Early (preweanling) recognition of alcohol's orosensory cues resulting from acute ethanol...

BEHAVIORAL AND NEURAL BIOLOGY 51, 307--325 (1989)

Early (Preweanling) Recognition of Alcohol's Orosensory Cues Resulting from Acute Ethanol Intoxication

JUAN CARLOS MOLINA, GABRIELA CHOTRO, AND NORMAN E . SPEAR l

Instituto de Investigacion Medica Mercedes y Martin Ferreyra, Cordoba, Argentina, and Center for Developmental Psychobiology, SUNY Binghamton,

Binghamton, New York 13901

Three experiments were conducted in order to analyze the possibility that during acute alcohol intoxication, preweanling rats process ethanol's orosensory cues. Intragastric administration of an ethanol dose equivalent to 3.0 g/kg resulted in peak blood alcohol levels greater than 250 mg% (Experiment 1). Twenty-four hours after receiving this dose, ll-day-old pups expressed an ethanol odor aversion in a locational test. This aversion response was not observed when pups were previously administered an ethanol dose resulting in blood alcohol levels lower than 150 mg% (Experiment 2). Nevertheless when subjects received such a lower dose and 30 to 60 min after administration suffered nociceptive stimulation, alcohol aversions were also detected in terms of alcohol ingestion patterns and ethanol odor aversions (Experiment 3). These results appear to indicate that orosensory ethanol processing takes place during acute ethanol intoxication probably due to ethanol elimination via respiration, salivation, and/or hematogenic olfaction processes. © 1989 Academic Press, Inc.

In infrahuman species, factors such as alcohol's orosensory cues and the postabsorptive effects of this drug have been found repeatedly to play a determinant role in establishing patterns of ethanol acceptance or rejection. Both mice and rats are able to discriminate different ethanol concentrations by relying upon the saliency of gustatory and olfactory cues of this pharmacological agent (Molina, Hoffman, Spear, & Spear, 1987; Waller, McBride, Gatto, Lumeng, & Li, 1984). At an electrophy- siological level of analysis, the rostral stimulation of the tongue with a solution of ethanol triggers a marked neuronal activation in the nucleus

This research was supported by Grant P.I.D. 681/86 from the Consejo de Investigaciones Cientificas y Tecnologicas de la Provincia de Cordoba (CONICOR) awarded to Juan Carlos Molina and by grants from the National Institute of Mental Health (1 R01 MH35219) and National Institute of Alcohol Abuse and Alcoholism (1 R01 AA06634) to Norman E. Spear. The authors express their appreciation to Dr. Hugo Carrer for his valuable considerations and to Teri Tanenhaus for preparation of the manuscript. Requests for reprints may be addressed to either Dr. Juan Carlos Molina, Instituto Ferreyra, Casilla de Correo 389, 5000 Cordoba, Argentina or Dr. Norman E. Spear, Department of Psychology, SUNY Binghamton, Binghamton, NY 13901.

307 0163-1047/89 $3.00

Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

308 MOLINA, CHOTRO, AND SPEAR

of the solitary tract. This neural response is different from that observed when gustatory stimulation is operationalized through the use of the four basic tastes (Di Lorenzo, Kiefer, Rice, & Garcia, 1986). From a behavioral perspective, it has been observed that mice avoid the ingestion of certain ethanol solutions due to the olfactory component of such solutions (Nach- man, Larue, & Le Magnen, 1971). Furthermore, Kahn and Stellar (1960) determined that bulbectomized rats exhibit postoperative changes in their preference toward alcohol. Other authors, when examining alcohol intake through intake preference tests or through operant paradigms where ethanol acts as a positive reinforcer, have also concluded that the orosensory cues inherent to this drug represent an important factor in de- termining discriminative recognition and/or consumption of alcohol (Meisch, 1977).

Alcohol preference is also regulated through postabsorptive consequences derived from ethanol administration. Intraperitoneal and intragastric administration of high doses of alcohol ranging from 2.0 to 5.15 g/kg paired with the ingestion of an originally innocuous and palatable substance induce a subsequent conditioned taste aversion (Berman & Cannon, 1974; Lester, Nachman, & Le Magnen, 1970). Similarly, when a distinctively flavored alcohol solution is rapidly ingested and hence, high blood-alcohol levels are achieved, the rat later exhibits an avoidance response toward the sensory elements of the solution (Eckardt, 1975). Forced drinking of larger quantities of alcohol has also been reported to promote associations between the taste and the odor of the drug and aversive consequences derived from the process of acute or chronic intoxication (Deutsh & Eisner, 1977).

Preweanling rats show a remarkable capacity in terms of modifying alcohol preference patterns as a function of previously learned and un- learned experiences with the odor of the drug (Molina, Hoffman, & Spear, 1986a; Molina, Serwatka, & Spear, 1984, 1986b). These animals also appear to avoid alcohol when rapid oral ingestion of a 5.6% (v/v) ethanol solution promotes behavioral signs of intoxication (Molina et al., 1984). This result seems to confirm the associative process between orosensory cues and postabsorptive consequences leading to the con- solidation of a conditioned taste and/or odor aversion. In this type of experiment as well as those performed with older rats, the organism is explicitly exposed to alcohol's orosensory cues during the ingestive episode. The sensory cues (conditioned stimulus, CS) are associated with internal consequences which, in terms of Pavlovian conditioning procedures, act as an unconditioned stimulus (US). From this point of view, it is obvious that in order to promote an associative process the organism should explicitly be exposed to paired experiences involving the CS and the US. Therefore, the sole exposure to the US should fail to promote an associative process between the elements under consideration. Never-

OROSENSORY CUES FROM INTOXICATION 309

theless, this hypothesis should not be accepted without further considerations. Although alcohol is mainly eliminated via oxidative hepatic processes, a small fraction of the drug is excreted unchanged through respiration, salivation, perspiration, and urine. In the rat this fraction of unmetabolized ethanol represents approximately 10-15% of a given dose (Abel, 1984; Hollstedt & Rydberg, 1985). This apparently small percentage might nevertheless be detected by the animal's sensory receptors. This allows the speculation that even under experimental circumstances in which ethanol is nonorally administered, the organism may associate postabsorptive consequences of the drug with sensory cues arising from the nonmetabolic elimination of the drug. The temporal properties of this hypothetical associative process follow the procedure known as simul- taneous conditioning which has been shown to be effective in terms of promoting learned responses in infrahuman species (Rescorla, 1980).

In the present study, preweanling rats were intragastrically administered with 1.5 or 3.0 g/kg of ethanol. This route of administration was employed in order to avoid explicit exposure to sensory components of the drug. As a first step, blood alcohol levels were assessed as a function of time after administration of the mentioned doses. In a subsequent experiment, infants were given an intragastric administration of an ethanol dose of 0.0, 1.5, or 3.0 g/kg and were tested 24 h later in a locational odor test in order to assess alcohol odor preference. We decided to use preweanling subjects due to several factors. At this developmental stage the rat is capable not only of establishing associations between gustatory and olfactory cues with different reinforcers, but also of transferring alcohol odor experience to taste experiences and vice versa, a transfer which is difficult to observe in adult rats (Spear & Molina, 1987). Furthermore it has been demonstrated that early perinatal experience involving ethanol's sensory properties and/or the drug's postabsorptive consequences exerts short- and long-lasting effects upon subsequent patterns of alcohol preference (Molina et al., 1984, 1986a, 1986b). Also, it has been observed recently that under a clear behaviorally defined state of intoxication, young rats are still capable of performing a previously acquired alcohol- odor aversion (Molina et al., 1984). Finally, and in agreement with this last result, acute alcohol intoxication in preweanlings does not necessarily disrupt the acquisition and expression of learning involving orosensory cues (Molina et al., 1986b).

EXPERIMENT 1

The aim of the present experiment was to determine, in ll-day-old rats, blood alcohol levels (BALs) resulting from prior administration of intragastric ethanol doses equivalent to 1.5 or 3.0 g/kg. These doses were selected in order to perform a second experiment directed toward the analysis of ethanol orosensory processing after alcohol administration.


BALs were assessed not only as a function of the administered dose, but also as a function of time after administration of each dose. In accordance with this goal animals were sacrificed 0, 30, 60, 90, 120, or 240 min after receiving the corresponding ethanol dose.

Method

Subjects and procedures. The subjects were 76 l 1-day-old male and female Wistar-derived rats sampled from 9 litters (litter size = 8-11 pups). All pups were born and reared at the breeding colony of the Instituto Ferreyra, Cordoba, Argentina. After birth and until commence- ment of the experiment, animals were housed with their parents and conspecifics in standard opaque maternity cages partially filled with wood shavings. All animals were maintained on a 14-h light/10-h dark illumination cycle where light onset occurred at 0700 h. Temperature in the colony ranged between 22 and 25°C. Parent animals had continuous access to rat lab chow (ADAB1C) and tap water delivered through automatic fluid dispensers.

A total of 3 pups were sacrificed by decapitation without receiving any prior treatment. The blood of these subjects was employed to determine standard blood alcohol concentrations. The remaining 73 infants were quasirandomly assigned to 1 of 12 groups defined as a function of the two independent variables under consideration, Ethanol Dose (1.5 or 3.0 g/kg) and Time after Administration (0, 30, 60, 90, 120, or 240 min). Attempts were made to equate the number of males and females as well as the number of pups representative of each litter for each dose and sacrifice time.

Before receiving the corresponding dose, each pup was weighed to the nearest 0.01 g. The 1.5 g/kg ethanol dose was achieved by administering 0.015 ml/g body weight of a 12.6% (v/v) alcohol solution, whereas the higher dose (3.0 g/kg) was achieved by administering a similar volume of 25.2% (v/v) alcohol solution (v/v dilutions refer to proportion mixed by volume of 95% alcohol/volume of vehicle). The vehicle for both solutions was tap water at room temperature. This vehicle was employed taking into account the nature of the experimental goals of the study. Since we were concerned with the possibility of orosensory processing during acute alcohol intoxication, we decided not to employ other com- monly used vehicles (such as physiological saline) in order to minimize the possible influence of nonalcohol gustatory or olfactory cues derived from such vehicles.

The intragastric administration was performed using polyethylene tubing (Clay Adams PE 10) attached to a 1.0-cc disposable syringe equipped with a 27 Gx 1/2 needle. This tubing was carefully placed in the oral cavity of the pup and then slowly pushed until the free end of the cannula

OROSENSORY CUES FROM INTOXICATION

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FIG. 1. Blood alcohol levels (mg%) as a function of ethanol dose (1.5 or 3.0 g/kg) and time of sacrifice (0, 30, 60, 90, 120, or 240 min after intragastric administration). Vertical bars represent standard errors of the mean.

was inserted approximately 3 cm beyond the infant 's tongue and into its stomach. This procedure normally required less than 20 s.

As previously stated pups were sacrificed by decapitation at different postadministration times. The numbers of subjects assigned to each dose and interval of time were as follows: for 1.5 g /kg-- immedia te ly , n = 6; 30 min, n = 7; 60 min, n = 7; 90 rain, n = 6; 120 rain, n = 5; 240 min, n = 6; and for 3.0 g /kg- -a l l intervals of time, n = 6.

Blood ethanol levels were analyzed by gas-liquid chromatography using the method originally described by Le Blanc (1968). Ethanol levels for each sample of trunk blood were computed from a standard curve using linear regression of known standards.

Results and Discussion

Blood alcohol concentrat ions (mg%) are depicted in Fig. 1. As could be expected, BALs varied as a function of the administered dose and time of sacrifice. Immediately after the intragastric administration, ethanol


was practically undetectable in blood among subjects given either dose. At 30, 60, 90, 120, or 240 rain postadministration time, subjects given 3.0 g/kg exhibited higher BALs than those given the lower dose. With this last dose, BALs peaked at 30 min and were stable until 120 min postadministration time. The peak BAL achieved with 3.0 g/kg, however, was observed at 90 min. Despite a clear decline in absolute values relative to shorter intervals, ethanol was still detectable 4 h after administration of either of the two doses.

These observations were confirmed by a two-way analysis of variance (ANOVA) which indicated that BALs were significantly affected by dose and time of assessment, F(1, 61) = 35.45, p < .001 and F(5, 61) = 17.39, p < .001, respectively. This ANOVA also revealed a borderline significant effect attributable to the interaction of the factors under consideration, F(5, 61) = 1.95, .05 < p < .10. In order to clarify the locus of this borderline interaction as well as specific features of the main effects, subsequent post hoc comparisons were performed using the Fisher test (probability of Type I Error set at 0.05; Keppel, 1982).

These post hoc comparisons revealed no significant differences in BALs when rats receiving the 1.5 or 3.0 g/kg dose were sacrificed immediately after innoculation. At 30, 60, 90, and 120 min, BALs resulting from both doses were significantly higher than those observed when rats were sacrificed immediately. At 240 min ethanol concentrations corresponding to 3.0 g/kg were also significantly higher than those observed immediately after sacrifice whereas with the lower dose this difference approached significance (.05 < p < . 10). At all intervals of time (30, 60, 90, 120, and 240 min) BALs corresponding to the higher dose (3.0 g/kg) were always significantly higher than those observed in animals receiving 1.5 g/kg. With this last dose, values corresponding to 30, 60, 90, and 120 min failed to differ significantly. Also with the 1.5 g/kg dose, at 240 min, BAL failed to differ significantly from that attained at the previous interval (120 min). With the higher dose, at 240 min, BAL was significantly higher than that registered immediately after sacrifice and significantly lower than those attained at 30, 60, and 90 min. When considering the results of this higher dose, BAL observed at 30, 60, 90, and 120 min failed to differ.

In summary, these results indicate that at 11 days of age BALs peaked at 30 min postinfusion and remained stable for approximately 90 min. At 240 min postadministration, these levels declined in relation to previous values. It is interesting to observe that the blood alcohol curve attained with the 1.5 g/kg dose is very similar to that described by Hollstedt, Olsson, and Rydberg (1980), for which animals of a weight class corresponding to an age comparable to the one here employed, received a slightly lower dose of ethanol (1.25 g/kg). Furthermore, in the present


experiment it is obvious that BALs vary as a function of the dose of ethanol administered. On the basis of these observations, and in terms of the measured pharmacological parameter, it may be concluded that the doses under consideration promote differential states of intoxication.

EXPERIMENT 2

As previously stated, high doses of ethanol have been employed as aversive unconditioned stimuli for conditioning procedures (Cappell & LeBlanc, 1977; Eckhardt, 1975). In addition, during an intoxicating episode induced with this drug, a fraction of ethanol is excreted unmetabolized through respiration and salivation (Abel, 1984; Goldstein, 1983). Therefore it is possible that under such a state of intoxication, the organism could associate ethanol's olfactory and/or gustatory cues with its aversive postadministration consequences. In turn this associative process should promote a subsequent expression of an aversion toward sensory consequences of the drug. This hypothesis was tested in ll-day-old rats that received intragastric administration of ethanol doses equivalent to 0.0, 1.5, or 3.0 g/kg body weight. One day later these animals were tested in an olfactory preference test where alcohol represented one of the odorants under consideration. It should be clear that unlike circumstances in which the proposed associative process takes place when the drug is orally ingested, so that ethanol's orosensory components are quite explicitly the sensory conditioned stimuli (Eckardt, 1975; Deutsh & Eisner, 1977), the present route of administration does not include such explicit pres- entation of these sensory properties during ethanol administration.

Method

Subjects. The subjects were 27 male and female 11-day-old Wistar- derived rats. Infants were sampled from 5 litters (litter size = 8-11 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 quasirandomly assigned to one of three treatments defined by their ethanol dose (0.0, 1.5, or 3.0 g/kg body weight). As in the first experiment, attempts were made to equate sex and number of pups from each litter across dose conditions.

Prior to intragastric administration procedures, pups were weighed ( + 0.01 g) and housed in opaque maternity cages according to the assigned treatment condition. These cages were partially filled with wood shavings. Temperature (34-36°C) in these holding cages was controlled through the use of heating pads.

The different ethanol doses were achieved by intragastric administration of 0.015 ml/g body weight of 0.0, 12.6, or 25.2% (v/v) ethanol solution. As in the first experiment tap water served as the vehicle. The admin-


istration procedure followed similar steps as those described in the first experiment. Prior to intragastric administration the cannula was carefully dried with paper towels in order to avoid the presence of ethanol or vehicle and thus prevent a direct stimulation of taste and or olfactory receptors.

Following this procedure, pups were separated according to the treatment condition in the different holding cages. Interaction among pups differ- entially treated was avoided since previous studies have demonstrated that taste aversions do occur in untreated animals that interact with conspecifics given interoceptive unconditioned stimuli (Coombes, Revusky, & Lett, 1980). Twenty-four hours after treatment, ethanol odor preferences were assessed in a locational olfactory test. This test was conducted in a clear Plexiglas chamber (33 × 14 x 15 cm) equipped with a stainless- steel grid floor (diameter of the bars, 0.3 mm; distance between bars, 10.0 mm). This apparatus was divided into two equal sections. Cotton scented with 5.0 cc of alcohol (190 proof ethanol) or 5.0 cc of lemon extract (Arcor Co.) was placed approximately 2.0 cm beneath the grid floor and proximal to either end section of the apparatus. For testing procedures, infants were individually placed in the center of the apparatus and time spent over each scented half section was recorded. The total duration of this test was 180 s. A rat was considered to be over a particular section (alcohol or lemon scented) when its nose and front paws were above that section.


One infant originally assigned to the 1.5 g/kg dose died immediately after intragastric administration. Therefore the numbers of subjects tested for ethanol odor preference were as follows: 0.0 g/kg, n = 9; 1.5 g/kg, n = 8; 3.0 g/kg, n = 9.

Immediately before and during ethanol olfactory testing procedures, no clear motor signs of alcohol intoxication were observed in any of the subjects. In terms of percentage body weight loss between the day of administration (11 days of age) and the testing day (12 days of age), no significant differences were observed when analyzing these values with a one-way ANOVA (F < 1.0, p > .1). Percentage body weight loss (weight of testing day - weight of administration day x 100/weight of administration day) for the three groups under consideration were as follows: 0.0 g/kg = 09.88 __- 0.36%; 1.5 g/kg = 10.07 --- 0.66%; 3.0 g/kg = 10.58 ___ 0.56%; mean __- standard error.

A one-way ANOVA was employed to analyze total time spent over alcohol odor as a function of prior ethanol dose administration. The analysis revealed a significant treatment effect, F(2, 23) = 3.54, p < .05.

O R O S E N S O R Y C U E S F R O M I N T O X I C A T I O N 315

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0.o G/KG 1.5 G/KG ~.0 G/KG

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FIG. 2. T ime spent over e thanol odor (s) as a funct ion of ethanol dose (0.0, 1.5, or 3.0 g/kg). Vertical bars represent s tandard errors of the mean.

Post hoc comparisons (Fisher test, p < .05) showed that infants administered 3.0 g/kg spent significantly less time over the ethanol odor than did those treated with vehicle or 1.5 g/kg ethanol. Furthermore, no significant differences were observed when comparing the scores of these last two groups. These results are depicted in Fig. 2.

According to the present results, intragastric administration of an ethanol dose of 3.0 g/kg promotes a subsequent alcohol-odor aversion which is expressed in a locational olfactory test. This result appears to be congruent with the hypothesis that during acute ethanol intoxication, sensory processing also takes place, leading to an association between the alcohol's orosensory cues and the drug's aversive interoceptive effects. In accord with the hypothesis, orosensory processing apparently can occur due to direct elimination of ethanol via respiration, salivation, urine, and perspiration.

In the present experiment intragastric administration of a mildly intoxicating ethanol dose (1.5 g/kg) failed to modify later olfactory preference for alcohol. Infants given this dose exhibited scores similar to those of vehicle-treated subjects. It is interesting to observe that in older rats a 1.5 g/kg dose has rarely been reported to act as an aversive unconditioned stimulus when examining the effects of one-trial learning. In preweanlings it has been observed that such a dose fails to affect olfactory processing


of nonethanol cues and also seems not to result in aversive learning with visual cues and/or nonalcohol olfactory cues (Molina et al., 1986b). A second possibility related to the lack of effect attained with 1.5 g/kg is that the amount of alcohol eliminated through nonmetabolic processes does not reach threshold levels in terms of allowing orosensory processing of ethanol. If orosensory processing does take place, then we should expect that under the state of intoxication induced by this dose, the presence of a potent aversive stimulus should facilitate a subsequent expression of ethanol aversion. In other words, this experimental approach may help not only to obtain new evidence of learning occurring during acute alcohol intoxication, but also may help explain why the 1.5 g/kg dose fails to promote by itself subsequent avoidance of ethanol's orosensory properties.

EXPERIMENT 3

According to the results of the first experiment, 30 to 60 min after receiving an ethanol dose equivalent to 1.5 g/kg, infants exhibit a peak and stable blood alcohol level. Normally, and especially with high alcohol doses, excretion of ethanol via respiration correlates positively with arterial blood concentration (Goldstein, 1983). Therefore we might expect that at this interval of time the probability of perceiving the sensory consequences of ethanol is maximized. In the present experiment and during this interval of time, rats administered 1.5 g/kg alcohol also experienced nociceptive peripheral stimulation (footshock). Exteroceptive electrical shock effectively promotes the acquisition and expression of olfactory or gustatory aversions in preweanlings of a similar age (e.g., Hoffmann & Spear, 1988; Kucharski & Spear, 1984). Furthermore, Lasiter and Braun (1981) have also demonstrated that taste aversions resulting from flavor-toxicosis pairings are facilitated when footshock is administered simultaneously with an unconditioned interoceptive stimulus such as lithium chloride. According to these observations it should be expected that if the taste and/or odor of alcohol is perceived during a mild state of intoxication, then the presence of a powerful aversive reinforcer during such a state should promote later rejection of the drug's sensory cues. In this experiment ethanol preference was not only assessed in terms of the previously employed odor locational test but also in terms of consumption of a palatable ethanol solution.

Method

Subjects and procedures. Fifty-two 1 l-day-old pups were sampled from 8 litters (litter size = 8-11 pups). Housing and rearing conditions for these subjects replicated those described for the previous experiments.

A factorial design included the between-group factors of Dose (vehicle


only (0.0 g/kg) or 1.5 g/kg alcohol) and Treatment (Baseline controls, B; dose unpaired with shock, UP; and dose paired with shock, P). The numbers of subjects quasirandomly assigned to each group were as follows: 0.0-B, n = 8; 0.0-UP, n = 9; 0.0-P, n = 9; 1.5-B, n = 8; 1.5-UP, n = 9; and 1.5-P, n = 9. Attempts were made to equate across conditions the number of males and females as well as the number of subjects representing a particular litter.

The procedure and materials for vehicle or alcohol intragastric administration were similar to those in the preceding experiments. Prior to intragastric administration, pups were separated, in accordance with their assigned condition, into clean maternity cages partially filled with wood shavings. Baseline animals (0.0-B and 1.5-B) at 11 days of age received only vehicle or alcohol administration. No further experimental manipulations were performed with these two groups prior to testing. Pups assigned to groups 0.0-P and 1.5-P were individually placed into clear Plexiglas chambers (15 x 7 x 15 cm) fitted with stainless-steel grid floors suitable for delivery of footshock from a shock generator (Perosio Ins.) beginning 30 min after their drug administration. For 30 consecutive min (30-60 min postadministration time) these subjects were exposed to nociceptive electrical stimulation (intensity = 0.8 mA, duration = 3.0 s, intershock interval = 10 s). Subjects assigned to groups 0.0- UP and 1.5-UP also received a 30-min trial of exposure to footshock using parameters and holding chambers identical to those employed for paired groups (0.0-P and 1.5-P). These unpaired animals, however, experienced nociceptive stimulation 90 min before receiving vehicle or alcohol intragastric administration. In other words, animals corresponding to 0.0-UP and 1.5-UP conditions experienced the last footshock 1 h before being exposed to drug treatment. Therefore, these subjects were treated with similar exteroceptive and drug stimuli as paired infants but with a different temporal arrangement of the stimuli.

Immediately following the above described treatments pups were housed in standard maternity cages in accordance with their experimental condition. These cages were maintained at 32-34°C by heating pads placed beneath them.

The following day (20 h after completion of the corresponding treatment) all pups were orally cannulated using a method similar to that described by Hall and Rosenblatt (1977) and Hoffman et al. (submitted for publication). Briefly, cannulae were constructed from 5-cm sections of polyethylene tubing (Clay Adams, PE-10, 0.28-mm internal diameter). The cannulae were heated at one end in order to form a small flange (external diameter: approx 1.2 mm). Cannulae were inserted using a 5-cm insect pin which was curved in order to achieve an angle of 45 ° . The pin was inserted into the nonflanged end of the cannulae and then was pulled through the medial internal surface of the right cheek. Therefore, the flanged end of


the cannula remained in the mouth while the remainder of the tubing exited from the oral cavity. Prior to this cannulation procedure all cannulas were rinsed with tap water to ensure that they were free from, extraneous particles. Cannulated pups were then returned to the holding cages where they remained for an additional 4-h period before being tested in terms of ethanol consumption patterns.

Testing took place in Plexiglas chambers (15 x 7 × 15 cm) fitted with grid floors and maintained at 32-34°C by heating pads placed beneath the grid floor. A 6.0% (v/v) alcohol solution was infused through the cheek cannula of each pup via a variable speed peristaltic pump (Sage Ins. Model 375A). This solution was delivered for 7.5 min divided into three 2.5-min trials (interval between trials = 60 s). Rate of infusion was set at 0.096 ml/min. The total amount of ethanol solution infused to all pups represented 5.49 ___ 0.12% (mean --- SE) of their preinfusion body weight. After infusion, subjects were placed in paper towels in order to remove noningested solution present in their pellage. Prior to testing, the preweanlings' bladders were voided and defecation was stimulated by stroking the anogenital region with a cotton swab. Preinfusion and postinfusion body weights were registered to the nearest 0.01 g. The amount of ingestion was expressed in terms of percentage body weight gain [100 × (postinfusion weight - preinfusion weight/preinfusion weight)].

Following the above described test, cheek cannulae were removed and pups were returned to their parents and conspecifics. Twenty-four hours later pups were tested for ethanol olfactory preference using the procedures and materials described in Experiment 2. The dependent variable under examination was total time spent over ethanol odor.


Ethanol ingestion (percentage body weight gain). The results are il- lustrated in Fig. 3. A two-way ANOVA revealed significant effects of the dose factor and of the interaction between Dose and Treatment, F(1, 46) = 4.04, p < .05 and F(2, 46) = 4.31, p < ~02, respectively. Post hoc comparisons (Fisher test with an o~ set at 0.05) demonstrated that animals receiving electrical stimulation during acute alcohol intoxication (group 1.5-P), when compared to the scores of the remaining five groups, exhibited significantly lower body weight gains resulting from ethanol infusion. No other differences were revealed.

Apparently the above-described significant effects are not attributable to differences in body weight loss between treatment and testing day. All groups exhibited similar body weight loss and therefore it is difficult to speculate that the experimental group, relative to the remaining control conditions, was more motivated to ingest the alcohol solution. A two- way ANOVA confirmed this observation by revealing nonsignificant effects


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FIG. 3. Ethanol ingestion (percentage body weight gain) as a function of ethanol dose (0.0 or 1.5 g/kg) and treatment (basal, unpaired, or paired). Vertical bars represent standard errors of the mean.

of the main factors under analysis and of the interaction between them (all Fs < 1.0). Body weight loss for each group was as follows: 0.0-B, 8.12 ± 0.55; 0.0-UP, 7.66 ± 0.70; 0.0-P, 9.11 _+ 0.86; 1.5-B, 8.87 _+ 0.51; 1.5-UP, 8.55 --- 0.36; 1.5-P, 8.11 ± 0.57 (mean ± SE percentage body weight loss between 11 and 12 days of age).

According to these results a mildly intoxicating alcohol dose (1.5 g/kg) does not, in itself, affect subsequent alcohol ingestion. This interpretation is based on the absence of significant differences in alcohol ingestion when comparing unpaired or baseline animals given this dose (1.5-B and 1.5-UP compared with those given only the vehicle, 0.0-B and 0.0-UP). This lack of effect replicates that observed in the preceding experiment when similar groups were tested in terms of ethanol olfactory preferences. Also, due to the similar consumption scores registered in baseline and unpaired conditions, it appears that electrical stimulation by itself also fails to promote changes in ethanol intake. Nevertheless when this aversive stimulus is presented 30 min after ethanol administration, an aversion is observed in terms of subsequent alcohol ingestion.

Ethanol olfactory preference (time spent over ethanol odor). Olfactory preference scores are depicted in Fig. 4. Animals given footshock 30-


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ETHANOL DOSE (G/KG)

Flc. 4. Time spent over ethanol odor (s) as a function of ethanol dose (0.0 or 1.5 g/kg) and treatment (basal, unpaired, or paired). Vertical bars represent standard errors of the mean.

60 min after intragastric administration of the ethanol dose (1.5-P) exhibited marked reductions in time spent over alcohol odor when compared to the remaining groups. This observation was confirmed by a two-way ANOVA as well as by Fisher post hoc comparisons (p < .05). The ANOVA demonstrated a significant effect of both main factors under analysis: Dose, F(1, 46) = 26.72, p < .001; Treatment, F(2, 46) = 23.88, p < .001. Additionally, this ANOVA also revealed a significant interaction between these factors, F(2, 46) = 19.89, p < .001. Finally, the Fisher tests indicated that group 1.5-P significantly differed from all the remaining groups (0.0-B, 0.0-UP, 0.0-P, 1.5-B, and 1.5-UP). No other significant differences were detected.

These results are very similar to those reported in terms of ethanol consumption. As previously described, and on the basis of present olfactory scores, ethanol administration per se does not influence subsequent ethanol odor preference. This null effect is consistent not only with what happened in terms of consumption but also when considering the absence of olfactory changes attained with this dose in Experiment 1. Also, and in agreement with the pattern of results for ethanol intake, footshock when presented in an unpaired fashion with postadministration effects of ethanol does not influence alcohol odor preference. This observation is based on the


lack of aversions evidenced in groups 0.0-UP and 1.5-UP when compared to baseline controls. Of major importance in this study is the aversion exhibited by animals which, under a mild state of alcohol intoxication, also received aversive exteroceptive stimulation. Apparently, this experimental procedure was effective not only in promoting a conditioned change in olfactory preferences but also in reducing the consumption of a solution that contained alcohol.

Overall, the results of the present experiment seem to suggest that the infant rat perceives orosensory cues during an interval of time in which blood alcohol concentrations are at a peak and stable level and can establish associations between such cues and an aver~ive reinforcer. This suggestion concerning perceptual and associative processes is primarily based on the expression of aversive responses not only in terms of rejection of an ethanol solution but also in terms of avoidance of the olfactory component of the drug under consideration.

GENERAL DISCUSSION

The results of these studies show that preweanling rats express an aversion toward ethanol odor if they previously were exposed to an intragastric ethanol administration of an alcohol dose (3.0 g/kg) that yields peak BALs higher than 250 mg% (Experiments 1 and 2). A lower dose (1.5 g/kg), resulting in BALs lower than 150 rag%, did not promote such an aversion (Experiments 1, 2, and 3). Nevertheless infants that were given this lower dose and 30-60 min later were given nociceptive stimulation exhibited alcohol aversions in ethanol consumption and locational tests of preference for ethanol odor (Experiment 3).

The behavioral data reported in Experiments 2 and 3 do not seem to be explainable in terms of explicit taste and/or odor cues resulting from oral introduction or withdrawal of the cannula, which in turn could be paired with either EtOH's peak physiological consequences or aversive exteroceptive stimulation. This possibility implies a conventional paradigm for conditioned flavor aversion, but several factors argue against it. As previously described (Experiment 1), the entire administration procedure took less than 20 s in order to be completed. The passage of the cannula into and out of the mouth, even when not specifically timed, took a minimum fraction of the entire temporal interval required for this procedure. Even if the animals sensed during this fraction of time the cues under consideration, it is difficult to view this short duration of the conditioned stimulus as sufficient to promote learning, particularly in circumstances in which the unconditioned stimulus is presented 30 rain later (Rudy & Cheatle, 1978; Westbrook, Bond, & Feyer, 1981): Furthermore, the delay between intragastric administration and footshock (Experiment 3) also seems to argue against gustatory or olfactory detection of EtOH at the time of intubation leading to the observed behavioral changes. Tests


have indicated that conditioning with a 0.5 h CS-US delay does not occur in animals of this age (Campbell & Alberts, 1979; Rudy & Cheatle, 1979; Rudy, Vogt, & Hyson, 1984). Additionally, such long trace conditioning seems to be particularly difficult to achieve when employing external nociceptive stimulation (Experiment 3) rather than internal toxic agents (Garcia et al., 1985).

As previously stated, rats exhibit alcohol aversions under experimental conditions in which the drug is orally ingested in quantities sufficient to act as an aversive unconditioned stimulus (Deutsh & Eisner, 1977; Eckardt, 1975). Explicit exposure to the orosensory properties of this pharmacological agent therefore appears to be associated easily with the toxic consequences of the drug. Furthermore, the aversive nature of alcohol has also been demonstrated in studies in which nonoral administration of alcohol promotes aversions to originally innocuous and palatable stimuli presented orally and paired with such administration (Cappell & LeBlanc, 1977). In the present set of experiments aversions toward ethanol's orosensory properties were also detected despite the fact that the drug was delivered intragastrically. Although this mode of administration follows a gastric route similar to that in voluntary or forced consumption, it avoids or minimizes explicit taste or odor stimulation. Recently the smell and taste of alcohol have been discarded as factors regulating self-administration of the drug given that ethanol was delivered intragastrically through the use oftransesophageal catheters (Waller et al., 1984). Perhaps this conclusion is inappropriate.

According to the present results, and specifically in infant rats, orosensory processing of some kind cannot be discarded when employing intragastric administration. Apparently the taste and smell of alcohol can become aversive if a high dose of ethanol is intragastrically administered or if a lower dose administered in the same way is followed by a nonethanol aversive stimulus. Both results may be attributed to the fact that intubated ethanol is not only metabolized into other subproducts but is also excreted through expired air, saliva, perspiration, and urine (Abel, 1984; Hollstedt & Rydberg, 1985). Especially during the first two forms of excretion (respiration and salivation), the organism might sense the odor and taste of the drug. This possibility is particularly important in young animals when considering not only the immaturity of the enzymatic systems involved in ethanol oxidation, but also their high respiratory rate (Hollstedt & Rydberg, 1985). In turn, ethanol's orosensory cues, derived through respiratory or salivary excretion of the drug, could be associated with inherent aversive properties of an ethanol-induced internal context or other aversive stimuli present at the time of excretion. It is interesting to observe that, while intoxicated with ethanol, the infant rat not only is still capable of recognizing alcohol odor in a preference test, but also is able to acquire and express pairings between odors and aversive stimuli


such as the one employed in Experiment 3 (footshock) (Molina et al., 1984, 1986b).

A second mechanism, involving alcohol distribution rather than elimination of alcohol through excretion processes, could also be involved in orosensory processing during the toxic state. Evidence has been reported for hematogenic or intravascular olfaction in humans, turtles, and rats (Hirai, Yashiki, Matsuzama, & Mima, 1981; Maruniak, Silver, & Moulton, 1983). This phenomenon indicates that certain substances present in vascular blood can act as odorants capable of stimulating olfactory receptors. Apparently blood-borne odorants stimulate the olfactory mucosa through direct diffusion from nasal capillaries. Therefore, alcohol present in vascular blood can be viewed as a stimulus affecting sites of olfactory stimulation and transduction.

In summary, the present results suggest that in physiologically immature rats, intragastric ethanol administration exerts a significant effect upon subsequent patterns of alcohol preference. Apparently this effect is me- diated through acquired association between ethanol's orosensory cues that arise from systemic elimination and/or hematogenic olfaction of the drug and other stimuli present during the state of alcohol intoxication. The ontogenetic character of these results remains to be determined. Furthermore, the proposed associative model implies that appetitive re- inforcement administered during ethanol intoxication should promote subsequent ethanol acceptance rather than ethanol rejection. Independently of these considerations, the above-described results in combination with previous data (Hayashi & Tadakoro, 1985; Molina et al., 1984, 1986a, 1986b) indicate that early in ontogeny, organisms are able to regulate patterns of alcohol acceptance as a function of prior experiences with this psychoactive agent.

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