Ricardo Marcos Pautassi

Adolescent, but not adult, rats exhibit ethanol-mediated appetitive second-order conditioning

BACKGROUND Adolescent rats are less sensitive to the sedative effects of ethanol than older animals. They also seem to perceive the reinforcing properties of ethanol. However, unlike neonates or infants, ethanol-mediated appetitive behavior is yet to be clearly shown in adolescents. Appetitive ethanol reinforcement was assessed in adolescent (postnatal day 33, P33) and adult rats (P71) through second-order conditioning (SOC). METHODS On P32 or P70, animals were intragastrically administered ethanol (0.5 or 2.0 g/kg) paired with intra-oral pulses of sucrose (CS(1), first-order conditioning phase). CS(1) delivery took place either 5-20 (early pairing) or 30-45 minutes (late pairing) following ethanol administration. The time interval between CS(1) exposure and ethanol administration was 240 minutes in unpaired controls. On P33 or P71, animals were presented the CS(1) (second-order conditioning phase) in a distinctive chamber (CS(2), second-order conditioning). Then they were tested for CS(2) preference. RESULTS Early and late paired adolescents, but not adults, had greater preference for the CS(2) than controls, a result indicative of ontogenetic variation in ethanol-mediated reinforcement. During the CS(1)-CS(2) associative phase, paired adolescents given 2.0 g/kg ethanol wall-climbed more than controls. Blood and brain ethanol levels associated with the 0.5 and 2.0 g/kg doses at the onset of each conditioning phase did not differ substantially across age, with mean blood ethanol concentration of 38 and 112 mg%. CONCLUSIONS These data indicate age-related differences between adolescent and adult rats in terms of sensitivity to ethanols motivational effects. Adolescents exhibited high sensitivity for ethanols appetitive effects. These animals also showed ethanol-mediated behavioral activation during the SOC phase. The SOC preparation provides a valuable conditioning model for assessing ethanols motivational effects across ontogeny.

Assessing appetitive, aversive, and negative ethanol-mediated reinforcement through an immature rat model.

The motivational effects of drugs play a key role during the transition from casual use to abuse and dependence. Ethanol reinforcement has been successfully studied through Pavlovian and operant conditioning in adult rats and mice genetically selected for their ready acceptance of ethanol. Another model for studying ethanol reinforcement is the immature (preweanling) rat, which consumes ethanol and exhibits the capacity to process tactile, odor and taste cues and transfer information between different sensorial modalities. This review describes the motivational effects of ethanol in preweanling, heterogeneous non-selected rats. Preweanlings exhibit ethanol-mediated conditioned taste avoidance and conditioned place aversion. Ethanols appetitive effects, however, are evident when using first- and second-order conditioning and operant procedures. Ethanol also devalues the motivational representation of aversive stimuli, suggesting early negative reinforcement. It seems that preweanlings are highly sensitive not only to the aversive motivational effects of ethanol but also to its positive and negative (anti-anxiety) reinforcement potential. The review underscores the advantages of using a developing rat to evaluate alcohols motivational effects.

Acute sensitivity and acute tolerance to ethanol in preweanling rats with or without prenatal experience with the drug

The present study examined behavioral sensitivity and acute tolerance to ethanol in infants with or without a moderate prenatal ethanol experience. During gestational days 17-20 dams received 0.0 or 2.0 g/kg ethanol. On postnatal day 13 pups were administered 0.0, 0.5 or 2.5 g/kg ethanol prior to assessment of locomotion. One third of the pups were evaluated at 5-10, 30-35 and 60-65 min after ethanol administration; another third was tested only during the last two post-administration periods; and the remaining third was tested only at 60-65 min. At 30-35 min blood ethanol levels were similar to those attained at 60-65 min. The main results of the study were: (a) The 2.5 g/kg ethanol dose induced biphasic motor effects: stimulation 5-10 min after drug administration and sedation after 30-35 or 60-65 min. (b) Infants exhibited acute tolerance to ethanols sedative effects. (c) Although pups prenatally treated with ethanol exhibited heightened locomotor activity levels, acute sensitivity and tolerance were not affected by prenatal treatment. In summary, infants are sensitive to biphasic motor consequences of ethanol and readily exhibit acute tolerance to ethanols sedative effects. In addition, moderate prenatal ethanol exposure was sufficient to induce hyper-reactivity in the offspring without affecting habituation.

Opioid antagonists block the acquisition of ethanol-mediated conditioned tactile preference in infant rats

It has been difficult to find conditioned preference for tactile cues paired with ethanol intoxication in rats. Toward understanding the ontogeny of ethanol reinforcement, we aimed at establishing a simple and reliable procedure for (1) assessing primary appetitive conditioning to ethanol in infant rats and (2) discerning the role the opioid system plays in ethanol-mediated conditioning at this age. Experiment 1 determined the parameters (i.e., dose, interval of conditioning) for assessing ethanol-mediated conditioning. Pups were then trained with differential Pavlovian conditioning (Experiments 2 and 3) in which ethanol intoxication (1.0-2.0 g/kg, intragastrically or intraperitoneally delivered) was paired with a tactile stimulus (sandpaper) while an alternative texture signaled the absence of ethanols effects. Unpaired control conditions were also used. Tactile preferences were assessed after two conditioning sessions. Paired rats spent significantly more time on sandpaper than unpaired controls, an effect that was greater after intragastric administration of 1.0 than 2.0 g/kg ethanol. This effect was replicated in Experiments 4a and 4c and found to be inhibited by pretreatment with general (naloxone [NAL]) or specific (d-Pen-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 [CTOP] and naltrindole) opioid antagonists. Blood ethanol levels at conditioning were not altered by NAL (Experiment 4b). The study outlines a procedure that reveals appetitive conditioning to ethanol by infant rats. The results are discussed in terms of a potential ethanol-induced activation of the endogenous opioid system during the onset of the intoxication process.

A comparison between taste avoidance and conditioned disgust reactions induced by ethanol and lithium chloride in preweanling rats

Adult rats display taste avoidance and disgust reactions when stimulated with gustatory stimuli previously paired with aversive agents such as lithium chloride (LiCl). By the second postnatal week of life, preweanling rats also display specific behaviors in response to a tastant conditioned stimulus (CS) that predicts LiCl-induced malaise. The present study compared conditioned disgust reactions induced by LiCl or ethanol (EtOH) in preweanling rats. In Experiment 1 we determined doses of ethanol and LiCl that exert similar levels of conditioned taste avoidance. After having equated drug dosage in terms of conditioned taste avoidance, 13-day-old rats were given a single pairing of a novel taste (saccharin) and either LiCl or ethanol (2.5 g/kg; Experiment 2). Saccharin intake and emission of disgust reactions were assessed 24 and 48 hr after training. Pups given paired presentations of saccharin and the aversive agents (ethanol or LiCl) consumed less saccharin during the first testing day than controls. These pups also showed more aversive behavioral reactions to the gustatory CS than controls. Specifically, increased amounts of grooming, general activity, head shaking, and wall climbing as well as reduced mouthing were observed in response to the CS. Conditioned aversive reactions but not taste avoidance were still evident on the second testing day. In conclusion, a taste CS paired with postabsorptive effects of EtOH and LiCl elicited a similar pattern of conditioned rejection reactions in preweanling rats. These results suggest that similar mechanisms may be underlying CTAs induced by LiCl and a relatively high EtOH dose.

Ethanol-mediated operant learning in the infant rat leads to increased ethanol intake during adolescence.

Recent studies indicate that the infant rat has high affinity for ethanol ingestion and marked sensitivity to the drugs reinforcing effects [Spear, N.E., Molina, J.C. Fetal or infantile exposure to ethanol promotes ethanol ingestion in adolescence and adulthood: a theoretical review. Alcohol Clin Exp Res 2005; 29: 909-29.]. A novel operant technique was developed to analyze reinforcing effects of ethanol delivery during the third postnatal week. The impact of this ethanol-reinforcement experience upon subsequent ethanol consumption during adolescence (postnatal weeks 5-6) was also examined. In Experiment 1, pups (postnatal days 14-17) were given an explicit contingency between nose-poking behavior and intraoral delivery of either water or 3.75% v/v ethanol (paired groups). Yoked controls (pups receiving either reinforcer independently of their behavior) were also included. Paired subjects reinforced with ethanol exhibited rapid and robust operant conditioning leading to blood ethanol concentrations in the 25-48 mg% range. In Experiment 2, a higher ethanol concentration (7.5% v/v) provided significant reinforcement. During adolescence, animals originally reinforced with 3.75% v/v ethanol exhibited greater ingestion of ethanol than control animals without prior ethanol reinforcement. These results indicate that, without extensive initiation to ethanol, infant rats rapidly learn to gain access to ethanol and that this experience has a significant impact upon later ethanol intake patterns.

Infant rats exhibit aversive learning mediated by ethanol's orosensory effects but are positively reinforced by ethanol's post-ingestive effects.

Previous work suggest aversive and appetitive hedonic effects of intraorally delivered EtOH in pre-weanling rats. Pups are reluctant to perform an operant response when reinforced with intraoral EtOH infusions, a result suggesting aversive orosensory properties of EtOH. Yet, post-absorptive effects of ethanol seem capable of supporting appetitive conditioning. Two experiments were conducted to test this phenomenon. Both included a pre-exposure phase (postnatal day 13, PD13) comprising intraoral stimulation with water or EtOH. In Experiment 1, pups were given pairings between a tactile conditioned stimulus (CS) and intraoral infusions of EtOH or water. A subsequent tactile preference test revealed that pups spent significantly less on the EtOH-related CS relative to time spent on the alternative CS. In Experiment 2 pups were exposed to a texture CS (sandpaper) while intraorally infused with EtOH or during a later EtOH post-infusion interval. A tactile locational test conducted on PD16 indicated that EtOH-pre-exposed animals that experienced sandpaper paired with EtOHs post-absorptive effects exhibited a significant preference for the CS, even relative to a control group that experienced non-reinforced exposure to the tactile CS during conditioning. These results confirm that intraoral ethanol acts as an aversive tastant. A brief pre-exposure to EtOH allows later expression of appetitive learning mediated by the drugs post-ingestive effects.

Domperidone interferes with conditioned disgust reactions but not taste avoidance evoked by a LiCl-paired taste in infant rats.

Rats exhibit taste avoidance and conditioned disgust reactions when stimulated with a tastant paired with lithium chloride (LiCl). Lithium-mediated activation of chemoreceptor nuclei at the brainstem appears to determine the acquisition of conditioned taste aversion (CTA) in adult rodents. Domperidone (DOM), an anti-emetic drug that does not cross the blood-brain barrier, was employed to analyze mechanisms underlying LiCl-mediated CTA in infant rats. On postnatal day 13 animals were given DOM followed by a pairing between intraoral saccharin and LiCl. Saccharin consumption at testing was lower in lithium-treated pups than in controls. DOM did not interfere with this LiCl-mediated taste avoidance but significantly decreased LiCl-mediated disgust reactions (head-shaking and wall climbing). Activation of the emetic system of the brainstem does not seem necessary for the acquisition of LiCl-mediated conditioned taste avoidance. Yet, these centers seem to be involved in the palatability shift resulting from taste-LiCl pairings. These results indicate an early dissociation between conditioned disgust reactions and conditioned taste avoidance.

Ethanol induces second-order aversive conditioning in adolescent and adult rats

Alcohol abuse and dependence are considered public health problems, with an etiological onset often occurring during late childhood and adolescence, and understanding age-related differences in ethanol sensitivity is important. Low to moderate ethanol doses (0.5 and 2.0 g/kg, intragastrically [i.g.]) induce single-trial, appetitive second-order place conditioning (SOC) in adolescent, but not adult, rats. Recent studies have demonstrated that adolescents may be less sensitive than adults to the aversive properties of ethanol, reflected by conditioned taste aversion. The present study assessed the aversive motivational effects of high-dose ethanol (3.0 and 3.25 g/kg, i.g., for adolescents and adults, respectively) using SOC. Experiment 1 revealed similar blood and brain ethanol levels in adolescent and adult rats given 3.0 and 3.25 g/kg ethanol, respectively. In Experiment 2, animals received ethanol or vehicle paired with intraoral pulses of sucrose (conditioned stimulus 1 [CS1]). After one, two, or three conditioning trials, the rats were presented with the CS1 while in a distinctive chamber (CS2). When tested for CS2 preference, ethanol-treated animals exhibited reduced preference for the CS2 compared with controls. This result, indicative of ethanol-mediated aversive place conditioning, was similar for adolescents and adults; for females and males; and after one, two, or three training trials. In conjunction with previous results, the present study showed that, in adolescent rats subjected to SOC, ethanols hedonic effects vary from appetitive to aversive as the ethanol dose increases. Adolescent and adult animals appear to perceive the postingestive effects of high-dose ethanol as similarly aversive when assessed by SOC.

Motivational effects of intraorally-infused ethanol in rat pups in an operant self-administration task

Motivational effects of self-administered ethanol have rarely been studied in preweanling rats due primarily to the lack of age-appropriate operant tasks. The present experiments assessed the hedonic effects of intraoral ethanol in infant rats self-administered by activating a touch sensor. On postnatal day (PD) 13 pups were pre-exposed to the drugs pharmacological and/or sensory effects. Operant sessions were conducted during PDs 14-16 (Experiments 1 and 2). Paired animals were placed in chambers equipped with a touch-sensitive disk and received an intraoral infusion of ethanol (3 or 5% v/v, 5 microl) after each sensor contact. Yoked controls were equated for number and distribution of ethanol infusions but had no control over the contingency between operant behavior and intraoral infusion. In Experiment 2, training trials were preceded by a non-reinforced phase. Paired pups performed fewer operant responses than controls and decreased their operant responses across sessions. These results suggest that intraoral self-administered ethanol has an aversive hedonic value in two-week old rats. Operant behavior seems to have been associated with aversive orosensory effects derived from intraoral ethanol infusion.