdale, N J: Erlbaum. Spear, N. E., & Molina, J. C. (1987). The role of sensory modality in the ontogeny of stimulus selection. In N. Krasnegor, N. Hofer, E. Blass, & W. Smotherman (Eds.), Perinatal development. A psychobiological perspective (pp. 83-110). New York: Academic Press. Spear, N. E., & Smith, G. J. (1978). Alleviation of forgetting in preweanling rats. Developmental Psychobiology, 11, 513-529. Vogt, M., & Rudy, J. (1984). Ontogenesis of learning. I. Variation in the rat's reflexive and learned responses to gustatory stimulation. Developmental Psychobiology, 17, 11-33.

Waller, M., McBride, W., Gatto, G., Lumeng, L., & Li, T. (1984). Intragastric self infusion of ethanol by ethanol prefering and nonprefering lines of rats. Science, 225, 78-80.