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

Alcohol 30 (2003) 45–60

Adolescent rats discriminate a mild state of ethanol intoxication likely to act as an appetitive unconditioned stimulus Juan M. Ferna´ndez-Vidala,*, Norman E. Spearb, Juan Carlos Molinaa,b,1 a Instituto de Investigacio´n Me´dica M. y M. Ferreyra, Co´rdoba, C.P. 5000, Argentina Center for Developmental Psychobiology, Binghamton University, Binghamton, NY 13902-6000, USA

b

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

Abstract Practically no information is available in relation to the capability of the adolescent animal in terms of discriminating postabsorptive effects of ethanol. Three experiments were conducted to analyze whether young, genetically heterogeneous rats discriminate different stages of the process of intoxication exerted by a low dose (0.5 g/kg) of ethanol. An ethanol pharmacokinetic profile was first examined to select two stages within the process of ethanol intoxication that, as a function of the corresponding blood ethanol concentrations (BECs), could represent two potentially discriminable drug states. In a second experiment, sucrose was available when the BECs of rats peaked or were of a lesser magnitude (5 and 30 min postadministration time, respectively). When animals were tested under similar or different drug states relative to the training procedure, no behavioral evidence indicative of differential sucrose expectancy was obtained. In Experiment 3, rats discriminated each of the previously defined ethanol states from a non-drug state. Unexpectedly, it was also found that the pharmacological effects of the 0.5-g/kg dose of ethanol are likely to support appetitive associative learning that involves the taste of sucrose as a conditioned stimulus. The apparent positive affective components of the state of ethanol intoxication have rarely been observed in genetically heterogeneous rats with rather brief experiences with the drug’s effects. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: Ethanol; State discrimination; Appetitive conditioning; Nose-poking; Sucrose; Adolescent rat

1. Introduction Interoceptive effects originated by drugs of abuse represent an integral stimulus that has a significant impact on subsequent seeking behavior and subsequent use of these substances (Duka et al., 1998). Drugs, such as amphetamines, barbiturates, and ethanol, have the capability to act as an interoceptive context that, when present during the acquisition and retrieval phases of a given learning situation, optimizes the expression of specific memories. That is, improvements in performance tend to occur when the interoceptive context is similar between training and test (Bruins Slot et al., 1999; Lowe, 1986), and a decrement in retention often occurs when the context of testing differs from that of training (Riccio et al., 1984; Spear & Riccio, 1994). This indicates that animals acquire information about not only

* Corresponding author. Instituto Ferreyra, Casilla de Correo 389, CP: 5000 Co´rdoba, Argentina. Tel.: ⫹54-351-468-1465; fax: ⫹54-351469-5163. E-mail address: [email protected] (J.M. Ferna´ndez-Vidal). 1 J.C. Molina is also working at Facultad de Psicologı´a, Universidad Nacional de Co´rdoba, CP: 5000 Co´rdoba, Argentina. Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00093-4

the reinforcing properties of a drug, but also the internal context provided by other effects of the drug. This phenomenon has been referred to frequently as state-dependent learning (SDL) (Bruins Slot et al., 1999; Deutsch & Roll, 1973; Lowe, 1986; Spear & Riccio, 1994) and has been verified even in early development (Hunt et al., 1990; McKinzie et al., 1994). The capability to encode the effects of a drug as an interoceptive state has also been tested frequently through the use of operant training procedures (Bruins Slot et al., 1999; Krimmer, 1992; Overton, 1979). In drug-discrimination procedures, animals learn to execute an instrumental response to obtain a given appetitive reinforcer or to avoid an aversive stimulus on the basis of the action of a drug or a non-drug state as a discriminative stimulus. In these paradigms, training procedures are long. Drug-discrimination capabilities can be tested only after behavioral shaping techniques take place (Colpaert & Koek, 1995); after strict performance criteria, in terms of operant responding, are met; and after specific training procedures, with drug-related interoceptive effects that act as discriminative stimuli, are conducted. The temporal brevity of some ontogenetic stages of development requires procedures that require correspondingly brief periods. In rats, for example, it is difficult to achieve

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consistent operant performance for drug-discrimination effects during infancy or adolescence because of the briefness of these developmental stages. Perhaps for this reason, very little is known regarding how young animals process discriminative properties of drugs such as ethanol. During early ontogeny, ethanol has been used frequently as a conditioned stimulus (CS) because of its particular chemosensory attributes [see Molina et al. (1985, 1986, 1999)] or as an unconditioned stimulus (US) capable of supporting associative learning [see Berman and Cannon (1974), Domı´nguez et al. (1994), and Hunt et al. (1991)]. Very rarely has this drug been used as an interoceptive context or as a discriminative stimulus that aids in the retrieval of early memories related to its postabsorptive effects. Numerous findings obtained from studies have demonstrated SDL generated by ethanol when focusing on adult animals (Bruins Slot et al., 1999; Holloway, 1972; Nakagawa & Iwasaki, 1995). In general in these studies, drug versus non-drug conditions as discriminative stimuli have been compared. Furthermore, drug-discrimination procedures can be defined through the use of differential doses of the same drug or effects derived from different psychotropic agents (Appel et al., 1999; Ja¨rbe & Swedberg, 1998) or from the combination of these agents (Mariathasan et al., 1999; Stolerman et al., 1999). The literature concerning discrimination of different stages within the development of the toxic process induced by a given ethanol dose is scarce [see, for example, Schechter (1989) and Shippenberg and Altshuler (1985)]. Differential effects across the state of intoxication, with the use of an ethanol dose, have been analyzed mainly through conditioning studies in which the postabsorptive effects of each particular stage of the toxic process are used as differential unconditioned stimuli (Cunningham & Prather, 1992; Krimmer, 1992; Risinger & Cunningham, 1992). It has been hypothesized that ethanol has positive-reinforcing qualities relatively soon after exposure, during the rising limb of the curve of blood ethanol concentrations (BECs), and aversive effects during the falling limb of the curve of BECs (Reid et al., 1985). This hypothesis has been supported mainly in terms of heightened ethanol self-administration patterns by mice or genetically selected rats. Heterogeneous strains of rats generally do not express appetitive contents of memories comprising postabsorptive effects of ethanol. On the contrary, these animals tend to encode primarily the aversive consequences of ethanol. Conditioned place and taste aversions are easily established with ethanol doses equal to or higher than 1 g of ethanol per kilogram of body weight (1 g/kg) (Co´rdoba et al., 1990; Cunningham et al., 1993; Gauvin & Holloway, 1992; Schechter & Krimmer, 1992). Lower doses rarely exert significant aversive or appetitive conditioning (Stewart & Grupp, 1985; van der Kooy et al., 1983). To attain ethanol-mediated conditioned preferences in heterogeneous rats, extensive training procedures (Bozarth, 1990), long-term preexposure to the interoceptive

effects of the drug (Reid et al., 1985), or concurrent presentation of other non-ethanol reinforcers (Marglin et al., 1988)— or a combination of these conditions—has been needed. In the current study, we tested whether adolescent rats can use the effects of a relatively low dose (0.5 g/kg) of ethanol as a discriminative state and whether they perceive differences in the effects of such a dose throughout the process of intoxication. An additional goal, but critical in terms of being able to analyze the above-stated possibilities, was to generate a technique that would enable us to verify drugdiscrimination capabilities after minimal behavioral training procedures. Obviously, this is a prerequisite to train and evaluate rats within the adolescent stage of development. In the rat, this stage lasts no more than 15 days [postnatal days (PNDs) 28–42] (Spear, 2000; Spear & Brake, 1983). In the current study, mildly dehydrated adolescent rats had easy access to a sucrose solution when sober or when intoxicated with ethanol. They were later tested in terms of sucrose-seeking behavior when in the state under which they were trained or in a different state. Although the study was focused on discriminative properties of a low dose of ethanol during adolescence, it was found that the training procedure allowed the expression of apparently appetitive consequences of a 0.5-g/kg dose of ethanol in the adolescent rat. In other words, beyond the original experimental intention, a brief, simple, and economical training procedure involving explicit pairings between a sweet solution and the interoceptive effects of ethanol resulted in the expression of behaviors that support the suggestion of appetitive contents of ethanolrelated memories. The original goals of the current study required that we first determine, in Experiment 1, pharmacokinetic profiles of ethanol in adolescents administered a low dose (0.5 g/ kg) of the drug. This experiment allowed identification, operationally, of distinctive states of intoxication as indicated by BECs. For the second and third experiments, we used this information to determine whether adolescents can discriminate such states of intoxication and/or an ethanol versus a non-ethanol state.

2. Experiment 1 Toward assessing the capacity of adolescents to discriminate acute states of ethanol intoxication, a pharmacokinetic analysis of BECs resulting from exposure to a low dose (0.5 g/kg) of ethanol was performed. The goal was to select postadministration intervals that yielded significantly different BECs. The dose was selected in accordance with preliminary results, supporting the suggestion that adolescent rats are capable of discriminating the interoceptive context generated by this dose from the state of sobriety (Godoy et al., 1999). More explicitly, the intention was to choose one postadministration interval in which BECs peaked and a second one with significantly lower BECs, still detectable as different from a non-drug state. This experiment was

J.M. Ferna´ndez-Vidal et al. / Alcohol 30 (2003) 45–60

considered necessary for a subsequent behavioral study that required two potentially different drug states. 2.1. Materials and methods 2.1.1. Subjects Fifteen Wistar-derived adolescent male rats (each weighing between 60 and 80 g) were used. The animals were born and reared in the animal colony at the Instituto Ferreyra. Rats were housed in standard opaque cages filled with pine shavings and maintained on a 14-h light/10-h dark cycle (light onset at 0700) and temperature-controlled (22ºC–24ºC) conditions. Unless specified, subjects had free access to rat chow (Cargill; Co´rdoba, Argentina) and water delivered through automatic dispenser valves. The day of birth was considered as PND 0. After delivery, on PND 1, pups were culled to eight per litter (four males and four females whenever possible) and weaned on PND 21. After weaning, animals from each litter were kept together in an environment similar to the original maternity cage. Only males were used for experimental purposes. In accordance with the preceding literature (Spear, 2000; Spear & Brake, 1983), adolescence in the rat takes place between 28 and 42 days of age. Taking this, as well as maturational parameters corresponding to the above-mentioned strain of rats, into account we ensured that all animals used in the current experiment were 34 days of age at the start of the experiment. Animal care and procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996). 2.1.2. Procedures Twenty-four hours before blood samples were obtained for analysis of BECs, rats were subjected to a surgical procedure aimed at placing a catheter in the right jugular vein. This surgery was necessary to allow serial blood sampling during the course of the ethanol toxicity process. The animals were anesthetized by means of an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) (Vetanarcol, Ko¨nig; Buenos Aires, Argentina) supplemented with xylazine hydrochloride (13.5 mg/kg) (Kensol, Ko¨nig; Buenos Aires, Argentina). An incision was then made in the ventral portion of the neck, and a catheter (filled with heparin diluted in physiological saline) was inserted into the jugular vein until reaching the cardiac atrium. The catheter was kept in this position by means of a suture procedure to maintain its attachment to the sternocleidomastoid muscle. The free end of the catheter was inserted subcutaneously until it reached the dorsal side of the neck. A small incision was made to express the free end of the cannula and attach it, with one suture stitch, to the skin. To diminish the possibility of postsurgical nociception, buprenorphine hydrochloride (0.03 mg/kg) (Temgesic, Schering-Plough; Buenos Aires, Argentina) was administered subcutaneously and used as an analgesic agent. All subjects were water deprived for 22 h during four consecutive days, the deprivation procedure to be used in

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subsequent behavioral experiments to test discrimination between states of intoxication (see Experiments 2 and 3). Four hours before intragastric administration of a 0.5-g/kg dose of ethanol (PND 34), the rats were also deprived of food. This ensured relatively equivalent stomach contents of food to minimize individual differences in absorption and distribution of the drug. To attain the 0.5-g/kg dose of ethanol, rats were subjected to intragastric administration of 0.015 milliliters per gram of body weight of a 4.2% [volume/ volume (vol./vol.)] ethanol solution. Blood samples (100 µl) were collected at each of five postadministration times (5, 15, 30, 60, and 90 min) after intubation of ethanol. Administration and sampling procedures took place between 1000 and 1200. Blood samples were subjected to head-space gas chromatography (Hachemberg & Schmidt, 1985; Molina et al., 1992). Samples were placed in microvials (total volume capacity: 700 µl) equipped with a rubber stopper. Each vial was placed on crushed ice to prevent ethanol vaporization. For assessment of BECs, samples were kept in a water bath at 60ºC for 30 min. Gas-tight syringes (Hamilton; Reno, Nevada; 10 µl) were used to collect the volatile component of the samples and to inject them into the gas chromatograph (Hewlett-Packard, Model 5890, Palo Alto, CA) column (Carbowax 20M; 10 m × 0.53 mm × 1.33 mm film thickness). Oven, injector, and detector temperatures were 60ºC, 150ºC, and 250ºC, respectively. Nitrogen served as the carrier gas (flow rate: 15 ml/min). Blood ethanol concentrations were computed by using linear regression analysis of known standards. Twenty microliters of butanol (52 mg/dl) was added to each blood sample to provide an internal standard control. Blood ethanol concentrations were expressed as milligrams of ethanol per deciliter of body fluid (mg/dl ⫽ mg%). 2.2. Results and discussion Blood ethanol concentrations resulting from the administration of a 0.5-g/kg dose of ethanol in adolescent rats can be seen in Fig. 1. The data were processed by using a oneway analysis of variance (ANOVA), with postadministration time as the factor under analysis. This ANOVA yielded a significant main effect [F(4,56) ⫽ 23.44, P ⬍ .001]. The BECs increased rapidly after administration of the drug and peaked [40.98 ⫾ 5.35 mg% (mean ⫾ S.E.M.)] 5 min after administration of the 0.5-g/kg dose of ethanol. These levels were similar to those encountered at the 15-min interval. Indeed, post hoc comparisons (Fisher least significant difference test, with an alpha level of .05) revealed that BECs at 5 and 15 min did not differ. Post hoc tests also indicated that BECs at 30 min were significantly lower than those registered in previous postabsorptive time intervals and also significantly higher than those obtained 60 and 90 min after ethanol administration. The scores in these two last intervals were not different from a 0 mg% theoretical value. Rate of ethanol elimination was estimated from individual slopes of the linear regression of ethanol concentrations

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

across 5-, 15-, 30-, and 60-min sampling points. The analysis was not conducted for the 90-min sampling period because all animals showed null levels of ethanol in blood. The elimination rate was equivalent to 46.13 ⫾ 5.9 mg%/h (mean ⫾ S.E.M). The rate of metabolism is relatively smaller when compared with the one reported by Silveri and Spear (2000) during periadolescence (PNDs 26 and 36). These investigators used Sprague–Dawley rats subjected to intraperitoneal administrations of higher ethanol doses (1.5 and 4.5 g/kg) relative to the dose used in the current experiment. The route of administration as well as genetic factors could be responsible for the differences observed across studies. Kelly et al. (1987) reported lower ethanol elimination rates in 30-day-old, male, Sprague–Dawley rats relative to the ones observed in the current experiment. In their study, a 2.5-g/kg dose of ethanol was administered intragastrically with the use of milk as a vehicle. This factor is probably responsible for lower rates of absorption and distribution of the drug, which will obviously have an impact on ethanol rate of elimination.

3. Experiment 2 Experiment 2 was conducted to assess whether adolescents can discriminate two temporally separate states of ethanol intoxication, operationally defined by significantly different amounts of ethanol in blood. In accordance with the results of Experiment 1, we decided to use the following postadministration intervals for Experiment 2: 5–15 versus 30–40 min. To assess discrimination between these drug states, we used a behavioral technique that allows completion

of training and testing within the adolescent stage of development. A very simple and naturally occurring behavior (nose poking) sustained by a sweet reinforcer (sucrose) was selected. Nose-poking behavior was easily completed by these adolescents to gain access to sucrose solution, during either peak or intermediate BECs derived from the same subnarcoleptic dose (0.5 g/kg) of ethanol used in Experiment 1. While still adolescents, animals were evaluated in terms of behavioral reactivity (nose poking) within the environment in which the sucrose solution was originally encountered. This reactivity was tested when the animals were under a similar or different drug state than that originally experienced during access to sucrose. 3.1. Materials and methods 3.1.1. Subjects Genetic as well as housing conditions of the animals replicated those described in Experiment 1. A total of 47 adolescent male rats were used. These animals were representative of 12 litters. At the beginning of the experiment, all subjects were 30 days of age. 3.1.2. Experimental design On the basis of results of Experiment 1, two ethanol postabsorptive states were defined: state A (5–15 min postadministration time) and state B (30–40 min postadministration time). Animals were quasirandomly assigned to one of two training groups: A⫹B⫺ (during state A, rats had access to sucrose, whereas during state B, rats did not have access to sucrose) and A⫺B⫹ (state A was experienced while sucrose was absent, and state B was experienced while sucrose was

J.M. Ferna´ndez-Vidal et al. / Alcohol 30 (2003) 45–60

present). The quasirandom distribution was executed to ensure equivalent litter representation and body weights across groups. Half the animals in each group were later evaluated when experiencing the same drug state experienced while having access to sucrose (evaluation group same; included training groups A⫹B⫺ tested in A and A⫺B⫹ tested in B). The remaining rats were evaluated under the state in which they did not have access to sucrose (evaluation group different; included training groups A⫹B⫺ tested in B and A⫺B⫹ tested in A). 3.1.3. Apparatus and procedures Black Plexiglas chambers (25 × 25 × 25 cm), which had a hole (diameter: 4 cm) in one of their lateral walls, were used. The center of this hole was 4.5 cm above the floor and equidistant from the adjacent walls. A transparent Plexiglas cup (volume capacity: 16 ml; diameter: 3.4 cm) was positioned in the external side of each chamber. The rim of the cup was in contact with the inferior border of the hole. This cup was removable. One of these devices was used to present a 10% [weight/volume (wt./vol.)] sucrose solution and a second one was always presented empty. Nose poking allowed the animals to have access to the empty or the sucrose-filled cup. The experiment had a total duration of 5 days (training: days 1, 2, 3, and 4, which correspond to PNDs 30, 31, 32, and 33, respectively; evaluation: day 5 or PND 34). Before the training and testing sessions, all subjects were water deprived during 22 consecutive hours. A pair of subjects was first weighed (⫾0.1 g) and subjected to intragastric administration of a 0.5-g/kg dose of ethanol. Five minutes later (state A), animals were placed in individual chambers: one with an empty cup (group A⫺B⫹) and the other one with a cup containing sucrose (group A⫹B⫺). Subjects remained in these chambers for 10 min. Immediately after, they were weighed and placed in individual holding cages where they stayed until postadministration time 30 min (state B). Under state B, animals were placed in the Plexiglas chambers where they remained 10 additional minutes. Animals in group A⫺B⫹ had access to the cup filled with sucrose, whereas animals in group A⫹B⫺ were exposed to an empty cup. Once again, body weights were registered before and after subjects were placed in the corresponding chambers. Two hours after ethanol administration, rats had free access to water for 100 consecutive minutes. They were then water deprived until commencement of the following training session. Fig. 2 summarizes the training and testing procedures that were used. Before evaluation procedures (day 5, PND 34), animals corresponding to each training group were subdivided into two evaluation groups: different and same. Different refers to the groups of rats that were tested under a different drug state than that previously associated with sucrose availability; that is, group A⫺B⫹ evaluated under A (5–15 min postadministration time, n ⫽ 12) and group A⫹B⫺ evaluated under B (30–40 min postadministration time, n ⫽ 12).

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Same refers to the groups of rats that were tested under the same drug state that previously signaled sucrose availability; that is, group A⫺B⫹ evaluated under B (30–40 min postadministration time, n ⫽ 11) and group A⫹B⫺ evaluated under A (5–15 min postadministration time, n ⫽ 12). Tests were conducted in a black Plexiglas box similar to the one used during training. All test sessions were conducted in absence of sucrose to evaluate sucrose-seeking behavior as operationalized through nose poking (see following section for details on this matter). During tests, animals were fluid deprived as when trained. Immediately after termination of the test, blood samples were collected from each adolescent after induction of ether anesthesia. Blood ethanol concentrations were determined by using the same procedures as those described in Experiment 1. The blood was collected through cardiac puncture with the use of a 271/2-gauge needle attached to a disposable syringe. 3.1.4. Collection of behavioral data All subjects were videotaped (Samsung SCA20) during the evaluation procedure. A real-time computer-based program served to determine parameters related to nose-poking behavior (duration, frequency, and latency to perform the first nose poke). The experimenter who used this computer program was blind relative to training and test conditions of the animals. Preliminary examination of the results of the current study (Experiments 2 and 3) and those corresponding to prior pilot experiments indicated that, very rapidly during the test session, animals decreased their nose-poke rate, probably because of the fact that they readily sensed the absence of sucrose. When considering both behavioral experiments of the current study (Experiments 2 and 3, overall n ⫽ 143 rats) we observed that, in terms of relative frequency of nose-poke behavior, 70.1% occurred during the first 4 min of the test. Hence, in the current and subsequent experiments, data subjected to inferential statistical analysis were mean nose-poke duration in the first 4 min of the test session. Latency to perform the first nose poke was also subjected to inferential analysis. These behavioral scores seem to represent the most sensitive indexes of sucrose-seeking behavior. 3.2. Results and discussion 3.2.1. Sucrose intake during training sessions Body weights at the start of the experiment did not differ between groups [A⫹B⫺: 61.96 ⫾ 1.33 g and A⫺B⫹: 65.58 ⫾ 2.27 g (mean ⫾ S.E.M.)]. Despite water-deprivation procedures, all animals gained weight across training days, and this increase was similar across groups [overall weight gain between days 1 and 4: 8.30 ⫾ 0.39 g (mean ⫾ S.E.M.)]. A two-way ANOVA showed significant increases in body weights across training days [F(3,135)⫽ 309.28, P ⬍ .001]. Post hoc Fisher tests indicated that body weights for each

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

specific day were always significantly higher relative to the ones recorded during the preceding day (data not shown). Percent body weight gains (%BWG) were calculated for each training session in which animals had explicit access to sucrose. This intake index was calculated by using the following formula: 100 × (postsession body weight ⫺ presession body weight)/ presession body weight This dependent variable was processed by using a two training group (A⫹B⫺ and A⫺B⫹) × 4 days of training two-way mixed ANOVA. This analysis indicated only a significant main effect of days [F(3,135) ⫽ 9.46, P ⬍ .001]. Post hoc Fisher least significant difference tests (P ⬍ .05) showed that sucrose intake was significantly lower during the first day in relation to the next three training days. These results have been depicted in Fig. 3. 3.2.2. Behavioral responsiveness during test Latency to perform the first nose poke when adolescents were evaluated by using an empty cup was analyzed through a two-way ANOVA. The factors under consideration were training (A⫹B⫺ or A⫺B⫹) and evaluation (same or different). Latency was observed to vary significantly only as a function of training [F(1,43) ⫽ 6.17, P ⬍ .01]. Animals assigned to group A⫹B⫺ showed significantly lower latencies than did subjects assigned to group A⫺B⫹. Mean duration of nose poking was analyzed through an ANOVA that incorporated the training and evaluation groups as independent factors. Neither the main effects nor the interactions between them were statistically significant.

As stated, BECs were determined after completion of the test. A two training group (A⫹B⫺ and A⫺B⫹) × two states at test (A, B) ANOVA revealed that animals tested under state A had significantly higher BECs than did animals tested under state B [F(1,43) ⫽ 43.7, P ⬍ .001]. This difference was not affected by prior training procedures, nor by the interactions of the factors under consideration. The difference between BECs of animals previously tested during state A or B was very similar to that observed in Experiment 1 when contrasting BECs at 5 and 30 min postadministration time. Latency to perform the first nose poke, mean duration of nose poke, and BECs at termination of test for the various treatment conditions are shown in Table 1. Clearly, the results of the current experiment failed to show any obvious behavioral sign indicative of discrimination between two drug states characterized by significantly different BECs. Performance during the test did not depend on the drug state present during the test. Behavior seemed essentially the same when the drug state was the same for training and when it was different. The only factor that had a significant impact on a specific dependent variable (latency to exhibit the first nose-poke behavior) was the nature of the training group. As stated, animals in group A⫹B⫺ more rapidly approached the cup previously associated with presence or absence of sucrose than did animals in group A⫺B⫹ (Table 1). Could this difference be due to differential unconditioned properties of a particular intoxication state that might favor appetitive or aversive conditioning? Under certain experimental conditions, ethanol has been found to exert biphasic hedonic effects during the course of the state

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

of intoxication (Cunningham & Prather, 1992; Krimmer, 1992; Risinger & Cunningham, 1992) or to act either as an appetitive or an aversive US (Co´rdoba et al., 1990; Stewart et al., 1988). If any of these possibilities was responsible for the observed latency differences in the test situation, probably some differences should also have been observed in sucrose-intake patterns as a function of the accumulation of training trials. This speculation is based on the possibility that sucrose sensory properties can act as a CS likely to be associated with ethanol’s toxic consequences (Berman & Cannon, 1974; Bormann & Cunningham, 1998; Co´rdoba et al., 1990; Hunt et al., 1990, 1991). Yet, sucrose-intake patterns, as a function of training procedures, did not differ between groups across the entire training procedure. An alternative explanation related to the observation that group A⫹B⫺ rats approached the cup faster than did group Table 1 Behavioral scores registered in Experiment 2 during evaluation phase and the corresponding blood ethanol concentrations (BECs) Training group

State at test

Nose-poke latency (s)

Nose-poke mean duration (s)

BECs after test (mg%)

A⫹B⫺

A B A B

4.23 ⫾ 0.90 3.53 ⫾ 0.66 7.74 ⫾ 1.80 6.21 ⫾ 1.27

1.82 ⫾ 0.19 1.63 ⫾ 0.15 1.80 ⫾ 0.18 2.28 ⫾ 0.22

41.15 ⫾ 1.74 26.58 ⫾ 2.35 38.07 ⫾ 2.75 22.72 ⫾ 2.03

A⫺B⫹

All values are expressed as mean ⫾ S.E.M. A ⫽ First of two ethanol postabsorptive states (on the basis of results of Experiment 1) defined as 5–15 min postadministration time; B ⫽ second of two ethanol postabsorptive states defined as 30–40 postadministration time; A⫹B⫺ ⫽ group that during state A had access (⫹) to sucrose but during state B did not have access (⫺) to sucrose; A⫺B⫹ ⫽ group that during state A did not have access to sucrose but during state B had access to sucrose.

A⫺B⫹ rats during test can be related to the sequence of sucrose presentation during the training phase of the experiment. It is likely that subjects that received sucrose the first time that they were placed in the training apparatus (A⫹B⫺) would exhibit shorter latencies in terms of seeking the sweet stimulus than those that received sucrose the second time that they were placed in the black Plexiglas chamber during training (A⫺B⫹). Animals encode not only exteroceptive and interoceptive information related to presence or absence of a particular salient stimulus, in this case a sucrose solution, but also information related to the sequence of access to such a stimulus. In other words, rats learned that sucrose was available during the first, but not the second, trial of a day, and the test was, in effect, the first trial of that day. Such learning has been appreciated for many years, primarily because of the work of E. J. Capaldi [see, for example, Capaldi et al. (1986) and La Fiette et al. 1994)].

4. Experiment 3 The intention of Experiment 3 was to analyze whether rats can discriminate either of the ethanol-induced states used in Experiment 2 with respect to a non-drug state (state of sobriety, S). In Experiment 2, adolescents did not indicate discrimination between an ethanol postabsorptive period characterized by peak BECs (state A) and a later period in which the contents of the drug were significantly lower (state B). In the current experiment, adolescent rats had access to sucrose while nonintoxicated (state S) or while experiencing peak BECs (state A) resulting from a 0.5-g/kg dose of ethanol (training groups S⫹A⫺ or S⫺A⫹, respectively). Two

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other additional groups were incorporated. In these groups, sucrose availability was associated with either the nondrug state or with levels of ethanol that characterize a time segment of the falling limb of the ethanol pharmacokinetic curve (training groups S⫹B⫺ or S⫺B⫹, respectively). Unexpectedly, and as will be described later in detail, there were clear indications supporting the suggestion that ethanol had a strong impact on sucrose-intake patterns after the first training session. 4.1. Materials and methods 4.1.1. Subjects Genetic and housing conditions of the animals replicated those described in Experiments 1 and 2. A total of 96 adolescent male rats representative of 24 litters were used. The age of animals ranged between 30 and 31 days at commencement of the experiment. 4.1.2. Experimental design Two factors defined the training conditions. The first factor comprised the two combinations of states that defined the training procedures. Animals were trained to discriminate either a non-drug state (state S) from that of a period of peak BECs (state A) or a non-drug state (state S) from that of a period during the falling limb of the pharmacokinetic curve (state B). Within each training procedure, the second independent factor was the state in which animals had access, or no access, to sucrose. Hence, the two independent factors in this study resulted in four different training groups: S⫺A⫹, S⫹A⫺, S⫺B⫹, and S⫹B⫺. During the test, each group was subdivided into two new groups (evaluation groups). Animals were evaluated under either the state in which they originally had access to sucrose (group same) or the state associated with the absence of sucrose (group different). Each of the eight independent groups included 11–13 adolescents. The animals were distributed across groups in a quasirandom manner. The constraints were to attain equivalent representation of litters and body weights across groups. 4.1.3. Apparatus and procedures The experiment included the same apparatus, schedule of deprivation, and drug administration procedure as in Experiment 2. The sole difference between these experiments was in the drug states to be discriminated. These included the A and B states, as defined in Experiment 2, and the S (sobriety) state. For the S state, animals were weighed and subsequently intubated intragastrically, but no solution was delivered into their stomachs (sham administration). Five minutes later, these animals were placed in the black Plexiglas chamber with access, or no access, to a 10% (wt./ vol.) sucrose solution. Under this non-drug state, animals remained in the chambers for 10 min. Intragastric fluid administration was not used to avoid an overload of the stomach, because all subjects were later subjected to intragastric administration of a 0.5-g/kg dose of ethanol. A second exposure to the black training chambers occurred 30 min after

completion of the initial experience within these chambers. Subjects assigned to group S⫺A⫹ or group S⫹A⫺ received the intragastric administration of ethanol 5 min before being exposed for the second time to the training chambers, whereas those assigned to group S⫺B⫹ or group S⫹B⫺ were intubated with ethanol 30 min before the second exposure to the training chambers. These administration parameters allowed training the animals under drug states homologous to states A and B, respectively, in Experiment 1. In summary, for some animals sucrose was available when they were not intoxicated with ethanol (groups S⫹A⫺ and S⫹B⫺), whereas in the remaining groups availability of sucrose was associated with peak BECs (group S⫺A⫹) or with significantly lower BECs (group S⫺B⫹). As in Experiment 2, tests had a total duration of 10 min and were executed by using similar chambers and deprivation schedules as those used during training. As specified in the Experimental Design section, rats were evaluated under either a similar or a different state in which they originally had access to sucrose. Once again, sucrose was not available in the evaluation phase. Latency to perform the first nose poke and mean duration of nose-poking behavior served as dependent variables. Fig. 4 provides a summary of the training and testing procedures that were used. 4.2. Results and discussion Body weights across groups were similar at commencement of the experiment [S⫹A⫺: 62.12 ⫾ 1.73 g; S⫺A⫹: 57.22 ⫾ 1.54 g; S⫹B⫺: 61.25 ⫾ 1.00 g; and S⫺B⫹: 59.51 ⫾ 1.11 g (mean ⫾ S.E.M.)]. As in Experiment 2, body weights increased significantly as a function of progression of days [F(3,276) ⫽ 850.59, P ⬍ .001]. This effect failed to interact significantly with the nature of the training procedures [overall body weight gain between training days 1 and 4: 7.41 ⫾ 0.21 g (mean ⫾ S.E.M.)]. Percent body weight gains (%BWG), as a function of sucrose intake during training, are shown in Fig. 5. A completely unexpected result was obtained. Adolescent rats exposed to sucrose under either peak (state A) or lower (state B) BECs drank significantly more sucrose than did those exposed to this stimulus under a non-drug state (state S). Apparently this difference did not exist during the first day of training. A 4 × 4 mixed ANOVA confirmed these observations. The independent factor under consideration was the training procedure (group S⫺A⫹, S⫹A⫺, S⫺B⫹, or S⫹B⫺). The repeated measures were derived from the days of training. This analysis showed significant main effects of training [F(3,92) ⫽ 14.89, P ⬍ .001] and of days of training [F(3,276) ⫽ 26.85, P ⬍ .001]. The interaction between these factors also achieved significance [F(9,276) ⫽ 4.30, P ⬍ .001]. The locus of the two-way interaction was analyzed with the use of post hoc Fisher least significant difference tests. All groups failed to differ in terms of sucrose intake during the first day of training. During the next 3 days, the intake scores of groups S⫺A⫹ and S⫺B⫹ were

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

significantly higher than those corresponding scores of groups S⫹A⫺ and S⫹B⫺. Furthermore, the intake scores of groups S⫺A⫹ and S⫺B⫹ during the second, third, and fourth training days were significantly higher than those shown by these same groups during the first day. This was not the case in groups S⫹A⫺ and group S⫹B⫺ animals; their

intake scores did not differ between groups, nor across training days. Latency to perform the first nose poke during test was analyzed by a two-way ANOVA defined by the following independent factors: training group and evaluation group (same or different, relative to the state paired with sucrose

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

during training). This ANOVA indicated only a significant main effect of evaluation [F(1,88) ⫽ 4.01, P ⬍ .05]. When animals were evaluated under the same state in which they originally had access to sucrose, latency to seek this stimulus was significantly shorter than when rats were evaluated under a different state (overall mean ⫾ S.E.M. collapsed across training groups: same ⫽ 3.51 ⫾ 0.36 s; different ⫽ 6.70 ⫾

1.59 s). Latency scores of the different groups have been illustrated in Fig. 6. Mean nose-poke duration during test was analyzed with the same form of ANOVA used for latencies. Only the training factor exerted a significant effect on nose-poke mean duration [F(3,88) ⫽ 3.09, P ⬍ .05]. Additional post hoc tests indicated that nose-poke duration was significantly higher in

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

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groups S⫺A⫹ and S⫺B⫹ in comparison with findings for groups S⫹A⫺ and S⫹B⫺, respectively. No differences were encountered when contrasting the values for groups S⫺A⫹ and S⫺B⫹ or for groups S⫹A⫺ and S⫹B⫺. These data have been illustrated in Fig. 7. As could be expected, differences in BECs corresponding to state A or B after test were consistent with the results obtained in Experiments 1 and 2. Those subjects that were tested 5 min after ethanol administration had significantly higher BECs than did adolescents tested at postadministration time 30 min (Student t test for independent groups: t ⫽ 9.56, df ⫽ 46, P ⬍ .001). Latency scores, mean nosepoke duration, and BECs for all independent groups have been incorporated in Table 2. In the current experiment, there was empirical evidence to support the possibility that adolescents are able to discriminate a moderate ethanol toxic state from a non-drug state. When the state associated with sucrose during training was replicated during the test, animals exhibited shorter nosepoke latencies than when the state varied between these phases of the experiment. Unexpectedly, those animals trained with access to sucrose while intoxicated with ethanol (groups S⫺A⫹ and S⫺B⫹) not only increased sucrose intake after the first association between these stimuli, but also exhibited a greater nose-poke mean duration than the other groups during test. The possibility that ethanol intoxication during training acts as an appetitive US seems to aid in the understanding of these results. This hypothesis obviously implies that the postabsorptive effects of a low dose of ethanol are

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Table 2 Behavioral scores registered in Experiment 3 during evaluation procedures and the corresponding blood ethanol concentrations (BECs) Training group

State at test

Nose-poke latency (s)

Nose-poke mean duration (s)

BECs after test (mg%)

S⫹A⫺

A S A S B S B S

5.70 ⫾ 3.06 2.82 ⫾ 0.60 3.66 ⫾ 0.87 9.31 ⫾ 3.56 3.58 ⫾ 0.96 3.80 ⫾ 0.61 3.74 ⫾ 0.78 8.45 ⫾ 4.36

2.18 ⫾ 0.27 2.02 ⫾ 0.23 2.36 ⫾ 0.29 3.06 ⫾ 0.35 1.96 ⫾ 0.23 2.05 ⫾ 0.23 2.88 ⫾ 0.41 2.41 ⫾ 0.28

42.68 ⫾ 3.43 0.00 44.17 ⫾ 2.15 0.00 22.32 ⫾ 1.49 0.00 21.95 ⫾ 1.20 0.00

S⫺A⫹ S⫹B⫺ S⫺B⫹

All values are expressed as mean ⫾ S.E.M. A ⫽ State in which BECs peaked (corresponding to 5–15 postadministration time); B ⫽ state occuring during the falling limb of the pharmacokinetic curve for BECs (corresponding to 30–40 min postadministration time); S ⫽ non-drug state (sobriety); “⫹” ⫽ access to sucrose; “⫺” ⫽ no access to sucrose.

an effective reinforcer, capable of supporting conditioning to sucrose’s sensory cues. 5. General discussion The original goal of the current study was to examine whether young rats are able to discriminate interoceptive effects of a low dose (0.5 g/kg) of ethanol after having relatively little experience with the drug. The first step was to select two postadministration periods, characterized by significantly different BECs, as distinctive states of intoxication (Experiment 1). These temporal parameters were used in a relatively simple behavioral study in which availability

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

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of a palatable sucrose solution through nose poking was associated with one state but not with the other. Tests of differential nose poking in the different ethanol-induced states associated with sucrose or no sucrose, however, provided no evidence that the adolescent rats discriminated these states (Experiment 2). When animals experienced either an ethanol-induced state associated with sucrose and a non-drug state associated with absence of this reinforcer or vice versa, however, behavioral evidence of state discrimination was obtained (Experiment 3). In Experiment 3, adolescents exhibited shorter nose-poke latencies when in the same state in which sucrose had been available. The results of Experiment 3 not only indicated discrimination between drug versus non-drug states, but also supported the suggestion that, at this age, the low dose (0.5 g/kg) of ethanol can support appetitive conditioning to the taste of sucrose. In the current study, a specific drug state was associated with sucrose availability, whereas an alternative state was not. This training procedure is analogous to the procedures used by Winter (1975) and York (1978), in which, ideally, animals learn to respond or to refrain from responding as a function of a given drug state that was or was not previously associated with delivery of an appetitive stimulus. The procedure was selected to rapidly train and test subjects within the ontogenetic stage of adolescence, and it was effective in the current study for discriminating either of two states of ethanol intoxication from a sober state. As mentioned, results of different studies, with the use of a wide range of training procedures, endorse the capability of adult rats to exhibit ethanol-mediated SDL (Bruins Slot et al., 1999; Holloway, 1972; Lowe, 1986; Nakagawa & Iwasaki, 1995). Nevertheless, Bruins Slot et al. (1999) found ethanol-induced SDL in adult rats only with a higher dose (1.25 g/kg, i.p.) relative to the one used in the current study. There were no indications of adult learning when ethanol doses equivalent to 0.32 or 0.64 g/kg were used. The findings of the current study indicate that periadolescents rapidly encode discriminative properties of the state of intoxication even when a 0.5-g/kg dose of ethanol is used. Also, Hunt et al. (1990) reported ethanol-mediated SDL in preweanling rats when a mild dose (0.4 g/kg, i.g.) of ethanol was used. Despite the need to consider different factors across studies (e.g., training procedures, modes of expression, and routes of administration), it seems that early in ontogeny the rat is highly responsive to discriminative properties of the acute state of intoxication induced by relatively low doses of ethanol. In terms of explicit comparisons (see Fig. 6), it seems that young rats that always experienced sucrose under either of the states of ethanol intoxication (groups S⫺A⫹ and S⫺B⫹) took longer to exhibit the first nose poke when tested under a sober state than when tested under the state associated with sucrose availability. A weaker effect of this kind occurred for rats that originally had access to sucrose while not intoxicated (groups S⫹A⫺ and S⫹B⫺). It is possible that state discrimination is more likely to occur for

subjects in which the toxic state was associated with sucrose than when a non-drug state was paired with this appetitive stimulus, similar to a common feature of discrimination learning, termed feature-positive effect (Sainsbury, 1971). Hence, the possibility of asymmetrical state discrimination should not be ruled out. During training, the contingency between ethanol intoxication and sucrose presence seems to be optimal in comparison with the contingency between non-drug state and sucrose availability. In other words, sobriety represents the animal’s most common state, not experienced only when the organism is placed in a particular environment in which an appetitive stimulus such as sucrose is available. It may also be significant that for animals in groups S⫹A⫺ and S⫹B⫺, state was confounded by temporal order in that the sober state was associated with sucrose and also with being placed in the training chamber for the first time during each conditioning session. As previously discussed, temporal cues (sequence) can be encoded readily as relevant signals that will affect the expression of a particular memory (Capaldi et al., 1986; La Fiette et al., 1994). In the test situation, all animals were placed in the apparatus only once, hence the “first” occasion in that session and so likely to yield maximal responsiveness in terms of sucroseseeking behavior. Therefore, both drug state and temporal sequence might have determined test performance. This hypothesis also seems to apply to the results of Experiment 2, in which animals were trained only while intoxicated and there was no evidence for discrimination of alternative ethanol-induced states. In this experiment, rats that experienced sucrose when first placed in the conditioning chamber during training (group A⫹B⫺) showed shorter latencies in the test situation than did animals that had access to this appetitive stimulus during the second phase of each training session (group A⫺B⫹). Ethanol has been described as a drug with unspecific pharmacological actions relative to other drugs, such as benzodiazepines and barbiturates. Results of drug-discrimination studies, in which animals have been trained to exhibit a given response under ethanol intoxication, have shown that this response also occurs when the animals are tested under several other drug states (e.g., benzodiazepines) but not vice versa (Barry, 1974; Barry & Krimmer, 1977; Rees et al., 1987). This result supports the suggestion that, as a discriminative stimulus, ethanol has diverse interoceptive attributes (Barry, 1991). Likewise, the pharmacological effects of ethanol have been defined as a stimulus complex with a redundant nature. In drug-discrimination procedures, this stimulus redundancy can determine overlapping qualities of ethanol across doses, postadministration intervals, or both (Grant, 1999). In other words, one interoceptive state derived from a given ethanol dose or postadministration interval could have an ample spectrum of effects that includes the discriminative effects of other ethanol-generated interoceptive states. This overlapping could impair the discrimination between such drug states. This phenomenon could help explain the discrimination failure observed for the two ethanol-induced

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states in Experiment 2. Furthermore, it is operationally impossible to avoid the effects of peak BECs (state A in Experiment 2) when the experimental design includes the need to present sucrose during the effects of lower BECs registered within the falling limb of the ethanol elimination curve (state B). Under these circumstances, the possibility exists relative to close temporal proximity between the first toxic state (A) and sucrose presentation during the following toxic state (B). This contiguity factor may be sufficient to support the association between the first state and the appetitive taste and hence impede the expression of discrimination across these hypothetically separable states. Results of Experiment 3 indicated that adolescents were able to discriminate an ethanol-induced state of intoxication from a non-drug state. Results also showed that ethanol was probably acting not only as a relatively redundant stimulus, but also as an appetitive US. Sucrose, which was used to establish the process of drug state discrimination, has a distinct flavor likely to become associated with unconditioned stimuli, such as lithium chloride (Eylam et al., 2000; Fouquet et al., 2001), morphine withdrawal (McDonald et al., 1997), and apomorphine (Wang et al., 1997). Both infant (Hunt et al., 1990) and adult (Co´rdoba et al., 1990) rats readily avoid this flavor when previously paired with postabsorptive consequences of ethanol doses (higher than the dose in the current study). In genetically heterogeneous rat strains, ethanol has generally been found to act as an aversive stimulus when associated with either chemosensory (Abate et al., 2001; Deutsch & Eisner, 1977; Eckardt, 1975) or tactile and visual (Cunningham et al., 1993; Gauvin & Holloway, 1992; Schechter & Krimmer, 1992) cues. Extensive training procedures (Bozarth, 1990), long-term preexposure to the interoceptive effects of the drug (Reid et al., 1985), concurrent presentation of other reinforcers [for example, food (Stewart & Grupp, 1985) or morphine (Marglin et al., 1988)], or stressful events (Matsuzawa et al., 2000) have been necessary to attain ethanolmediated conditioned preferences in heterogeneous rats. It is interesting to note that, in Experiment 3, the possibility of a relatively mild stressor could have favored heightened sucrose-intake patterns in groups S⫺A⫹ and S⫺B⫹. The sham intragastric administration procedure can imply stressrelated effects that could be alleviated through the anxiolytic effects of ethanol (Pohorecky, 1981) and, hence, facilitate sucrose intake. Under this perspective, negative-reinforcing or appetitive-reinforcing effects (or both types of effects) of ethanol could have determined sucrose-preference patterns in these groups relative to pertinent controls and modulated subsequent seeking behavior of the taste originally paired with the drug. It was recently suggested that characteristics of the adolescent brain predispose the adolescent to be highly responsive to appetitive properties of different drugs of abuse, including ethanol (Spear, 2000). The age factor, in conjunction with different procedural characteristics of the current experiments, might help explain the unexpected outcome in

57

relation to the US properties of ethanol. The dose (0.5 g/ kg) of ethanol used in the current study is lower than the doses reported to support aversive conditioning (normally, equal to or higher than 1 g/kg). Also, the strength of the association between sucrose and ethanol was probably favored by at least two factors. First, when appetitive conditioning became evident (Experiment 3), animals had been trained in a distinct environment under a non-drug condition in which sucrose was explicitly absent and in the same environment under the effects of ethanol paired with this tastant. Preexposure to the exteroceptive cues in which sucrose–ethanol pairings took place probably diminishes the possibility that other cues will interfere with the establishment of the associative memory between sucrose and the ethanol intoxication (Ferna´ndez-Vidal & Molina, 2001). Second, the lack of interference effects on the acquisition of this memory is probably also favored by a rather short process of intoxication. One hour after administration of the 0.5-g/kg dose of ethanol, the drug was practically undetectable in blood (Experiment 1). Hence, because of the low ethanol dose and its pharmacokinetic profile, the possibility that other cues will become associated with the toxic effects of ethanol seems minimized. With regard to this last issue, it is also important to note that, after training, adolescents were placed in a holding chamber that was very similar to the home environment. This implies a lack of salient cues that might retroactively compete with the signaling properties of sucrose relative to the effects of ethanol. It could be argued that the results of Experiment 3 do not reflect the reinforcing properties of ethanol but, rather, indicate the development of ethanol-mediated conditioned taste aversions (Cappell et al., 1973; Cunningham, 1979; Hunt et al., 1990). According to this perspective, groups S⫹A⫺ and S⫹B⫺ could be drinking less sucrose relative to the amount consumed by groups S⫺A⫹ and S⫺B⫹ because in animals in the former group the sweet solution sensed when first exposed to the black box becomes associated with later aversive effects derived from the state of ethanol intoxication. The following considerations argue against this possibility. Results of different studies have demonstrated that low doses (lower than 0.8 g/kg) of ethanol paired with flavors or alternative environmental cues fail to establish conditioned aversions (Cappell et al., 1973; Cunningham et al., 1993; Lester et al., 1970; Sherman et al., 1983; Stewart & Grupp, 1985; van der Kooy et al., 1983). Furthermore, results of a recently conducted study (unpublished observations, J. M. Ferna´ndez-Vidal, N. E. Spear, & J. C. Molina, 2003) also seem to argue against the possibility of conditioned taste aversions. We examined whether the heightened sucrose intake observed in S⫺A⫹ rats is more likely to occur when alternative training procedures are used. The complete design included groups S⫺A⫹ and S⫹A⫺, as well as S⫹ and A⫹ alone groups. The S⫹ rats received sucrose in the black box, followed 5 h later by intragastric administration of ethanol (0.5 g/kg) in the

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home cage. The A⫹ rats received sucrose paired with intoxication in the black box, followed 5 h later by a sham intragastric intubation in the home cage. These procedures allowed us to keep ethanol-related experiences and intubations constant across groups. The S⫹ group implies a longer delay between the CS and US than the one used in group S⫹A⫺. There were no differences in sucrose intake between groups during the first training day. In subsequent days, S⫺A⫹ subjects drank significantly more than did subjects in any remaining conditions. If conditioned taste aversions are derived from forward pairings between sucrose and ethanol, it would be logical to expect lesser consumption in S⫹A⫺ rats when compared with findings for the S⫹ alone group because of notable differences in delays between the CS and US. These groups failed to differ between them and both drank significantly less than did S⫺A⫹ periadolescents. Interestingly, A⫹ rats also failed to exhibit enhanced sucrose-consumption patterns. This latter result argues against nonspecific activating or dipsogenic effects of ethanol leading to increases in intake behavior and supports the suggestion that discriminative procedures could facilitate acquisition of ethanol-mediated sucrose-acceptance patterns. In Experiment 2, sucrose was presented when BECs either were at peak or during the falling limb of the pharmacokinetic curve. The design of this experiment does not allow focusing on the possibility of ethanol as an appetitive reinforcer. All groups (A⫹B⫺ and A⫺B⫹) tasted sucrose while experiencing the postabsorptive effects of ethanol. Interestingly, in this experiment, sucrose consumption increased after the first day of training. Nevertheless, this increase seems to be of a lesser magnitude than the one observed in Experiment 3 in group S⫺A⫹ or group S⫺B⫹ (for explicit comparisons, see Figs. 2 and 3). In these last groups (S⫺A⫹ and S⫺B⫹), the contingency between sucrose and the pharmacological effects of ethanol in a distinctive training environment seems optimal. Under a non-drug state, sucrose was absent, whereas under the state of intoxication, sucrose was present. In Experiment 2, comparable contingencies are less optimal: Although these rats also were placed into the training environment on two occasions, on both of these they were under the effects of ethanol and only once was sucrose available. These results indicate that, within relatively brief periods, the adolescent rat is capable of learning about specific characteristics of the acute state of intoxication. Under appropriate experimental circumstances, these young organisms seem highly sensitive to positive hedonic consequences of the state of intoxication. This ontogenetic consideration should be tempered by the fact that appetitive effects of ethanol have been described in food-deprived adult rats as a consequence of the caloric properties of ethanol (Sherman et al., 1983). It cannot be completely dismissed that a similar mechanism was responsible for the effects reported in this article, despite the fact that the regimen of fluid deprivation and, hence, possible partial food deprivation is markedly minor when compared with the one used by Sherman et al.

(1983). Yet, it is necessary to note that periadolescence is characterized by a unique rate of growth associated with the greatest caloric intake relative to body weight of any time in the life span (Nance, 1983; Spear, 2000). It remains to be determined whether the age factor, the present discrimination procedures, or both facilitated the emergence of phenomena reported in this article that have been rarely observed in heterogeneous strains of rats.

Acknowledgments This work was supported by grant PICT 5-7053 from Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, grant Ramo´n Carrillo and Arturo On˜ativia from Ministerio de Salud, Argentina (J.C.M.), and grant RO1AA10223 and grant RO1AA11960 from NIAAA (N.E.S.), as well as by fellowship from Fundacio´n Interior Argentina and fellowship from Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas awarded to J.M.F.V. We wish to express our gratitude to Beatriz Haymal and Teri Tanenhaus for their technical assistance.

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