Adolescent forced swim stress increases social anxiety-like behaviors and alters kappa opioid receptor function in the basolateral amygdala of male rats.

Adolescence is a developmental period marked by robust neural alterations and heightened vulnerability to stress, a factor that is highly associated with increased risk for emotional processing deficits, such as anxiety. Stress-induced upregulation of the dynorphin/kappa opioid receptor (DYN/KOP) system is thought to, in part, underlie the negative affect associated with stress. The basolateral amygdala (BLA) is a key structure involved in anxiety, and neuromodulatory systems, such as the DYN/KOP system, can 1) regulate BLA neural activity in an age-dependent manner in stress-naïve animals and 2) underlie stress-induced anxiety in adults. However, the role of the DYN/KOP system in modulating stress-induced anxiety in adolescents is unknown. To test this, we examined the impact of an acute, 2-day forced swim stress (FSS - 10 min each day) on adolescent (~postnatal day (P) 35) and adult Sprague-Dawley rats (~P70), followed by behavioral, molecular and electrophysiological assessment 24 h following FSS. Adolescent males, but not adult males or females of either age, demonstrated social anxiety-like behavioral alterations indexed via significantly reduced social investigation and preference when tested 24 h following FSS. Conversely, adult males exhibited increased social preference. While there were no FSS-induced changes in expression of genes related to the DYN/KOP system in the BLA, these behavioral alterations were associated with alterations in BLA KOP function. Specifically, while GABA transmission in BLA pyramidal neurons from non-stressed adolescent males responded variably (potentiated, suppressed, or was unchanged) to the KOP agonist, U69593, U69593 significantly inhibited BLA GABA transmission in the majority of neurons from stressed adolescent males, consistent with the observed anxiogenic phenotype in stressed adolescent males. This is the first study to demonstrate stress-induced alterations in BLA KOP function that may contribute to stress-induced social anxiety in adolescent males. Importantly, these findings provide evidence for potential KOP-dependent mechanisms that may contribute to pathophysiological interactions with subsequent stress challenges.

Stress, κ manipulations, and aversive effects of ethanol in adolescent and adult male rats.

Elevated ethanol use during adolescence, a potentially stressful developmental period, is accompanied by insensitivity to many aversive effects of ethanol relative to adults. Given evidence that supports a role for stress and the kappa opioid receptor (KOR) system in mediating aversive properties of ethanol and other drugs, the present study assessed the role of KOR antagonism by nor-binaltorphimine (nor-BNI) on ethanol-induced conditioned taste aversion (CTA) in stressed (exposed to repeated restraint) and non-stressed male rats (Experiment 1), with half of the rats pretreated with nor-BNI before stressor exposure. In Experiment 2, CTA induced by the kappa agonist U62,066 was also compared in stressed and non-stressed adolescents and adults. A highly palatable solution (chocolate Boost) was used as the conditioned stimulus (CS), thereby avoiding the need for water deprivation to motivate consumption of the CS during conditioning. No effects of stress on ethanol-induced CTA were found, with all doses eliciting aversions in adolescents and adults in both stress conditions. However, among stressed subjects, adults given nor-BNI before the repeated stressor displayed blunted ethanol aversion relative to adults given saline at that time. This effect of nor-BNI was not seen in adolescents, findings that support a differential role for the KOR involvement in ethanol CTA in stressed adolescents and adults. Results from Experiment 2 revealed that all doses of U62,066 elicited aversions in non-stressed animals of both ages that were attenuated in stressed animals, findings that support a modulatory role for stress in aversive effects of KOR activation. Collectively, these results suggest that although KOR sensitivity appears to be reduced in stressed subjects, this receptor system does not appear to contribute to age differences in ethanol-induced CTA under the present test circumstances.

Newborn rats' first suckling experience: taste differentiation and suckling plasticity.

The present study examined responsiveness of newborn rats to a surrogate nipple providing fluids with basic tastes (sweet, sour, bitter and salty) and assessed the effects of this first gustatory experience on subsequent responding to the nipple itself (empty nipple) or the nipple providing water. Responsiveness (attachment to and ingestion from a surrogate nipple providing saccharin, saline, quinine or ammonium chloride) was compared with that toward a nipple providing water. Compared to water, saline and quinine significantly reduced attachment to and ingestion from the nipple, while saccharin and milk significantly increased attachment behavior. Ammonium chloride increased attachment but not ingestive behavior. Suckling experience with saline, quinine and ammonium chloride attenuated both attachment and ingestive behaviors when subjects were tested 1 h later with an empty surrogate nipple or a nipple providing water. Experience with saccharin and milk (but not water) increased both measures. The data suggest that in newborn rats, as early as a few hours after birth, mechanisms of gustatory detection have control over suckling behavior. Initial experience with the tastants available from the nipple in the first suckling episode may alter further responsiveness to the nipple itself, mediated perhaps by mechanisms of appetitive and aversive conditioning.

Acute effects of ethanol on behavior of adolescent rats: role of social context.

BACKGROUND: First experiences with alcohol in humans occur predominantly in adolescence, and to a large extent the attractiveness of alcohol at this age is based on its ability to facilitate certain forms of social behavior (social facilitation). Adolescence is strongly marked by a focus on peer relationships, and the social nature of the situation plays an important role in responsiveness to alcohol. Peer-directed social activity of adolescent rats may be a valuable experimental model for the study of ethanol-induced changes in social behavior and assessment of the role of the social context in responsiveness to ethanol. METHOD: In the present study we used a modified dyad social interaction test to characterize acute effects of ethanol on different forms of social behavior (social investigation, contact behavior, and play) and social motivation (preference/avoidance of a peer) in adolescent rats. Ethanol effects on behavior directed toward a peer were compared with those induced by exposure to an inanimate novel object. RESULTS: In the social context, the effects of ethanol were dose-dependent and biphasic. Low doses of ethanol (0.25-0.75 g/kg) produced apparent social facilitation (increased social activity and enhanced social preference), whereas higher doses (3 and 4 g/kg) caused social inhibition (decreased social activity and avoidance of a peer). This pattern was not observed for a nonsocial stimulus: Although overall activity in the nonsocial context was suppressed by 2 and 3 g/kg of ethanol, 0.5 g/kg of ethanol did not activate overall activity directed to the inanimate object. CONCLUSIONS: These findings demonstrate that the social nature of the testing situation plays an important role in responsiveness to alcohol in adolescence, especially to its activating effects. The results suggest also that the study of ethanol effects on social behavior of adolescent rats may be an effective tool for the study of adolescent alcohol use and abuse.

Ethanol as a reinforcer in the newborn's first suckling experience.

BACKGROUND: Recent evidence suggests that human infants prefer alcohol-flavored milk when fed through a bottle. Animal models also indicate a surprising predisposition for neonatal and infant rats to voluntarily and willingly ingest ethanol. These findings suggest high susceptibility to the reinforcing properties of ethanol early in ontogeny. METHODS: A surrogate nipple technique-a highly effective tool for investigation of the reinforcing properties of different fluids-was applied in the present study. Tests of ethanol reinforcement were accomplished in terms of two basic paradigms of Pavlovian conditioning. In one paradigm, the conditioned stimulus (CS) was the surrogate nipple, and in the other, the CS was a novel odor. RESULTS: Newborn rats showed sustained attachment to the nipple providing 5% ethanol, and later reproduced this behavioral pattern toward the empty nipple (CS alone). Ingestion of ethanol yielding appetitive reinforcement was accompanied by detectable blood alcohol concentrations, with most in the range of 20-30 mg/dl. The reinforcing efficacy of ethanol was also confirmed in the classical olfactory conditioning paradigm: following pairing with intraoral ethanol infusions, the odor (CS) alone elicited sustained attachment to an empty nipple. Females showed better olfactory conditioning with low concentrations of ethanol, whereas males were effectively more conditioned to high concentrations. Although there were no reinforcing consequences of intraperitoneally injected ethanol [as an unconditioned stimulus (US)] when a neutral odor was the CS, when paired with ingestion of water from a nipple, the injection of ethanol had a reinforcing effect. CONCLUSIONS: The present series of experiments revealed ethanol reinforcement in the newborn rat. Two varieties of Pavlovian conditioning established that ethanol can serve as an effective US, and hence reinforcer, in such a way as to increase the approach and responsiveness toward stimuli paired with that US, indicating appetitive reinforcement.

Self-administration of ethanol and saccharin in newborn rats: effects on suckling plasticity.

Responsiveness to a surrogate nipple providing water, saccharin, 5% ethanol, or 10% ethanol was tested in newborn rats naive to suckling (3-5 hr old) on Postnatal Day (P) 0 and in older neonates with regular suckling experience on P1 or P2. At all ages, pups demonstrated greater nipple attachment for saccharin or 5% ethanol than for water. Intake of saccharin and 5% ethanol was high in newborns, far exceeding that of water. At P1 and P2, pups exhibited a preference for saccharin, but not for 5% ethanol. Preexposure to a nipple providing ethanol or saccharin (but not a nipple alone or fluids alone) increased subsequent responsiveness toward an empty surrogate nipple (no fluid), both 1 hr and 24 hr after preexposure. Although, during preexposure, pups responded most positively to the nipple providing saccharin, the longest time spent on an empty nipple was observed in pups preexposed to 10% ethanol.

The first suckling episode in the rat: the role of endogenous activity at mu and kappa opioid receptors.

The present study examined the role of endogenous activity at mu and kappa opioid receptors in attachment to and ingestion of milk from a surrogate nipple in cesarean-delivered newborn rats prior to regular suckling experience. Selective opioid antagonist drugs were injected into the cisterna magna (IC administration) or lateral ventricles (ICV administration). Blockade of endogenous activity at mu opioid receptors by IC administration of the selective antagonist CTOP reduced attachment time and markedly increased disengagements from the nipple. CTOP also increased the intensity of suckling measured as milk intake per min attached to the nipple, when milk was available from the nipple in a free-access regime, and enhanced intake when milk was infused through an intraoral cannula aside from the suckling context. The ICV administration of the selective kappa antagonist nor-BNI considerably increased latency to grasp the surrogate nipple, while time on the nipple and milk intake were decreased. The presented data suggest that populations of mu and kappa receptor-containing neurons, differentiable by the route of antagonist administration, play an important role in initiation and maintenance of suckling behavior in the newborn rat during its first encounter with the nipple and milk. The kappa opioid system is predominantly involved in the initiation of the newborn's behavior directed toward the nipple providing milk. The role of the mu opioid system seems more complicated: it transforms initial oral grasp responses into sustained attachment to the nipple and maintains the intake of milk at a certain physiological level.

Classical conditioning of responses to an artificial nipple in the rat fetus: mu and kappa opioid systems.

The rat fetus exhibits oral grasp responses when presented with an artificial nipple in utero. Preexposure to milk produces a transient decrease in oral grasp responses. The effect of milk on oral grasping is mediated by endogenous activity in brain opioid systems. Specifically, milk triggers mu activity in rostral brain regions and kappa opioid activity in caudal brain regions to decrease oral grasping of the artificial nipple. Reexposure to the artificial nipple after it has been paired with milk during a classical conditioning trial results in a conditioned reduction in oral grasping that is evident when fetuses are reexposed to the nipple. The classically conditioned decrease in oral grasping is mediated by mu opioid activity in rostral brain regions and kappa opioid activity in caudal brain regions. Endogenous opioid systems, activated by exposure to milk and the nipple, may regulate the processing of sensory information during suckling in the rat.

Acute effects of ethanol and the first suckling episode in the newborn rat.

BACKGROUND: In humans, early postnatal experience with alcohol is far from rare and includes exposure to alcohol through breast milk or through the bottle to attain sedative effects (Croce, 1987). Exposure to alcohol though mother's milk alters the infant's suckling behavior. However, acute effects of alcohol on suckling behavior using animal models remain to be investigated. METHODS: The present study was designed to examine the effects of neonatal alcohol exposure on attachment to a surrogate nipple and ingestion of milk in rat pups, naive both to suckling and to maternal care. Cesarean-delivered rat pups were pre-exposed to four dosages of ethanol (0, 1, 2, and 3 g/kg intragastrically) and tested 30 min after ethanol administration. RESULTS: Suckling behavior was impaired only in pups exposed to a dosage of 3 g/kg of ethanol. Pups in this group demonstrated longer latency to attach followed by less efficient suckling--the pups maintained contact with the nipple but showed decreased ingestion of milk from it. Pups treated with 1 g/kg of ethanol showed the greatest suckling efficiency, ingesting far more milk per minute attached to the surrogate nipple than pups in all other groups. At the same time, nonevoked motor activity was significantly suppressed by all three dosages of ethanol. Blood alcohol levels showed a linear relationship with dose at 30 min postintubation. CONCLUSIONS: These findings suggest a dissociation between acute ethanol effects on suckling and overall motor activity, with suckling apparently less sensitive to suppressive and more sensitive to activating effects of ethanol.

Rapid and robust olfactory conditioning with milk before suckling experience: promotion of nipple attachment in the newborn rat.

An olfactory conditioning paradigm tested the hypothesis that newborn rats are able to learn about events associated with their first experience with milk as early as 3-5 hr after birth. Exposure to lemon odor (conditioned stimulus, [CS]) paired with intraoral milk infusions (unconditioned stimulus, [US]) resulted in strong conditioning: In the presence of the CS, sustained attachment occurred to an empty nipple as if it provided milk, whereas pups in control conditions showed little attachment. A single CS-US pairing was sufficient for strong conditioning, which was evident with a trace interval as long as 60 s. Conditioning was robust enough to promote attachment to a nipple providing saline, which is aversive to the newborn rat, and comparably strong conditioning occurred with sucrose or saccharin as the US. These findings suggest that olfactory conditioning has the potential to modify suckling behavior.

Social behavior and social motivation in adolescent rats: role of housing conditions and partner's activity.

The present study investigated 1) the effects of individual and grouped housing on social investigation, social contact behavior, and play behavior in adolescent rats tested with low socially active (grouped) and high socially active (isolated) play partners; and 2) the effects of long-term (8 days) and short-term (24 h) isolation on social behavioral manifestations and social motivation in terms of preference or avoidance of play partners. Social isolation of adolescent rats activated play behavior and social behaviors different from play, but play was predominantly affected under the conditions of this study. Long-term isolation was more effective than short-term, and resulted in greater manifestation of play and social preference. Adolescent rats were able to modify their social behaviors in response to social activity of the play partner: in isolated animals exposed to low socially active group-housed partners, play behavior was transformed into social activities unrelated to play; exposure of group-housed adolescents to high socially active previously isolated partners resulted in an increase of play behavior. Testing that allowed avoidance of social contacts revealed a dissociation between manifestations of play behavior and social motivation: group-housed play partners of isolated animals showed elevated levels of play behavior but a tendency to avoid their isolated pairmates.

A new model of ethanol self-administration in newborn rats: gender effects on ethanol ingestion through a surrogate nipple.

BACKGROUND: Ethanol intake in the context of suckling may have distinct and potentially long-lasting consequences for further responsiveness to and acceptance of ethanol, compared to other, more indirect and less natural ways of ethanol exposure early in ontogeny. METHOD: Our findings presented in this paper show that a surrogate nipple technique can be used for the study of early ethanol intake in cesarean-derived rat pups tested before any suckling experience. RESULTS: Neonatal rats attached to and voluntary ingested ethanol through the surrogate nipple as early as 4 hr after birth. Moderate concentrations of ethanol (2% and 5%, v/v) promoted substantial initial suckling behavior, including sustained attachment to the nipple. Higher concentrations (10% and 15%) were not effective in sustaining suckling. Females responded less positively to 10% ethanol than did males. High concentrations of ethanol were less effective in eliciting suckling behavior, probably due to the aversiveness of ethanol odor. However, when ethanol was presented in solution with milk, newborn pups attached to the nipple and ingested even 15% ethanol. Contamination of milk with 15% ethanol was more aversive for females than for males. Newborn rat pups demonstrated similar patterns of nipple attachment and ingestive behavior for 5% ethanol and milk. Initial experience with milk in the context of suckling did not prevent further voluntary ethanol ingestion from the same nipple; furthermore, initial exposure to 5% ethanol did not impair subsequent responsiveness to milk. CONCLUSIONS: Gender differences in responsiveness or sensitivity to ethanol can be detected in rat pups as early as a few hours after birth. The results suggest a leftward shift in the dose-response curve for females compared with males, indicating that female neonates are more sensitive or more responsive than males to ethanol. The similarity of suckling behaviors produced by moderate concentrations of ethanol and milk suggests a certain unity in their reinforcing mechanisms in the context of the first suckling episode.

Sustained attachment to the nipple in the newborn rat depends on experience with the nipple, milk, and the expression of oral grasping.

Newborn rats showed mouthing, licking, and oral grasping when presented with a surrogate nipple. These responses changed after the pup expressed an oral grasp response and experienced milk at the nipple. Newborn pups that ingested milk from the surrogate nipple showed brief oral grasp responses and, when tested 1 hr later, showed sustained attachment to an empty surrogate nipple. Contact with the nipple, oral grasping of the nipple, and experience with milk altered subsequent behavioral responses to the nipple. Classical and instrumental conditioning may play a role in transforming brief oral grasp responses into longer oral grasp responses and sustained attachment to the nipple.

Thermal, olfactory, and tactile stimuli increase oral grasping of an artificial nipple by the newborn rat.

Caesarean-delivered rat pups tested before any suckling experience show oral grasp responses after stimulation with an artificial nipple. Manipulating the sensory stimuli present at the time of testing alters behavioral responses to the nipple. Specifically, when the nipple is warm, when pups are tested in the presence of amniotic fluid or milk odor, or when pups are tested in the presence of a conspecific, oral grasping of the artificial nipple is increased. Pups respond to the nipple with a shorter latency, show more oral grasp responses, and the individual grasp responses are longer in duration. The experiments suggest that the newborn rat pup exhibits a basic set of behaviors in response to the nipple early in development and that sensory stimuli normally present during the expression of suckling increase oral appetitive behaviors evoked by the nipple.

Endogenous opioids and the first suckling episode in the rat.

Endogenous activity at opioid receptors affects the appetitive behavior of Caesarean-delivered rat pups during presentation of a surrogate nipple that provides milk. Blockade of opioid receptors by peripheral injection of naloxone has no effect on responses evoked by the surrogate nipple. Similarly, blockade of caudal brain opioid receptors by injection of naloxone into the cisterna magna has no effect on the pup's behavior in response to the surrogate nipple. However, blockade of rostral opioid receptors by injection of naloxone into the cerebral ventricles increases the latency to the first oral grasp response, decreases total time on the nipple, and virtually eliminates ingestion of milk from the surrogate nipple (Experiment 1). Blockade of endogenous opioid activity does not affect responses to a nipple that provides distilled water (Experiment 2) or to an empty surrogate nipple (Experiment 3). These data indicate that during the initial suckling episode endogenous opioids in rostral brain regions affect the pup's behavioral responses to the nipple. The results are consistent with the hypothesis that milk engages opioid systems during the first suckling and that endogenous opioids play a role in early suckling.

Endogenous mu opioid systems and suckling in the neonatal rat.

Localized injections of the mu antagonist CTOP into intracisternal (i.c.) or intracerebroventricular (i.c.v.) sites altered the behavior of 1-day-old rat pups during continuous exposure to an artificial nipple. Blockade of mu opioid receptors by i.c. injection decreased oral responsiveness to the nipple, while blockade of receptors by i.c.v. injection of CTOP increased oral responsiveness. The injection of CTOP into the i.c. site produced a transient reduction in body weight gain in pups suckling from their mother, while injection into the i.c.v. site had no effect. When cesarean-delivered pups were tested prior to suckling, injection of CTOP into the i.c. site increased latency of the first nipple attachment and decreased total time attached to a surrogate nipple providing milk. Injection of CTOP into the i.c.v. site decreased latency to the first nipple attachment. The results indicate that there is a caudal population of opioid receptors that is involved in the initiation of suckling behavior and a rostral population that plays a role in decreasing responsiveness at the nipple.

The newborn rat ingests fluids through a surrogate nipple: a new technique for the study of early suckling behavior.

In this report we describe an apparatus and procedure that permits a newborn rat pup to ingest test fluids including milk through a surrogate nipple. The surrogate nipple represents a new testing situation for the experimental study of sensory and neurochemical controls of suckling behavior immediately after birth.

Endogenous mu opioid systems and perioral responsiveness in the rat fetus.

Pharmacological manipulation of mu opioid receptors located in rostral and caudal parts of the brain produces distinctive changes in perioral responsiveness to nipple-like tactile stimulation in the E20 rat fetus. Blockade of caudal mu opioid receptors by intracistema magna (I.C.) injection of the selective mu antagonist drug CTOP reduces appetitive responses directed toward the artificial nipple. In contrast, blockade of mu opioid receptors in the rostral part of the brain by intracerebroventricular (I.C.V.) administration of CTOP increases fetal responsiveness to perioral cutaneous stimulation including oral capture and grasping of the artificial nipple. This pattern of the results suggests that there are at least two functionally different neuronal populations of mu opioid receptor-containing neurons that are involved in the regulation of the perioral responsiveness in the E20 rat fetus. The caudal part of this mu opioid system increases perioral responsiveness while the rostral part of the system decreases responsiveness to nipple-like perioral stimulation. These findings suggest the possibility that mu opioid systems may play a functional role in regulating neonatal behavior at the nipple.

Oral grasping of a surrogate nipple by the newborn rat.

Newborn rat pups exhibit oral appetitive behaviors when presented with an artificial nipple. These behaviors include mouthing and licking movements and expression of stereotyped oral grasp response. Caesarean-delivered pups show increased responding to the nipple over the first 5 h after birth that is independent of experience with the nipple. Mimicking maternal licking by stimulating the anogenital region of the newborn rat with a soft paintbrush increases response to the nipple. Pups tested after 24 hr of normal experience respond to the artificial nipple when tested immediately after separation from the mother. However, oral grasping of the nipple is more frequent in 1-day-old pups tested 3 or 5 hr after separation from the mother. Study of behavioral responses to the artificial nipple promises to provide information about sensory and neurochemical controls of the initial suckling episode.