Transgenerational Transmission of the Effect of Gestational Ethanol Exposure on Ethanol Use-Related Behavior.

BACKGROUND: Prenatal alcohol exposure (PAE) enhances the risk for alcoholism by increasing the propensity to consume alcohol and altering neurophysiological response to alcohol challenge. Trans-generationally transmittable genetic alterations have been implicated in these behavioral changes. To date, transgenerational transmission of PAE-induced behavioral responses to alcohol has never been experimentally investigated. Therefore, we explored the transgenerational transmission of PAE-induced behavioral effects across 3 generations. METHODS: Pregnant Sprague Dawley dams received 1 g/kg ethanol (EtOH) or water daily on gestational days 17 through 20 via gavage, or remained untreated in their home cages. To produce second filial (F2) or F3 generations, similarly treated adult F1 or F2 offspring were mated and left undisturbed through gestation. On postnatal day (PND) 14, male and female F1, F2, and F3 offspring were tested for consumption of 5% (w/v) EtOH (in water), or water. Using the loss of righting reflex (LORR) paradigm on PND 42, F1 and F2 adolescent male offspring were tested for sensitivity to acute EtOH-induced sedation-hypnosis at 3.5 or 4.5 g/kg dose. F3 male adolescents were similarly tested at 3.5 g/kg dose. Blood EtOH concentration (BEC) was measured at waking. RESULTS: EtOH exposure increased EtOH consumption compared to both water and untreated control groups in all generations. EtOH-treated group F1 and F2 adolescents displayed attenuated LORR duration compared to the water group. No attenuated LORR was observed in the F3 generation. BEC at waking corroborated with the significant LORR duration differences while also revealing differences between untreated control and water groups in F1 and F2 generations. CONCLUSIONS: Our results provide novel behavioral evidence attesting that late gestational moderate EtOH exposure increases EtOH intake across 3 generations and may alter sensitivity to EtOH-induced sedation-hypnosis across 2 generations.

Prenatal ethanol increases ethanol intake throughout adolescence, alters ethanol-mediated aversive learning, and affects µ but not δ or κ opioid receptor mRNA expression.

Animal models of prenatal ethanol exposure (PEE) have indicated a facilitatory effect of PEE on adolescent ethanol intake, but few studies have assessed the effects of moderate PEE throughout adolescence. The mechanisms underlying this facilitatory effect remain largely unknown. In the present study, we analysed ethanol intake in male and female Wistar rats with or without PEE (2.0 g/kg, gestational days 17-20) from postnatal days 37 to 62. The results revealed greater ethanol consumption in PEE rats than in controls, which persisted throughout adolescence. By the end of testing, ethanol ingestion in PEE rats was nearly 6.0 g/kg. PEE was associated with insensitivity to ethanol-induced aversion. PEE and control rats were further analysed for levels of µ, δ and κ opioid receptor mRNA in the infralimbic cortex, nucleus accumbens shell, and ventral tegmental area. Similar levels of mRNA were observed across most areas and opioid receptors, but µ receptor mRNA in the ventral tegmental area was significantly increased by PEE. Unlike previous studies that assessed the effects of PEE on ethanol intake close to birth, or in only a few sessions during adolescence, the present study observed a facilitatory effect of PEE that lasted throughout adolescence. PEE was associated with insensitivity to the aversive effect of ethanol, and increased levels of µ opioid receptor transcripts. PEE is a prominent vulnerability factor that probably favors the engagement of adolescents in risky trajectories of ethanol use.

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.

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.

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.

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.

Ontogeny of ethanol intake in alcohol preferring (P) and alcohol nonpreferring (NP) rats.

There is a scarcity of research on ethanol affinity in alcohol-preferring (P) rats before weaning and it is unknown if neonate P rats exhibit ethanol intake preferences comparable to those observed in adult P rats. This study examined ethanol intake in P and alcohol-nonpreferring (NP) rats 3 hr after birth (Experiment 1, surrogate nipple test), at postnatal days (PD) 8, 12, and 18 (Experiment 2, consumption from the floor procedure) and at adolescence (Experiment 3, two-bottle choice test at PD32). The high-preference genotype was readily expressed 3 hr after birth. P neonates drank twice as much ethanol as their NP counterparts. This heightened ethanol preference transiently reversed at P8, reemerged as weaning approached (P18) and was fully expressed during adolescence. These results help to clarify the ontogeny of genetic predisposition for ethanol. Genetic predisposition for higher ethanol intake in P than in NP rats seems to be present immediately following birth.

High ethanol dose during early adolescence induces locomotor activation and increases subsequent ethanol intake during late adolescence.

Adolescent initiation of ethanol consumption is associated with subsequent heightened probability of ethanol use disorders. The present study examined the relationship between motivational sensitivity to ethanol initiation in adolescent rats and later ethanol intake. Experiment 1 determined that ethanol induces locomotor activation shortly after administration but not if tested at a later post-administration interval. In Experiment 2, adolescent rats were assessed for ethanol-induced locomotor activation on postnatal Day 28. These animals were then evaluated for ethanol-mediated conditioned taste aversion and underwent a 16-day-long ethanol intake protocol. Ethanol-mediated aversive effects were unrelated to ethanol locomotor stimulation or subsequent ethanol consumption patterns. Ethanol intake during late adolescence was greatest in animals initiated to ethanol earliest at postnatal Day 28. Females that were more sensitive to ethanol's locomotor-activating effects showed a transient increase in ethanol self-administration. Blood ethanol concentrations during initiation were not related to ethanol-induced locomotor activation. Adolescent rats appeared sensitive to the locomotor-stimulatory effects of ethanol. Even brief ethanol exposure during adolescence may promote later ethanol intake.

Offspring of male rats exposed to binge alcohol exhibit heightened ethanol intake at infancy and alterations in T-maze performance.

Alcohol use is associated with a variety of negative consequences, including heightened likelihood of cognitive impairment, proclivity to alcohol use disorders (AUD), and alterations in the drinker's offspring. Children and rodents exposed to alcohol during pregnancy, or those whose fathers consumed alcohol prior to mating, often exhibit neurodevelopmental, physiological, and behavioral deficits. The present study assessed cognitive function and alcohol intake in male and female rats that were offspring of alcohol-exposed fathers. Adult male rats were exposed to alcohol or vehicle (0.0 or 2.0 g/kg, respectively; twice daily for 2 days followed by a rest day, for a total of eight alcohol or vehicle exposure days), or were left untreated and then mated with non-manipulated females. The offspring were assessed for alcohol intake, via intraoral infusion, followed by cognitive assessment via an alternating T-maze task. The results indicated that paternal ethanol exposure, prior to breeding, resulted in offspring that consumed significantly more ethanol than vehicle or untreated controls. Furthermore, the offspring of alcohol-exposed fathers exhibited a significant failure to initiate and complete the T-maze performance tests. Although, when they did engage in the tests they performed at the level of controls (i.e., 80% correct). The present results add to a growing body of literature suggesting that paternal pre-conception alcohol exposure can have deleterious effects on the offspring.

Restraint stress exacerbates cell degeneration induced by acute binge ethanol in the adolescent, but not in the adult or middle-aged, brain.

Restraint stress (RS) induces neurotoxicity in the hippocampus, yet most of the studies have employed protracted RS (i.e., ≈ 21 days). Binge ethanol can induce brain toxicity, an effect affected by age. It could be postulated that RS may facilitate ethanol-induced neurotoxicity, perhaps to a greater extent in adolescent vs. older subjects. We analyzed whether adolescent, adult or middle-aged male rats exposed to five episodes of RS followed, 72h later, by binge ethanol (i.e., two administrations of 2.5 g/kg ethanol) exhibited hippocampal neurotoxicity. Adolescents, but not adult or middle-aged rats, exhibited sensitivity to the neurotoxic effects of ethanol at dorsal CA2, ventral CA3 and ventral DG, and a neurotoxic effect of stress at dorsal CA1. Moreover, the combination of ethanol and stress exerted a synergistic effect upon cell degeneration at ventral CA1 and CA2, which was restricted to adolescents. Ethanol also increased cell degeneration, irrespective of age or stress, in dorsal CA3 and in dorsal DG; and ethanol and stress had, across all ages, a synergistic effect upon cell degeneration at the dorsal CA1. The greater neurotoxic response of adolescents to ethanol, stress, or ethanol+stress can put them at risk for the development of alcohol problems.

Influence of prenatal pre-exposure to an odor on intake behavior of an aversive solution in newborn rats.

Early pre- or postnatal sensory experiences significantly influence flavor preference and food intake, and can induce liking for innately unpalatable flavors. Previous work found that newborn rats stimulated with an odor experienced shortly after birth exhibited heightened intake and seeking towards an artificial nipple containing quinine. This result suggests that odors made familiar trough early postnatal pre-exposure can shift the motivational value of unconditional stimuli. The objective of the current study was to assess the effect of an odor (lemon) experienced in-utero on the first intake responses towards an artificial nipple supplying quinine. The hypothesis, which was corroborated, was that stimulation with the olfactory stimulus experienced in-utero would increase the newborn's intake and grasp responses to the artificial nipple containing quinine. Exposure to the odor that had been pre-exposed in utero increased quinine intake and seeking (i.e., latency to grasp and total time in contact with the nipple, as well as number of and mean duration of nipple grasps) in 3-h-old pups. These results replicate those previously found with postnatal odor pre-exposure, and extend the phase for pre-exposure to the prenatal stage.

Central reinforcing effects of ethanol are blocked by catalase inhibition.

Recent studies have systematically indicated that newborn rats are highly sensitive to ethanol's positive reinforcing effects. Central administrations of ethanol (25-200mg %) associated with an olfactory conditioned stimulus (CS) promote subsequent conditioned approach to the CS as evaluated through the newborn's response to a surrogate nipple scented with the CS. It has been shown that ethanol's first metabolite, acetaldehyde, exerts significant reinforcing effects in the central nervous system. A significant amount of acetaldehyde is derived from ethanol metabolism via the catalase system. In newborn rats, catalase levels are particularly high in several brain structures. The present study tested the effect of catalase inhibition on central ethanol reinforcement. In the first experiment, pups experienced lemon odor either paired or unpaired with intracisternal (IC) administrations of 100mg% ethanol. Half of the animals corresponding to each learning condition were pretreated with IC administrations of either physiological saline or a catalase inhibitor (sodium-azide). Catalase inhibition completely suppressed ethanol reinforcement in paired groups without affecting responsiveness to the CS during conditioning or responding by unpaired control groups. A second experiment tested whether these effects were specific to ethanol reinforcement or due instead to general impairment in learning and expression capabilities. Central administration of an endogenous kappa opioid receptor agonist (dynorphin A-13) was used as an alternative source of reinforcement. Inhibition of the catalase system had no effect on the reinforcing properties of dynorphin. The present results support the hypothesis that ethanol metabolism regulated by the catalase system plays a critical role in determination of ethanol reinforcement in newborn rats.

Strain-specific programming of prenatal ethanol exposure across generations.

Behavioral consequences of prenatal alcohol exposure (PAE) can be transmitted from in utero-exposed F1 generation to their F2 offspring. This type of transmission is modulated by genetic and epigenetic mechanisms. This study investigated the intergenerational consequences of prenatal exposure to a low ethanol dose (1 g/kg) during gestational days 17-20, on ethanol-induced hypnosis in adolescent male F1 and F2 generations, in two strains of rats. Adolescent Long-Evans and Sprague-Dawley male rats were tested for sensitivity to ethanol-induced hypnosis at a 3.5-g/kg or 4.5-g/kg ethanol dose using the loss of righting reflex (LORR) paradigm. We hypothesized that PAE would attenuate sensitivity to ethanol-induced hypnosis in the ethanol-exposed animals in these two strains and in both generations. Interestingly, we only found this effect in Sprague-Dawley rats. Lastly, we investigated PAE related changes in expression of GABAA receptor α1, α4, and δ subunits in the cerebral cortex of the PAE sensitive Sprague-Dawley strain. We hypothesized a reduction in the cerebral cortex GABAA receptor subunits' expression in the F1 and F2 PAE groups compared to control animals. GABAA receptor α1, α4, and δ subunits protein expressions were quantified in the cerebral cortex of F1 and F2 male adolescents by western blotting. PAE did not alter cerebral cortical GABAA receptor subunit expressions in the F1 generation, but it decreased GABAA receptor α4 and δ subunits' expressions in the F2 generation, and had a tendency to decrease α1 subunit expression. We also found correlations between some of the subunits in both generations. These strain-dependent vulnerabilities to ethanol sensitivity, and intergenerational PAE-mediated changes in sensitivity to alcohol indicate that genetic and epigenetic factors interact to determine the outcomes of PAE animals and their offspring.

Restraint stress enhances alcohol intake in adolescent female rats but reduces alcohol intake in adolescent male and adult female rats.

Adolescents may be more sensitive to stress-induced alcohol drinking than adults, which would explain the higher prevalence of alcohol abuse and dependence in late adolescence than in adulthood. The present study analyzed the impact of restraint stress on the initiation of alcohol intake across 2 weeks of intermittent, two-bottle choice intake in male and female adolescent rats and adult female rats. Restraint stress significantly increased alcohol intake and preference in female adolescent rats but decreased alcohol intake and preference in male adolescent and female adult rats. The effects of restraint stress on alcohol intake were mitigated in adolescent females following administration of the κ opioid receptor antagonist norbinaltorphimine. Adolescent but not adult female rats that were subjected to restraint stress spent more time on the open arms of the elevated plus maze. Female adolescents exposed to stress also exhibited greater risk-taking behaviors in a concentric square field test compared with non-stressed controls. These results indicate age- and sex-related differences in the sensitivity to alcohol-stress interactions that may facilitate the initiation of alcohol use in female adolescents. The facilitatory effect of stress on alcohol intake was related to greater exploratory and risk-taking behaviors in young females after stress exposure.

Reinforcing properties of ethanol in neonatal rats: involvement of the opioid system.

Toward understanding why infant rats ingest high levels of ethanol without initiation procedures, the authors tested effects of mu and kappa receptor antagonists on ethanol reinforcement in neonatal rats. After an intracisternal injection of CTOP (micro antagonist), nor-Binaltorphimine (kappa antagonist), or saline, newborn (3-hr-old) rats were given conditioning pairings of an odor with intraorally infused ethanol or a surrogate nipple with ethanol administered intraperitoneally (to minimize ethanol's gustatory attributes). In each case, these opioid antagonists reduced or eliminated ethanol's reinforcement effect. The same effects occurred with saccharin as the reinforcer in olfactory conditioning. The results imply that activation of mu and kappa receptors, apparently acting jointly, is necessary for reinforcement or that antagonists of this activity impair basic conditioning.

Dynorphin A (1-13) and responsiveness of the newborn rat to a surrogate nipple: immediate behavioral consequences and reinforcement effects in conditioning.

The role of endogenous opioid system in learning (Pavlovian conditioning) and reinforcement was studied in newborn rats by pairing central injection of dynorphin A (1-13), an endogenous ligand with high affinity for kappa opioid receptors, with a surrogate nipple. For conditioning, the nipple served as the conditioned stimulus and dynorphin as the unconditioned stimulus. Dynorphin was found to elicit an immediate unconditioned, dose-dependent increase in the neonatal responsiveness to a surrogate nipple providing water, an effect apparently mediated through the kappa opioid receptors. This immediate effect of dynorphin was evident in the context of suckling behavior but not in the context of adult-like drinking when water was delivered through an intra-oral cannula. One hour after injection, the unconditioned stimulatory effect of dynorphin had dissipated and no longer affected responsiveness to the nipple. However, pairing of centrally injected dynorphin and suckling on a nipple delivering water substantially enhanced responsiveness to a nipple 1h later. This suggested conditioning, and hence reinforcement, by the centrally injected dynorphin. The conditioned sustained nipple attachment was mediated through both kappa and micro opioid receptors. These experiments confirm a central role for the brain opioid systems in reinforcement of neonatal behavior on the nipple.

Age-related effects of chronic restraint stress on ethanol drinking, ethanol-induced sedation, and on basal and stress-induced anxiety response.

Adolescents are sensitive to the anxiolytic effect of ethanol, and evidence suggests that they may be more sensitive to stress than adults. Relatively little is known, however, about age-related differences in stress modulation of ethanol drinking or stress modulation of ethanol-induced sedation and hypnosis. We observed that chronic restraint stress transiently exacerbated free-choice ethanol drinking in adolescent, but not in adult, rats. Restraint stress altered exploration patterns of a light-dark box apparatus in adolescents and adults. Stressed animals spent significantly more time in the white area of the maze and made significantly more transfers between compartments than their non-stressed peers. Behavioral response to acute stress, on the other hand, was modulated by prior restraint stress only in adults. Adolescents, unlike adults, exhibited ethanol-induced motor stimulation in an open field. Stress increased the duration of loss of the righting reflex after a high ethanol dose, yet this effect was similar at both ages. Ethanol-induced sleep time was much higher in adult than in adolescent rats, yet stress diminished ethanol-induced sleep time only in adults. The study indicates age-related differences that may increase the risk for initiation and escalation in alcohol drinking.

Can low-level ethanol exposure during pregnancy influence maternal care? An investigation using two strains of rat across two generations.

Gestational alcohol use is well documented as detrimental to both maternal and fetal health, producing an increase in offspring's tendency for alcoholism, as well as in behavioral and neuropsychological disorders. In both rodents and in humans, parental care can influence the development of offspring physiology and behavior. Animal studies that have investigated gestational alcohol use on parental care and/or their interaction mostly employ heavy alcohol use and single strains. This study aimed at investigating the effects of low gestational ethanol dose on parental behavior and its transgenerational transmission, with comparison between two rat strains. Pregnant Sprague Dawley (SD) and Long Evans (LE) progenitor dams (F0) received 1g/kg ethanol or water through gestational days 17-20 via gavage, or remained untreated in their home cages. At maturity, F1 female offspring were mated with males of the same strain and treatment and were left undisturbed through gestation. Maternal behavior was scored in both generations during the first six postnatal days. Arch-back nursing (ABN) was categorized as: 1, when the dam demonstrated minimal kyphosis; 2, when the dam demonstrated moderate kyphosis; and 3, when the dam displayed maximal kyphosis. Overall, SD showed greater amounts of ABN than LE dams and spent more time in contact with their pups. In the F0 generation, water and ethanol gavage increased ABN1 and contact with pups in SD, behaviors which decreased in treated LE. For ABN2, ethanol-treated SD dams showed more ABN2 than water-treated dams, with no effect of treatment on LE animals. In the F1 generation, prenatal exposure affected retrieval. Transgenerational transmission of LG was observed only in the untreated LE group. Strain-specific differences in maternal behavior were also observed. This study provides evidence that gestational gavage can influence maternal behavior in a strain-specific manner. Our results also suggest that the experimental procedure during gestation and genetic variations between strains may play an important role in the behavioral effects of prenatal manipulations.

Change in the hedonic value of an aversive stimulus in the presence of a pre-exposed odor.

Rats exhibit a sensitive period from the time of birth until postnatal day 10 during which they develop preferences for odors even if those odors are paired with a moderately aversive stimulus. It is still unknown whether pre-exposure to an odor produces alterations on intake responses of basic tastants, and on other patterns that indicate a change in the hedonic value of reward, such as nipple grasping behavior. The current study assessed the effect of pre-exposure to an odor immediately after birth on intake responses of appetitive and aversive tastants. The objectives were to assess if 3-hour-old rats adjust their behaviors to obtain different values of appetitive and aversive rewards in the presence of a familiar odor. Specifically we wanted to determine whether the intake of saccharin or quinine, administered through the artificial nipple, increases in the presence of the familiar odor. Results showed that 3-hour-old rats differentially respond to two different concentrations of saccharin and two concentrations of quinine. In the presence of the pre-exposed odor newborn rats increased intake and grasp responses to the artificial nipple containing quinine. This effect disappeared with a higher concentration of quinine. These results suggest that the pre-exposed odor generated a change in the hedonic value of the aversive reward.

Olfactory aversive conditioning in the newborn (3-hr-old) rat impairs later suckling for water and milk.

An olfactory conditioning paradigm tested whether newborn rats can acquire a conditioned aversion to olfactory events associated with their first postnatal meal 3-5 hr after birth. Exposure to lemon odor (conditioned stimulus [CS]) paired with intraoral infusions of 0.1% quinine (unconditioned stimulus) resulted in explicit conditioning. Responsiveness to a surrogate nipple providing water in the presence of the CS was significantly lower than the 3 control conditions. The conditioning dramatically suppressed responsiveness to a surrogate nipple providing milk, which normally is expressed voraciously in terms of sustained nipple attachment and milk intake. These findings suggest that as early as 3-5 hr after birth newborn rats are capable of aversive conditioning to odors in the context of suckling behavior.