The Role of Acetaldehyde in the Increased Acceptance of Ethanol after Prenatal Ethanol Exposure.

Recent studies show that acetaldehyde, the first metabolite in the oxidation of ethanol, can be responsible for both, the appetitive and the aversive effects produced by ethanol intoxication. More specifically, it has been hypothesized that acetaldehyde produced in the periphery by the liver is responsible for the aversive effects of ethanol, while the appetitive effects relate to the acetaldehyde produced centrally through the catalase system. On the other hand, from studies in our and other laboratories, it is known that ethanol exposure during the last gestational days (GD) consistently enhances the postnatal acceptance of ethanol when measured during early ontogeny in the rat. This increased liking of ethanol is a conditioned appetitive response acquired by the fetus by the association of ethanol's flavor and an appetitive reinforcer. Although this reinforcer has not yet been fully identified, one possibility points to acetaldehyde produced centrally in the fetus as a likely candidate. This hypothesis is supported by data showing that very early in the rat's ontogeny brain catalases are functional, while the liver's enzymatic system is still immature. In this study, rat dams were administered on GD 17-20 with water or ethanol, together with an acetaldehyde-sequestering agent (D-penicillamine). The offspring's responses to ethanol was then assessed at different postnatal stages with procedures adequate for each developmental stage: on day 1, using the "odor crawling locomotion test" to measure ethanol's odor attractiveness; on day 5, in an operant conditioning procedure with ethanol as the reinforcer; and on day 14 in an ethanol intake test. Results show that the absence of acetaldehyde during prenatal ethanol exposure impeded the observation of the increased acceptance of ethanol at any age. This seems to confirm the crucial role of acetaldehyde as a reinforcer in the appetitive learning occurring during prenatal ethanol exposure.

Neonatal sensitization to ethanol-induced breathing disruptions as a function of late prenatal exposure to the drug in the rat: modulatory effects of ethanol's chemosensory cues.

Preclinical and clinical studies have systematically demonstrated abrupt changes in fetal respiratory patterns when the unborn organism is exposed to the effects of maternal ethanol intoxication. In subprimates, chronic exposure to this drug during gestation and infancy results in marked alterations of the plasticity of the respiratory network. These alterations are manifested in terms of an early incapability to overcome deleterious effects of hypoxic events as well as in terms of sensitization to ethanol's depressant effects upon breathing patterns. It has also been demonstrated that near term rat fetuses process ethanol's chemosensory cues when the drug contaminates the amniotic fluid and that associative learning processes occur due to the temporal contiguity existing between these cues and different ethanol-related physiological effects. In the present study during the course of late gestation (gestational days 17-20), pregnant rats were intragastrically administered with either 0.0 or 2.0 g/kg ethanol. Seven-day-old pups derived of these dams were evaluated in terms of respiration rates (breaths/min) and apneas when subjected to different experimental conditions. These conditions were defined by postnatal exposure to the drug (intragastric administrations of either 0.0, 0.5, 1.0 or 2.0 g/kg ethanol), postadministration time of evaluation (5-10 or 30-35 min) and olfactory context at test (no explicit ambient odor or ethanol ambient odor). The results, obtained via whole body plethysmography, indicated that brief prenatal experience with the drug sensitized the organisms to ethanol's depressant effects particularly when employing the higher ethanol doses. In turn, presence of ethanol odor at test potentiated the above mentioned respiratory alterations. Prenatal treatment with ethanol was not found to alter pharmacokinetic profiles resulting from postnatal exposure to the drug or to affect different morphometric parameters related with lung development. These results indicate that even brief exposure to the drug during late gestation is sufficient to sensitize the organism to later disruptive effects of the drug upon breathing responsiveness. These deficits are potentiated through the re-exposure to the olfactory context perceived in utero which is known to be associated with ethanol's unconditioned effects. As a function of these observations it is possible to suggest a critical role of fetal sensory and learning capabilities in terms of modulating later ethanol-related breathing disruptions.

Brief prenatal ethanol exposure alters behavioral sensitivity to the kappa opioid receptor agonist (U62,066E) and antagonist (Nor-BNI) and reduces kappa opioid receptor expression.

BACKGROUND: Approximately 10 to 15% of women consume alcohol (ethanol [EtOH]) during pregnancy in the United States. Even low amounts of EtOH consumption during pregnancy can elicit long-term consequences. Prenatal experience with as few as 3 drinks has been associated with increase problem drinking in adulthood. Such effects are corroborated in rodents; however, the underlying neural adaptations contributing to this effect are not clear. In the current set of experiments, we investigated whether changes in EtOH responding following prenatal EtOH exposure involved kappa opioid receptor activation and expression. METHODS: Sprague-Dawley rats were prenatally exposed to low levels of alcohol (1.0 g/kg) during late gestation (gestational days 17 to 20 [GD17-20]) via intragastric intubation of pregnant dams. Following birth, EtOH intake, kappa- and mu-opioid-induced place conditioning, and kappa opioid receptor expression in mesolimbic brain regions were assessed in infant rats (postnatal days 14 to 15 [PD14-15]) that were offspring of dams given EtOH, vehicle, or untreated, during pregnancy. RESULTS: Animals exposed to prenatal alcohol drank more alcohol later in life and exhibited significant changes in the kappa opioid system. While control subjects found kappa opioid activation aversive, animals exposed to EtOH prenatally exhibited either no aversion or appetitive responding. Further analysis revealed that synaptosomal kappa opioid receptor expression was significantly decreased in brain areas implicated in responding to EtOH. CONCLUSIONS: Overall, these data suggest that prenatal EtOH affects kappa opioid function and expression and that these changes may be involved in increased drinking later in life.

New evidence of ethanol's anxiolytic properties in the infant rat.

Ethanol induces appetitive, aversive, and anxiolytic effects that are involved in the development of ethanol use and dependence. Because early ethanol exposure produces later increased responsiveness to ethanol, considerable effort has been devoted to analysis of ethanol's appetitive and aversive properties during early ontogeny. Yet, there is a relative scarcity of research related to the anxiolytic effects of ethanol during early infancy, perhaps explained by a lack of age-appropriate tests. The main aim of this study was to validate a model for the assessment of ethanol's anxiolytic effects in the infant rat (postnatal days 13-16). The potentially anxiolytic effects of ethanol tested included: i) amelioration of conditioned place aversion, ii) ethanol intake in the presence of an aversive conditioned stimulus, iii) the inhibitory behavioral effect in an anxiogenic environment, and iv) innate aversion to a brightly illuminated area in a modified light/dark paradigm. Ethanol doses employed across experiments were 0.0, 0.5, and 2.0 g/kg. Results indicated that a low ethanol dose (0.5 g/kg) was effective in attenuating expression of a conditioned aversion. Ethanol intake, however, was unaffected by simultaneous exposure to an aversive stimulus. An anxiogenic environment diminished ethanol-induced locomotor stimulation. Finally, animals given 0.5 g/kg ethanol and evaluated in a light/dark box showed increased time spent in the illuminated area and increased latency to escape from the brightly lit compartment than rats treated with a higher dose of ethanol or vehicle. These new results suggest that ethanol doses as low as 0.5 g/kg are effective in ameliorating an aversive and/or anxiogenic state in preweanling rats. These behavioral preparations can be used to assess ethanol's anxiolytic properties during early development.

Prenatal ethanol increases sucrose reinforcement, an effect strengthened by postnatal association of ethanol and sucrose.

Late prenatal exposure to ethanol recruits sensory processing of the drug and of its motivational properties, an experience that leads to heightened ethanol affinity. Recent studies indicate common sensory and neurobiological substrates between this drug and sweet tastants. Using a recently developed operant conditioning technique for infant rats, we examined the effects of prenatal ethanol history upon sucrose self-administration (postnatal days, PDs 14-17). Prior to the last conditioning session, a low (0.5 g/kg) or a high (2.5 g/kg) ethanol dose were paired with sucrose. The intention was to determine if ethanol would inflate or devalue the reinforcing capability of the tastant and if these effects are dependent upon prenatal ethanol history. Male and female pups prenatally exposed to ethanol (2.0 g/kg) responded more when reinforced with sucrose than pups lacking this antenatal experience. Independently of prenatal status, a low ethanol dose (0.5 g/kg) enhanced the reinforcing capability of sucrose while the highest dose (2.5 g/kg) seemed to ameliorate the motivational properties of the tastant. During extinction (PD 18), two factors were critical in determining persistence of responding despite reinforcement omission. Pups prenatally exposed to ethanol that subsequently experienced the low ethanol dose paired with sucrose, showed higher resistance to extinction. The effects here reported were not associated with differential blood alcohol levels across prenatal treatments. These results indicate that fetal ethanol experience promotes affinity for a natural sweet reinforcer and that low doses of ethanol are also capable of enhancing the positive motivational consequences of sucrose when ethanol and sucrose are paired during infancy.

Central effects of ethanol interact with endogenous mu-opioid activity to control isolation-induced analgesia in maternally separated infant rats.

Endogenous opioid activity plays an important role in ethanol consumption and reinforcement in infant rats. Opioid systems are also involved in mediation and regulation of stress responses. Social isolation is a stressful experience for preweanling rats and changes the effects of ethanol through opioid-dependent mechanisms. The present study assessed effects of intracisternal (i.c.) administration of a selective mu-opioid antagonist (CTOP) and i.p. administration of a nonspecific opioid antagonist (naloxone) on voluntary intake and behavior in socially isolated 12-day-old (P12) pups treated with 0.5 g/kg ethanol. Voluntary intake of 0.1% saccharin or water, locomotion, rearing activity, paw licking and grooming were assessed during short-term isolation from littermates (STSI; 8-min duration). Thermal nociceptive reactivity was measured before and after this intake test, with normalized differences between pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). Results indicated several effects of social isolation and ethanol mediated through the mu-opioid system. Effects of low dose ethanol (0.5 g/kg) and voluntary consumption of saccharin interacted with endogenous mu-opioid activity associated with STSI. Blockade of mu-opioid receptors on saccharin consumption and paw licking-grooming affected intoxicated animals. Low dose ethanol and ingestion of saccharin blunted effects of CTOP on rearing behavior and nociceptive reactivity. Central injections of CTOP stimulated paw licking and grooming dependent on ethanol dose and type of fluid ingested. Ethanol selectively increased saccharin intake during STSI in females, naloxone and CTOP blocked ethanol-mediated enhancement of saccharin intake. We suggest that enhancement of saccharin intake by ethanol during STSI is the product of synergism between isolation-induced mu-opioid activity that increases the pup's sensitivity to appetitive taste stimulation and the anxiolytic effects of 0.5 g/kg ethanol that decreases behaviors otherwise competing with independent ingestive activity.

Binge ethanol intoxication heightens subsequent ethanol intake in adolescent, but not adult, rats.

A question still to be answered is whether ethanol initiation has a greater effect on ethanol consumption if it occurs during adolescence than in adulthood. This study assessed the effect of ethanol initiation during adolescence or adulthood on voluntary ethanol consumption when animals were still within the same age range. Adolescent or adult rats were given 5, 2, or 0 ethanol exposures. The animals were tested for ethanol consumption through two-bottle choice tests, before undergoing a 1-week deprivation. A two-bottle assessment was conducted after the deprivation. Adolescents, but not adults, given two ethanol administrations during initiation exhibited significantly higher ethanol intake during the pre-deprivation period. These adolescents also exhibited a threefold increase in ethanol intake after 7 days of drug withdrawal, when compared with controls. These findings suggest that very brief experience with binge ethanol intoxication in adolescence, but not in adulthood, impacts later predisposition to drink.

Prenatal exposure to ethanol during late gestation facilitates operant self-administration of the drug in 5-day-old rats.

Prenatal ethanol exposure modifies postnatal affinity to the drug, increasing the probability of ethanol use and abuse. The present study tested developing rats (5-day-old) in a novel operant technique to assess the degree of ethanol self-administration as a result of prenatal exposure to low ethanol doses during late gestation. On a single occasion during each of gestational days 17-20, pregnant rats were intragastrically administered ethanol 1 g/kg, or water (vehicle). On postnatal day 5, pups were tested on a novel operant conditioning procedure in which they learned to touch a sensor to obtain 0.1% saccharin, 3% ethanol, or 5% ethanol. Immediately after a 15-min training session, a 6-min extinction session was given in which operant behavior had no consequence. Pups were positioned on a smooth surface and had access to a touch-sensitive sensor. Physical contact with the sensor activated an infusion pump, which served to deliver an intraoral solution as reinforcement (Paired group). A Yoked control animal evaluated at the same time received the reinforcer when its corresponding Paired pup touched the sensor. Operant behavior to gain access to 3% ethanol was facilitated by prenatal exposure to ethanol during late gestation. In contrast, operant learning reflecting ethanol reinforcement did not occur in control animals prenatally exposed to water only. Similarly, saccharin reinforcement was not affected by prenatal ethanol exposure. These results suggest that in 5-day-old rats, prenatal exposure to a low ethanol dose facilitates operant learning reinforced by intraoral administration of a low-concentration ethanol solution. This emphasizes the importance of intrauterine experiences with ethanol in later susceptibility to drug reinforcement. The present operant conditioning technique represents an alternative tool to assess self-administration and seeking behavior during early stages of development.

Maternal isolation during the first two postnatal weeks affects novelty-induced responses and sensitivity to ethanol-induced locomotor activity during infancy.

Animals exposed to chronic maternal separation (MS) exhibit enhanced ethanol self-administration and greater hormonal and behavioral responsiveness to stress in adulthood. Whether the effects of MS are immediately evident in infancy or whether they appear only later on development is still an unanswered question This study tested sensitivity to ethanol's behavioral stimulating effects in infant rats that experienced MS from postnatal Day 1-14. MS infants exhibited significantly greater reactivity to the motor stimulating effects of 1.25 g/kg ethanol than control animals, yet greater motor suppression after 2.5 g/kg ethanol. Baseline level of response to novelty was altered in MS infants, in a nor-binaltorphimine insensitive manner, that is, despite modified activity of the kappa-opioid system. These results indicate that the consequences of chronic maternal isolation emerge early in ontogeny, affecting ethanol sensitivity in infancy.

Olfactory preference for ethanol following social interaction with an intoxicated peer in adolescent rats exposed to ethanol in-utero.

BACKGROUND: Prenatal exposure to ethanol and later socially mediated exposure predicts ethanol intake in human adolescents. Animal rat models indicate that brief interactions with an ethanol-intoxicated peer result in heightened preference for ethanol odor and ethanol intake. METHODS: This study assessed preference for ethanol odor in adolescent male rats (observers) following social interaction with an ethanol intoxicated peer (demonstrators) as a function of prenatal ethanol exposure (gestational days 17-20, 1.0 g/kg, intragastric). Social behavior and locomotion during social interaction was also measured. RESULTS: Social investigation was greater in observers that interacted with an intoxicated demonstrator in comparison to those that interacted with a sober peer. Social contact increased when the demonstrator was under the effects of ethanol, but only if the observer had experienced ethanol prenatally. Ethanol inhibited locomotion in the demonstrators. Finally, social interaction with an intoxicated peer during adolescence as well as prenatal ethanol experience increased preference for ethanol odor. CONCLUSIONS: Fetal exposure to ethanol mediated by maternal intoxication at late gestation or by interaction with an intoxicated peer at adolescence heightens preference for the chemosensory cues of the drug.

Acetaldehyde reinforcement and motor reactivity in newborns with or without a prenatal history of alcohol exposure.

Animal models have shown that early ontogeny seems to be a period of enhanced affinity to ethanol. Interestingly, the catalase system that transforms ethanol (EtOH) into acetaldehyde (ACD) in the brain, is more active in the perinatal rat compared to adults. ACD has been found to share EtOH's behavioral effects. The general purpose of the present study was to assess ACD motivational and motor effects in newborn rats as a function of prenatal exposure to EtOH. Experiment 1 evaluated if ACD (0.35 µmol) or EtOH (0.02 µmol) supported appetitive conditioning in newborn pups prenatally exposed to EtOH. Experiment 2 tested if prenatal alcohol exposure modulated neonatal susceptibility to ACD's motor effects (ACD dose: 0, 0.35 and 0.52 µmol). Experiment 1 showed that EtOH and ACD supported appetitive conditioning independently of prenatal treatments. In Experiment 2, latency to display motor activity was altered only in neonates prenatally treated with water and challenged with the highest ACD dose. Prenatal EtOH experience results in tolerance to ACD's motor activity effects. These results show early susceptibility to ACD's appetitive effects and attenuation of motor effects as a function of prenatal history with EtOH, within a stage in development where brain ACD production seems higher than later in life.

Age-dependent effects of stress on ethanol-induced motor activity in rats.

RATIONALE: It is important to study age-related differences that may put adolescents at risk for alcohol-related problems. Adolescents seem less sensitive to the aversive effects of ethanol than adults. Less is known of appetitive effects of ethanol and stress modulation of these effects. OBJECTIVES: This study aims to describe the effects of acute social or restraint stress on ethanol-precipitated locomotor activity (LMA), in adolescent and adult rats. Effects of activation of the kappa system on ethanol-induced LMA were also evaluated. METHODS: Adolescent or adult rats were restrained for 90 min, exposed to social deprivation stress for 90 or 180 min or administered with the kappa agonist U62,066E before being given ethanol, and assessed for LMA. RESULTS: Adolescents were significantly more sensitive to the stimulating, and less sensitive to the sedative, effects of ethanol than adults. Basal locomotion was significantly increased by social deprivation stress in adult, but not in adolescent, rats. U62,066E significantly reduced basal and ethanol-induced locomotion in the adolescents. Corticosterone and progesterone levels were significantly higher in adolescents than in adults. CONCLUSIONS: Adolescents exhibit greater sensitivity to ethanol-induced LMA and reduced sensitivity to ethanol-induced motor sedation than adult rats. Ethanol's effects on motor activity were not affected by acute stress. Unlike adults, adolescents were insensitive to acute restraint and social deprivation stress but exhibited motor depression after activation of the endogenous kappa opioid receptor system.

Participation of the nociceptin/orphanin FQ receptor in ethanol-mediated locomotor activation and ethanol intake in preweanling rats.

Activation of nociceptin/orphanin FQ (NOP) receptors seems to attenuate ethanol-induced reinforcement in adult rodents. Since early ethanol exposure results in later increased responsiveness to ethanol, it is important to analyze NOP receptor modulation of ethanol-related behaviors during early ontogeny. By measuring NOP involvement in ethanol intake and ethanol-induced locomotor activation, we analyzed the specific participation of NOP receptors on these ethanol-related behaviors in two-week-old rats. In each experiment animals were pre-treated with the endogenous ligand for this receptor (nociceptin/orphanin FQ at 0.0, 0.5, 1.0 or 2.0 µg) or a selective NOP antagonist (J-113397 at 0.0, 0.5, 2.0 or 5.0 mg/kg). Results indicated that activation of the nociceptin receptor system had no effect on ethanol or water intake, while blockade of the NOP receptor has an unspecific effect on consummatory behavior: J-113397 increased ethanol (at a dose of 0.5 mg/kg) and water intake (at 0.5 and 5.0 mg/kg). Ethanol-mediated locomotor stimulation was attenuated by activation of the NOP system (nociceptin at 1.0 and 2.0 µg). Nociceptin had no effect on basal locomotor activity. Blockade of NOP receptors did not modify ethanol-induced locomotor activation. Contrary to what has been reported for adult rodents, nociceptin failed to suppress intake of ethanol in infants. Attenuation of ethanol-induced stimulation by activation of NOP receptor system suggests an early role of this receptor in this ethanol-related behavior.

Prenatal ethanol exposure increases ethanol intake and reduces c-Fos expression in infralimbic cortex of adolescent rats.

Prenatal ethanol exposure significantly increases later predisposition for alcohol intake, but the mechanisms associated with this phenomenon remain hypothetical. This study analyzed (Experiment 1) ethanol intake in adolescent inbred WKAH/Hok Wistar rats prenatally exposed to ethanol (2.0g/kg) or vehicle, on gestational days 17-20. Subsequent Experiments (2, 3 and 4) tested several variables likely to underlie the effect of gestational ethanol on adolescent ethanol preference, including ethanol-induced locomotor activation (LMA), ethanol-induced emission of ultrasonic vocalizations (USVs) after exposure to a rough exteroceptive stimulus, and induction of the immediate early gene C-fos in brain areas associated with processing of reward stimuli and with the retrieval and extinction of associative learning. Prenatal ethanol induced a two-fold increase in ethanol intake. Adolescents exhibited significant ethanol-induced LMA, emitted more aversive than appetitive USVs, and postnatal ethanol administration significantly exacerbated the emission of USVs. These effects, however, were not affected by prenatal ethanol. Adolescents prenatally exposed to ethanol as fetuses exhibited reduced neural activity in infralimbic cortex (but not in prelimbic cortex or nucleus accumbens core or shell), an area that has been implicated in the extinction of drug-mediated associative memories. Ethanol metabolism was not affected by prenatal ethanol. Late gestational exposure to ethanol significantly heightened drinking in the adolescent offspring of an inbred rat strain. Ethanol-induced LMA and USVs were not associated with differential ethanol intake due to prenatal ethanol exposure. Prenatal ethanol, however, altered basal neural activity in the infralimbic prefrontal cortex. Future studies should analyze the functionality of medial prefrontal cortex after prenatal ethanol and its potential association with predisposition for heightened ethanol intake.

Ethanol-induced locomotor activity in adolescent rats and the relationship with ethanol-induced conditioned place preference and conditioned taste aversion.

Adolescent rats exhibit ethanol-induced locomotor activity (LMA), which is considered an index of ethanol's motivational properties likely to predict ethanol self-administration, but few studies have reported or correlated ethanol-induced LMA with conditioned place preference (CPP) by ethanol at this age. The present study assessed age-related differences in ethanol's motor stimulating effects and analyzed the association between ethanol-induced LMA and conventional measures of ethanol-induced reinforcement. Experiment 1 compared ethanol-induced LMA in adolescent and adult rats. Subsequent experiments analyzed ethanol-induced CPP and conditioned taste aversion (CTA) in adolescent rats evaluated for ethanol-induced LMA. Adolescent rats exhibit a robust LMA after high-dose ethanol. Ethanol-induced LMA was fairly similar across adolescents and adults. As expected, adolescents were sensitive to ethanol's aversive reinforcement, but they also exhibited CPP. These measures of ethanol reinforcement, however, were not related to ethanol-induced LMA. Spontaneous LMA in an open field was, however, negatively associated with ethanol-induced CTA.

The role of acetaldehyde in ethanol reinforcement assessed by Pavlovian conditioning in newborn rats.

RATIONALE: Animal studies indicate that central acetaldehyde, dependent on catalase metabolism of ethanol (EtOH), modulates ethanol reinforcement. Brain catalase activity and acetaldehyde (ACD) production are significantly higher in rat pups compare d with adults. Interestingly, infant rats show high EtOH affinity for alcohol consumption and are particularly sensitive to the drug's reinforcing effects. OBJECTIVES: We tested whether central ACD is necessary and sufficient to induce appetitive conditioning in newborn rats through the artificial nipple technique. METHODS: Vehicle, EtOH (100 mg%), and acetaldehyde (0.35 µmol) were administered into the cisterna magna (1 µl). Half of the animals also received a central administration of 75 µg (experiment 1) or 40 µg of D-penicillamine (experiment 2). Afterwards, pups were exposed to an olfactory cue (conditioned stimulus). One hour later, neonates were tested with an artificial nipple in the presence of the conditioned cue. Nipple attachment duration, mean grasp duration, and number of nipple disengagements served as dependent variables. RESULTS: Positive responses to the scented nipple occurred in neonates conditioned with EtOH or ACD (experiments 1 and 2). In experiment 1, there were indications that D-penicillamine weakened the reinforcing effects of EtOH and ACD. In experiment 2, D-penicillamine (40 µg) significantly inhibited appetitive conditioned responses dependent upon EtOH or ACD. CONCLUSIONS: Appetitive conditioning was observed when employing either central EtOH or ACD as unconditioned stimuli. Central abduction of ACD inhibited conditioned appetitive responsiveness to the surrogate nipple. Central ACD is involved in the determination or modulation of EtOH's motivational properties during early stages in development.

µ-Opioid blockade reduces ethanol effects on intake and behavior of the infant rat during short-term but not long-term social isolation.

Numerous findings in adult and infant rats have shown that the endogenous opioid system is involved in control of ethanol consumption and its reinforcing effects. Opioid systems are also involved in reactivity to social isolation with several factors (age, duration, and type of isolation) affecting this modulation. The present study investigated the effects of a selective mu-opioid antagonist CTOP (0, 0.1, 0.5mg/kg), ethanol (0, 0.5 g/kg), and the interaction of the two drugs on the behavioral consequences of two types of social isolation given to preweanling rats: 1) short-term social isolation from littermates (STSI, duration 8 min) and 2) relatively long-term (5h) isolation (LTSI) from the dam and littermates. Voluntary intake of saccharin, locomotion, rearing activity, paw licking, and grooming were assessed during an 8-min. intake test. Thermal nociceptive reactivity was also measured before and after the testing session with normalized differences in pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). The results indicate that pharmacological blockade of mu-opioid receptors by CTOP substantially attenuated ethanol's anxiolytic effects on the developing rat's reactions to social isolation. Some of these stress-attenuating effects of CTOP were observed only in animals exposed to short-term isolation (STSI) but not in pups isolated for 5h (LTSI). Ethanol selectively increased saccharin intake during STSI in females and CTOP blocked this effect. Ethanol decreased the magnitude of analgesia associated with STSI but had no effect on pain reactivity during LTSI. CTOP by itself did not affect IIA or saccharin intake in sober animals. The findings of the present experiments indicate that the anxiolytic effects of 0.5 g/kg ethanol on pups exposed to STSI are modulated by endogenous opioid activity.

Role of mu, delta and kappa opioid receptors in ethanol-reinforced operant responding in infant rats.

We recently observed that naloxone, a non-specific opioid antagonist, attenuated operant responding to ethanol in infant rats. Through the use of an operant conditioning technique, we aimed to analyze the specific participation of mu, delta, and kappa opioid receptors on ethanol reinforcement during the second postnatal week. In Experiment 1, infant rats (PDs 14-17) were trained to obtain 5, 7.5, 10, or 15% ethanol, by operant nose-poking. Experiment 2 tested blood ethanol levels (BELs) attained by operant behavior. In Experiment 3, at PDs 16-18, rats received CTOP (mu antagonist: 0.1 or 1.0 mg/kg), naltrindole (delta antagonist: 1.0 or 5.0 mg/kg) or saline before training. In Experiment 4, rats received nor-binaltorphimine (kappa antagonist: 10.0 or 30.0 mg/kg, a single injection after completion of PD 15 operant training), spiradoline mesylate (kappa agonist: 1.0 or 5.0 mg/kg; at PDs 16-18) or saline (PDs 16-18), before the conditioning. Experiments 5 and 6 assessed possible side effects of opioid drugs in locomotor activity (LA) and conditioned taste aversion (CTA). Ethanol at 7.5 and 10% promoted the highest levels of operant responding. BELs were 12-15 mg/dl. In Experiment 3 naltrindole (dose-response effect) and CTOP (the lowest dose) were effective in decreasing operant responding. Nor-binaltorphimine at 10.0 mg/kg and spiradoline at 5.0 mg/kg also blocked ethanol responding. The effects of opioid drugs on ethanol reinforcement cannot be explained by effects on LA or CTA. Even though particular aspects of each opioid receptor require further testing, a fully functional opioid system seems to be necessary for ethanol reinforcement, during early ontogeny.

Prenatal ethanol exposure leads to greater ethanol-induced appetitive reinforcement.

Prenatal ethanol significantly heightens later alcohol consumption, but the mechanisms that underlie this phenomenon are poorly understood. Little is known about the basis of 'this effect of prenatal ethanol on the sensitivity to ethanol's reinforcing effects. One possibility is that prenatal ethanol exposure makes subjects more sensitive to the appetitive effects of ethanol or less sensitive to ethanol's aversive consequences. The present study assessed ethanol-induced second-order conditioned place preference (CPP) and aversion and ethanol-induced conditioned taste aversion (CTA) in infant rats prenatally exposed to ethanol (2.0 g/kg) or vehicle (water) or left untreated. The involvement of the κ opioid receptor system in ethanol-induced CTA was also explored. When place conditioning occurred during the ascending limb of the blood-ethanol curve (Experiment 1), the pups exposed to ethanol in utero exhibited greater CPP than untreated controls, with a shift to the right of the dose-response curve. Conditioning during a later phase of intoxication (30-45 min post-administration; Experiment 2) resulted in place aversion in control pups exposed to vehicle during late gestation but not in pups that were exposed to ethanol in utero. Ethanol induced a reliable and similar CTA (Experiment 3) in the pups treated with vehicle or ethanol during gestation, and CTA was insensitive to κ antagonism. These results suggest that brief exposure to a moderate ethanol dose during late gestation promotes ethanol-mediated reinforcement and alters the expression of conditioned aversion by ethanol. This shift in the motivational reactivity to ethanol may be an underlying basis of the effect of prenatal ethanol on later ethanol acceptance.

Ontogenetic differences in ethanol's motivational properties during infancy.

Pairing a conditioned stimulus (CS) with ethanol generally produces aversion for that CS in adult rodents. However, infant rats (PD1-PD3) exposed to ethanol demonstrate appetitive reinforcement to ethanol (Nizhnikov, Varlinskaya, Petrov, & Spear, 2006; Petrov, Varlinskaya, & Spear, 2003). This sensitivity to the appetitive properties of ethanol during infancy may be transient, as during the second postnatal week rat pups tend to exhibit conditioned aversions to flavors paired with ethanol. The present study examined changes in the motivation properties of ethanol through ontogeny and the neurobiology underlying these changes. Rat pups were exposed to a taste conditioning procedure on PD4 or PD12. Rat pups were intraorally infused with 2.5% of their body weight of saccharin solution (0.1%) and immediately after injected intraperitoneolly (i.p.) with one of six doses of ethanol (0.0-2.0 g/kg). A day later pups were given saccharine infusions and percent body weight gain was used as an index of ethanol's reinforcing effects. PD4 pups expressed appetitive reinforcement to ethanol, as indicated by greater saccharin intake, as compared to control counterparts and to the older PD12 pups. Subsequent experiments revealed that PD4 pups were less sensitive to the aversive properties of the drug than PD12 pups. The older pups found high doses of ethanol aversive while PD4 rat pups did not condition aversions to this dose of ethanol after a single trial. A similar pattern of results was observed between the low doses of ethanol and the highest doses of a kappa opioid agonist. The PD12 animals did not condition to the kappa opioid agonist, while the younger rats expressed an appetitive response. These results illustrate an ontogenetic change in the motivational properties of ethanol, with sensitivity to its appetitive properties declining and responsiveness to the aversive properties increasing with age during early infancy.