Contextual fear memory modulates PSD95 phosphorylation, AMPAr subunits, PKMζ and PI3K differentially between adult and juvenile rats.

It is well known that young organisms do not maintain memories as long as adults, but the mechanisms for this ontogenetic difference are undetermined. Previous work has revealed that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAr) subunits are trafficked into the synaptic membrane following memory retrieval in adults. Additionally, phosphorylated PSD-95-pS295 promotes AMPAr stabilization at the synapse. We investigated these plasticity related proteins as potential mediators in the differential contextual stress memory retrieval capabilities observed between adult and juvenile rats. Rats were assigned to either pedestal stress (1 h) or no stress control (home cage). Each animal was placed alone in an open field for 5 min at the base of a 6 × 6 sq inch pedestal (4ft high). Stress subjects were then placed on this pedestal for 1hr and control subjects were placed in their home cage following initial exploration. Each animal was returned to the open field for 5 min either 1d or 7d following initial exposure. Freezing postures were quantified during the memory retrieval test. The 1d test shows adult (P90) and juvenile (P26) stressed rats increase their freezing time compared to controls. However, the 7d memory retrieval test shows P90 stress rats but not P26 stress rats freeze while in the fear context. Twenty minutes after the memory retrieval test, hippocampi and amygdala were micro-dissected and prepared for western blot analysis. Our results show that 1d fear memory retrieval induced an upregulation of PSD-95 and pS295 in the adult amygdala but not in the juvenile. However, the juvenile animals upregulated PKMζ, PI3K and GluA2/3, GluA1-S845 in the dorsal hippocampus (DH), but the adults did not. Following the 7d memory retrieval test, adults upregulated GluA2 in the amygdala but not the juveniles. In the DH, adults increased PSD-95 and pS295 but not the juveniles. The adults appear to preferentially increase amygdala-driven processing at 1d and increase DH-driven context specific processing at 7d. These data identify molecular processes that may underlie the reduced fear-memory retrieval capability of juveniles. Together these data provide a potential molecular target that could be beneficial in treatment of anxiety disorders and PTSD.

Repeated recall and PKMζ maintain fear memories in juvenile rats.

We examined the neural substrates of fear memory formation and maintenance when repeated recall was used to prevent forgetting in young animals. In contrast to adult rats, juveniles failed to show contextual fear responses at 4 d post-fear conditioning. Reconsolidation sessions 3 and 6 d after conditioning restored contextual fear responses in juveniles 7 d after initial training. In juveniles that received reconsolidation sessions, protein kinase M zeta (PKMζ) increased in the amygdala, but not in the hippocampus. These data suggest that repeated reminders and increased PKMζ maintain fear responses in juvenile animals that otherwise would not exhibit this behavior.

Isolation-induced vocalization in the infant rat depends on the nucleus accumbens.

Mammalian infants vocalize when socially isolated. Vocalization guides the return of the caregiver and thereby maintains an environment critical to the infant's survival. Although the role of the periaqueductal gray area (PAG) in these vocalizations is established, other aspects of the relevant neural circuitry remain under-studied. Here we report that output from the nucleus accumbens (Acb) is necessary for isolation-induced vocalizations of infant rats aged postnatal days (PND) 11-13. Local inhibition via infusion of the GABAA agonist muscimol (.8 µg/side) of the Acb, but not the dorsolateral striatum, blocked isolation-induced vocalizations, independent of whether the isolation was at room temperature, followed a brief reunion with the dam, or occurred in a cool (10 °C) environment. These findings highlight a possible anatomical area mediating the mammalian infant response to social separation and, more generally, to the development of social attachment.

Vocal coselection in rat pup ultrasonic vocalizations.

Selective breeding and natural selection that select for one trait often bring along other correlated traits via coselection. Selective breeding for an infantile trait, high or low call rates of isolation-induced ultrasonic vocalization of rat pups, also alters functions of some brain systems and emotional behaviors throughout life. We examined the effect of breeding for call rate on acoustic parameters that are of communicative significance. Selecting for higher call rate produced calls of significantly increased amplitude and bandwidth relative to a randomly bred line. Selecting for lower rate produced calls of decreased duration. These nonmorphological, functional trait changes demonstrate enhanced communicatory potential and energy expenditure for the High line and the opposite for the Low line. This demonstration of coselection in a communicatory system suggests an underlying heritable suite of linked acoustic vocalization characteristics that in noisy environments could enhance dam-pup communication and lead to selection of emotionality traits with beneficial responses to stress.

Prairie vole pups show potentiated isolation-induced vocalizations following isolation from their mother, but not their father.

Vocalizations can be markers of emotional social communication. Maternal potentiation was originally described as an increased rate of vocalization by isolated rat pups following an interaction with their mothers, but not with other social companions. Here we asked if potentiation in prairie voles, a species with pair-bonding and bi-parental rearing, is parent-specific. We found that isolated, 8-11-day-old voles exhibited potentiation following reunions with the dam, but not the sire. These responses were present whether parents were anesthetized or active during the reunion. There were no significant correlations between parental behaviors during reunions and pup vocalization rates during re-isolation. The absence of potentiation to the sire contrasts to findings in bi-parentally reared rat pups, which do potentiate vocalizations to the sire. We interpret these results to be consistent with the idea that potentiation reflects disruption of mother-infant coregulation and is dependent upon the unique biology of mothering. © 2016 Wiley Periodicals, Inc. Dev Psychobiol 58:687-699, 2016.

Effects of maternal deprivation and the duration of reunion time on rat pup ultrasonic vocalization responses to isolation: possible implications for human infant studies.

In a paradigm that may serve as a translational model for maternal separation experiences of human infants in neonatal intensive care units, we examined how the duration of reunion with the dam influenced the phenomenon of maternal potentiation of ultrasonic vocalizations, in which isolated rat pups increase rates of vocalization following brief interactions with dams. We report that maternal potentiation in 12-13 day-old rats did not occur after reunions with their anesthetized dam that lasted longer than 15-min. However, after 18 hr maternal separation, isolated pups given reunions with their anesthetized dam increased vocalization rate even with reunions as long as 3 hr. Using a split-cage apparatus that prevented physical contact, the impact of 18 hr separations on maternal potentiation was partially offset by experiencing olfactory and/or auditory stimuli of the mother. These results suggest that maintaining partial maternal sensory exposure during prolonged maternal separation can reduce responses elicited by subsequent maternal separation.

Parental potentiation of vocalization as a marker for filial bonds in infant animals.

Maternal and paternal potentiation of vocalization are two parts of a promising model of early life social bonds that has been and can be a useful tool in research. Most mammalian infants vocalize when isolated. Interactions with adult females just before isolation have been found to increase vocalizations in several species. Interactions with littermates and other social stimuli do not. In guinea pigs and pigs, the response is specific to the dam. In rats and octagon degus, an unrelated adult female from the colony is sufficient. The presence of an intact adult male in the test chamber with dam-reared pups evokes behavioral inhibition, a fear response. Previous exposure to the male in the home cage, biparental rearing, dramatically transforms the response of the pup. The pup treats the adult male as it does its dam, including potentiation of vocalization during a subsequent isolation. This article outlines the methods, advantages, and disadvantages of parental potentiation as a research tool, as well as a brief review of the evidence supporting its use as a marker for filial attachment. Future research directions are outlined.

Medial prefrontal cortex processes threatening stimuli in juvenile rats.

To survive, all mammalian species must recognize and respond appropriately to threatening stimuli. In adults, the prelimbic medial prefrontal cortex (mPFC) appears to be involved in fear expression, whereas the infralimbic mPFC mediates fear extinction. In juvenile rats (PN26), the mPFC receives information on potential predators but does not act on it. To test whether the prefrontal cortex is capable of fear regulation in the young organism, we exposed juvenile rats to a threatening or nonthreatening stimulus and assessed fear and brain Fos activation of the mPFC subdivisions, amygdala and periaqueductal gray (PAG). In response to the threat, juveniles froze more, spent more time far from the threat, and had elevated numbers of Fos-positive cells in the prelimbic mPFC, the medial amygdala, and ventral PAG. To test the hypothesis that the mPFC has a dual role in modulating the amygdala and PAG in juveniles, we pharmacologically disinhibited each of the two subdivisions of the mPFC and assessed freezing and downstream activation to the threat. Juvenile rats infused with picrotoxin into the prelimbic mPFC and exposed to a threatening stimulus froze less, spent less time far from the threat, and increased Fos expression. Infusion of picrotoxin into the infralimbic mPFC also reduced fear responding to the threatening stimulus but had no effect on Fos expression. In sum, it appears that the mPFC can process threatening stimuli in juveniles at this age, even though it is normally not involved in the fear responses.

Cutting the vagus nerve below the diaphragm prevents maternal potentiation of infant rat vocalization.

In maternal potentiation, the rate of vocalization by a young organism during isolation is greatly enhanced if that isolation has been immediately preceded by an interaction with the mother (or other adult female in the case of rats). The enhancement in isolation-induced vocalization rate does not occur if the young animal had an interaction with other social companions like littermates or with familiar inanimate stimuli like home cage shavings. The present study demonstrates that pups whose vagus nerve is cut below the diaphragm do not potentiate vocalization after an interaction with their dam. The vocalization rates of denervated pups in a first isolation, in the presence of the dam, and during cold exposure do not differ from control pups. Their non-vocal behaviors also appear unaffected by the surgery. Similar to what has been shown in studies of fever-induced behavioral changes, an intact vagus nerve from the gut is necessary for young rat pups to show normal social mediation of their isolation-induced vocal responses.

Enrichment materials do not negatively affect reproductive success and offspring survival and weight in mice.

Environmental enrichment is designed to improve the overall welfare of laboratory animals, including mice. Few studies have directly assessed the effects of different types of enrichment on mouse offspring survival and growth. The authors examined how survival and growth of C57BL/6 mouse pups are affected by three kinds of cage enrichment materials: compressed cotton squares, two-ply tissues and plastic igloos. During the last week of gestation and the first two weeks postpartum, the authors observed cages with litters and noted use of the enrichment materials, quality of nest construction, number of pups per litter and weight of pups. Both the first and second litters were evaluated for each dam. Dams and pups had continuous contact with the enrichment materials, especially cotton squares and tissues. Neither the presence nor the type of enrichment material influenced the survival and weight of offspring, suggesting that the use of such materials does not negatively impact reproductive success or offspring survival.

The role of the medial prefrontal cortex in innate fear regulation in infants, juveniles, and adolescents.

In adult animals, the medial prefrontal cortex (mPFC) plays a significant role in regulating emotions and projects to the amygdala and periaqueductal gray (PAG) to modulate emotional responses. However, little is known about the development of this neural circuit and its relevance to unlearned fear in pre-adulthood. To address these issues, we examined the mPFC of 14-d-old (infants), 26-d-old (juveniles), and 38- to 42-d-old (adolescents) rats to represent different developmental and social milestones. The expression patterns of the neuronal marker FOS were used to assess neurological activity. Muscimol, a GABA agonist, was used to inactivate the prelimbic and infralimbic mPFC subdivisions (400 ng in 200 nl). Animals were exposed to either a threatening or nonthreatening stimulus that was ecologically relevant and age specific. Freezing was measured as an indicator of innate fear behavior. The data indicated that the mPFC is neither active nor responsive to innate fear in infant rats. In juveniles, the prelimbic mPFC became responsive in processing aversive sensory stimulation but did not regulate freezing behavior. Finally, during adolescence, inactivation of the prelimbic mPFC significantly attenuated freezing and decreased FOS expression in the ventral PAG. Surprisingly, across all ages, there were no significant differences in FOS levels in the medial and basolateral/lateral amygdala when either mPFC subdivision was inactivated. Together, unlearned fear has a unique developmental course with different brain areas involved in unlearned fear in the immature animal than the adult. In particular, the mPFC neural circuitry is different in young animals and progressively develops more capacities as the animal matures.

Sex and experience influence behavioral responses of adult rats to potentiated and nonpotentiated ultrasonic vocalizations of pups.

Responsiveness of adult rodent caretakers to infant rodents is necessary for their survival and proper development. Both olfactory and auditory cues are known to influence adult behavior toward the young. In the present study, we found that adults respond differentially to a recording of potentiated vs. non-potentiated ultrasound vocalizations of a 12-day-old rat pup, either in the presence or absence of olfactory cues. The combination of olfactory and potentiated ultrasonic vocalization produced the greatest effect. Adult behavior was also affected by the adults' sex and parental experience in an interaction. Parental experience of females made them more responsive to the type of ultrasonic vocalization; males were little affected. Female experience, of course, includes the hormonal changes due to pregnancy and lactational state. The results are consistent with a communicatory function for isolation-induced ultrasonic vocalization.

Dopamine's role in social modulation of infant isolation-induced vocalization: I. Reunion responses to the dam, but not littermates, are dopamine dependent.

Rat pups' vocalization during social separation and the cessation of vocalization upon social reunion (contact quieting) model early life affiliative relationships. The present study examined the roles of dopamine (DA) receptors in regulating contact quieting. Contact quieting to the dam, but not to littermates, was disrupted by either blockade or exogenous stimulation of DA D1-like receptors. The D2 antagonist raclopride also prevented the quieting effect of reunion with the dam and had a lesser effect on the quieting properties of littermates. In contrast, the D2 agonist quinpirole permitted or enhanced contact quieting. Combined systemic and local striatal administration of D2 ligands showed that stimulation of striatal D2 receptors can enhance, but is not necessary for, contact quieting to the dam. These results are consistent with the literature linking the neural mechanisms of affiliation and reinforcement. This is also the first demonstration that the neurochemical substrates of an infant comfort response to dams differ from a behaviorally similar response to siblings.

Dopamine's role in social modulation of infant isolation-induced vocalization: II. Maternally modulated infant separation responses are regulated by D1- and D2-family dopamine receptors.

Mammalian infant behavior directed toward caregivers is critical to survival and may play a role in establishing social bonds. Most mammalian infants vocalize when isolated. Rat pups vocalize at a higher rate when isolated following an interaction with an adult female than after an interaction with littermates, a phenomenon termed maternal potentiation. We previously reported that the D2 receptor family agonist quinpirole disrupts maternal potentiation at a dose that does not alter vocalization rate following contact with littermates. Here we further examine the role of dopamine in maternal potentiation by testing effects of both D1 and D2 receptor family ligands, alone and in combination, on maternal potentiation. We tested the drugs' effects on isolation vocalization subsequent to littermate contact and then another isolation preceded by a brief "reunion" period of exposure either to the anesthetized dam or a handling-only "pickup" condition. D2 receptor stimulation blocked the increase in vocalizations following reunion with the dam. The D2 agonist effect in the dam-reunion condition was much larger than its small effect in the pickup condition, providing further evidence that D2 receptors exert a selective modulation of maternal potentiation. On the other hand, systemic administration of the D1 agonist SKF81297 reduced isolation vocalizations nonspecifically, across all the experimental conditions. Finally, the D1 and D2 receptor dual antagonist, alpha-flupenthixol, increased isolation vocalizations and disrupted potentiation, but at doses that also inhibited locomotion. We conclude that D2 receptor family activation has a more selective effect of disrupting maternal potentiation than D1 receptor family activation.

Ventral striatum dopamine D2 receptor activity inhibits rat pups' vocalization response to loss of maternal contact.

Most mammalian infants vocalize when isolated. The vocalization promotes caregiver proximity, which is critical to survival. If, before isolation, a rat pup has contact with its dam, its isolation vocalization rate is increased (maternal potentiation) relative to isolation preceded only by littermate contact. Prior work showed that systemic administration of a D2 receptor agonist blocks maternal potentiation at doses that do not alter baseline vocalization. In this study, infusion of quinpirole (2 microg/side) into the nucleus accumbens also blocks maternal potentiation. Infusion of the accumbens with the D2 antagonist raclopride (4 microg/side) prevents systemic quinpirole from blocking potentiation. Quinpirole infusion in the dorsal striatum did not affect maternal potentiation and infusion of raclopride in the dorsal striatum did not reverse the block of maternal potentiation by systemic quinpirole. Vocalization results after a second vehicle infusion on a given day are no different than the results following an initial vehicle infusion, so experimental design can not account for the effects of drug infusions. Because activity level was increased by both dorsal and ventral striatum infusions, activity level can not account for the results.

Acquisition and expression of a socially mediated separation response.

Separation and reunion responses have been used to investigate social relationships in many species, including humans. When isolated from their mothers and siblings, infant rats vocalize in the ultrasonic range. An isolated pup reduces its rate of vocalization when placed in contact with familiar stimuli, particularly social ones such as its dam or littermates. The isolated pup's vocalization is greatly increased if the pup has been in contact with its mother immediately before isolation, a phenomenon called maternal potentiation. Early experience can play a role in the acquisition of potentiation. If rat pups are reared by both dam and sire, or even reared by the dam in the presence of the sire's odor, the pups show potentiation to the sire instead of the fear-related behavioral inhibition. Littermates, home cage shavings, and other familiar stimuli from the rearing environment do not elicit increased vocalizations during a subsequent isolation. The neurobiological mechanisms by which the sire becomes capable of potentiating vocalization are unknown, but are hypothesized to depend on the processes underlying development of an odor preference. Expression of potentiation is hypothesized to be related to reward processes in part because dopamine activity plays a regulatory role. Activation of dopamine type 2 receptors in the nucleus accumbens blocks maternal potentiation without altering vocalization rate in an initial isolation. The modulation of isolation-induced vocalization by social interactions provides a paradigm for investigating the neurobehavioral mechanisms underlying acquisition and expression of early life social bonds.

Brief exposure to the biological mother "potentiates" the isolation behavior of precocial Guinea pig pups.

When isolated rat pups are briefly reunited with a lactating female, her subsequent removal leads to a dramatic increase in the emission of ultrasonic vocalizations, but not other behaviors. Whether this socially induced augmentation of isolation behavior (i.e., "potentiation") is characteristic only of altricial rodents is not known. Therefore, we examined precocial guinea pig pups in a potentiation paradigm. Ten-day-old guinea pigs were isolated in a test cage for 10 min, at which time they were then placed into a second cage for 5 min that either contained a companion or, for controls, was empty. Pups were then isolated again in the test cage for a second 10-min period. Control pups showed a significant reduction in vocalizing and locomotor activity from the first to second isolation period. Exposure to the biological mother prevented the decline in both behaviors (Experiment 1), whereas exposure to a familiar littermate (Experiment 2) had no effect, and exposure to an unfamiliar lactating female (Experiment 3) had only a minimal effect on locomotor activity. The results show that potentiation of isolation behaviors is not limited to altricial rodents, and suggest that specific characteristics of the effect (i.e., its magnitude, the specific behaviors affected, and the selectivity of the response to particular social partners) varies with the abilities and requirements of the young, as well as the behavioral ecology of the species in question.

Maternally modulated infant separation responses are regulated by D2-family dopamine receptors.

Although dopamine is necessary for mammalian adult pair-bond formation and maternal behavior, its function in infant social behavior and attachment has been less thoroughly explored. The vocalization rate of an isolated rat pup is influenced by recent social contact. Interactions with the dam potentiate vocalization rate. Interactions with littermates or adult males do not. Systemic administration of the D2-family agonist quinpirole specifically blocked maternal potentiation at doses that did not alter vocalization rate in an isolation prior to dam contact. This result was not explained by quinpirole's effects on body temperature or locomotion. The results are consistent with a role for dopamine in infant social behavior.

Blood pressure responses to feeding in infancy: spin-offs of serendipity.

This article summarizes results from a program of research that has focused on understanding the characteristics of, and factors that shape, acute cardiovascular responses to feeding in infants. The work developed from behavioral observations in rats suggesting a linkage between variations in maternal behavior and adult blood pressure. However, the hypothesis that specific types of interactions, in particular those associated with feeding, might alter cardiovascular function was greatly influenced by a serendipitous observation made by Myron Hofer several years earlier. Myron discovered that nutrient availability is a key regulator of cardiovascular function in newborn rats. Our spin-off studies established that, in newborn rats and human infants, ingestion of nutrient is associated with robust increases in heart rate and blood pressure.

Development of the baroreflex in the young rat.

The baroreceptor-heart period reflex was assessed in conscious, freely behaving rat pups on postnatal days 6 and 14. The baroreceptor-heart period reflex was elicited using the alpha1-adrenergic agonist phenylephrine to increase blood pressure and the vasodilator, sodium nitroprusside, to decrease blood pressure. The autonomic effects of the baroreceptor manipulations were determined using pharmacological autonomic blockade. The data demonstrate that vasoconstriction produces a potent baroreflex-mediated bradycardia as early as postnatal day 6, which had previously been demonstrated only in anesthetized pups. In the anesthetized pup, the bradycardia is mediated by vagal activation, while we demonstrate that both vagal activation and sympathetic withdrawal occur in unanesthetized animals. In addition, the results replicate previous findings in rats demonstrating minimal cardiac sympathetic activation or vagal withdrawal following vasodilation during the first week of life, but substantial baroreflex-mediated tachycardia by the second week.