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.

Variations in maternal behavior in rats selected for infant ultrasonic vocalization in isolation.

Individual differences in maternal behavior in rodents are associated with altered physiology and behavior in offspring across their lifespan and across generations. Offspring of rat dams that engage in high frequencies of high-arched-back nursing and pup-licking (High-LG) show attenuated stress responses compared to those engaging in lower frequencies (Low-LG). Selective breeding also produces widespread alterations in physiology and behavior that are stable over generations. To examine processes underlying generational and developmental influences on anxiety in an animal model, we developed two lines of rats that emit either extremely high (High-USV) or low (Low-USV) rates of 45kHz ultrasonic vocalizations in isolation at postnatal day 10. Compared to the Low-USV line, High-USV rats display increased indices of anxiety- and depression-like behavior in adulthood. The current study assessed maternal behaviors as well as oxytocin and vasopressin receptor density in High-USV and Low-USV dams to determine if selective breeding had produced differences that paralleled those found in Low- and High-LG dams. We found that Low-USV dams engage in more high-arched nursing and pup-licking than High-USV dams. Differences in oxytocin and vasopressin receptor levels were not widespread throughout the brain, with line differences in the piriform cortex and nucleus accumbens. This research illustrates the potential interplay between genetically determined (USV line) and environmental (postnatal mother-infant interactions) factors in accounting for the phenotypes associated with maternal separation induced postnatal vocalizations.

Family Nurture Intervention in the Neonatal Intensive Care Unit improves social-relatedness, attention, and neurodevelopment of preterm infants at 18 months in a randomized controlled trial.

BACKGROUND: Preterm infants are at high risk for adverse neurodevelopmental and behavioral outcomes. Family Nurture Intervention (FNI) in the Neonatal Intensive Care Unit (NICU) is designed to counteract adverse effects of separation of mothers and their preterm infants. Here, we evaluate effects of FNI on neurobehavioral outcomes. METHODS: Data were collected at 18 months corrected age from preterm infants. Infants were assigned at birth to FNI or standard care (SC). Bayley Scales of Infant Development III (Bayley-III) were assessed for 76 infants (SC, n = 31; FNI, n = 45); the Child Behavior Checklist (CBCL) for 57 infants (SC, n = 31; FNI, n = 26); and the Modified Checklist for Autism in Toddlers (M-CHAT) was obtained for 59 infants (SC, n = 33; FNI, n = 26). RESULTS: Family Nurture Intervention significantly improved Bayley-III cognitive (p = .039) and language (p = .008) scores for infants whose scores were greater than 85. FNI infants had fewer attention problems on the CBCL (p < .02). FNI improved total M-CHAT scores (p < .02). Seventy-six percent of SC infants failed at least one of the M-CHAT items, compared to 27% of FNI infants (p < .001). In addition, 36% of SC infants versus 0% of FNI infants failed at least one social-relatedness M-CHAT item (p < .001). CONCLUSIONS: Family Nurture Intervention is the first NICU intervention to show significant improvements in preterm infants across multiple domains of neurodevelopment, social-relatedness, and attention problems. These gains suggest that an intervention that facilitates emotional interactions between mothers and infants in the NICU may be key to altering developmental trajectories of preterm infants.

Communal nesting exerts epigenetic influences on affective and social behaviors in rats selectively bred for an infantile trait.

Communal nesting (CN) is a mouse model of early social enrichment during pregnancy and lactation. In this study, a rat model of CN was developed to determine if CN exerts an epigenetic effect in rats selectively bred for an infantile affective trait (high and low rates of ultrasonic distress calls). High and Low offspring from CN groups were compared to standard reared (SN) offspring on five measures of social and affective behavior at three critical ages. A differential effect of the CN paradigm on High and Low lines was seen in measures of anxiety and arousal, but not in measures of depression or social behavior. Neonatal CN subjects emitted fewer distress calls than SN subjects when separated from their dams, and the High line subjects were more affected by the CN procedure. As juveniles, CN subjects showed increased social behaviors in tests of juvenile parenting and play compared to SN subjects. In adulthood, CN differentially increased the activity of Low line subjects. All CN subjects displayed less anxiety behavior in an open field compared to SN subjects; High line subjects were more anxious than Lows. CN reduced immobility and increased attempts to escape on the Porsolt forced swim task relative to SN subjects. These results extend the usefulness of this early enrichment paradigm from mice to rats, and found some rodent species differences in outcomes dependent on the behavioral test. They also emphasize the importance of social contact during pregnancy and lactation on offspring's optimal development across behaviors and ages.

Family nurture intervention (FNI): methods and treatment protocol of a randomized controlled trial in the NICU.

BACKGROUND: The stress that results from preterm birth, requisite acute care and prolonged physical separation in the Neonatal Intensive Care Unit (NICU) can have adverse physiological/psychological effects on both the infant and the mother. In particular, the experience compromises the establishment and maintenance of optimal mother-infant relationship, the subsequent development of the infant, and the mother's emotional well-being. These findings highlight the importance of investigating early interventions that are designed to overcome or reduce the effects of these environmental insults and challenges. METHODS: This study is a randomized controlled trial (RCT) with blinded assessment comparing Standard Care (SC) with a novel Family Nurture Intervention (FNI). FNI targets preterm infants born 26-34 weeks postmenstrual age (PMA) and their mothers in the NICU. The intervention incorporates elements of mother-infant interventions with known efficacy and organizes them under a new theoretical context referred to collectively as calming activities. This intervention is facilitated by specially trained Nurture Specialists in three ways: 1) In the isolette through calming interactions between mother and infant via odor exchange, firm sustained touch and vocal soothing, and eye contact; 2) Outside the isolette during holding and feeding via the Calming Cycle; and 3) through family sessions designed to engage help and support the mother. In concert with infant neurobehavioral and physiological assessments from birth through 24 months corrected age (CA), maternal assessments are made using standard tools including anxiety, depression, attachment, support systems, temperament as well as physiological stress parameters. Quality of mother-infant interaction is also assessed. Our projected enrolment is 260 families (130 per group). DISCUSSION: The FNI is designed to increase biologically important activities and behaviors that enhance maternally-mediated sensory experiences of preterm infants, as well as infant-mediated sensory experiences of the mother. Consequently, we are enlarging the testing of preterm infant neurodevelopment beyond that of previous research to include outcomes related to mother-infant interactions and mother-infant co-regulation. Our primary objective is to determine whether repeated engagement of the mother and her infant in the intervention's calming activities will improve the infant's developmental trajectory with respect to multiple outcomes. Our secondary objective is to assess the effectiveness of FNI in the physiological and psychological co-regulation of the mother and infant. We include aspects of neurodevelopment that have not been comprehensively measured in previous NICU interventions. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01439269.

Moderate recurrent hypoglycemia during early development leads to persistent changes in affective behavior in the rat.

Recurrent hypoglycemia is a common problem among infants and children that is associated with several metabolic disorders and insulin-dependent diabetes mellitus. Although studies have reported a relationship between a history of juvenile hypoglycemia and psychological health problems, the direct effects of recurrent moderate hypoglycemia have not been fully determined. Thus, in this study, we used an animal model to examine the effects of recurrent hypoglycemia during the juvenile period on affective, social, and motor function (assessed under euglycemic conditions) across development. To model recurrent hypoglycemia, rats were administered 5 U/kg of insulin or saline twice per day from postnatal day (P)10 to P19. Body weight gain was retarded in insulin-treated rats during the treatment period, but recovered by the end of treatment. However, insulin-treated rats displayed increases in affective reactivity that emerged early during treatment and persisted after treatment into early adulthood. Specifically, insulin-treated pups showed increased maternal separation-induced vocalizations as infants, and an exaggerated acoustic startle reflex as juveniles and young adults. Moreover, young adult rats with a history of recurrent juvenile hypoglycemia exhibited increased fear-potentiated startle and increases in behavioral and hormonal responses to restraint stress. Some of these effects were sex-dependent. The changes in affective behavior in insulin-exposed pups were accompanied by decreases in adolescent social play behavior. These results provide evidence that recurrent, transient hypoglycemia during juvenile development can lead to increases in fear-related behavior and stress reactivity. Importantly, these phenotypes are not reversed with normalization of blood glucose and may persist into adulthood.

Effects of gestational allopregnanolone administration in rats bred for high affective behavior.

The anxiolytic neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one or 3alpha,5alpha-THP) has been proposed to play a developmental role in emergent neural regulation of affective behavior. This experiment examined whether allopregnanolone administered during the last week of gestation in rats would alter neonatal and adult offspring behaviors in the selectively-bred High vocalizing line, who have low levels of allopregnanolone and high levels of anxious/depressive behaviors. Dams were injected twice a day with the neurosteroid or vehicle, or handled as controls, and were tested on the elevated plus maze just before parturition. Maternal behavior was assessed throughout the first week of life, and affective behavior in the offspring was tested at one week of age (ultrasonic vocalizations test) and as adults (plus maze and forced swim tests). Offspring prenatally exposed to allopregnanolone were less anxious as neonates and less depressed as adults compared to both control groups. Only male adult offspring, however, revealed less anxious behavior on the plus maze. Neither the dams' anxiety behavior measured in late gestation nor their postnatal maternal behavior was altered compared to controls, suggesting a direct, long-lasting effect of gestational allopregnanolone on the developing fetal brain independent of mediating maternal factors. These results are discussed in light of new evidence about the developmental role of the GABA-A receptor prenatally.

PRX-00023, a selective serotonin 1A receptor agonist, reduces ultrasonic vocalizations in infant rats bred for high infantile anxiety.

To address the development of early anxiety disorders across the lifespan, the High USV line of rats was bred based on rates of infant ultrasonic vocalization in the 40-50 kHz range of predominant frequencies (USV) to maternal separation at postnatal day (P) 10. In this study, rates of USV in High line infants (pups: Postnatal Day 11+/-1) were compared to those of randomly-bred controls in response to EPIX compound PRX-00023, a unique serotonin (5-HT) agonist, acting exclusively at the 5-HT1A receptor, or buspirone, a nonspecific 5HT1A agonist. After testing, pups were examined for sedation and other drug-related effects. The results indicated that all doses of buspirone reduced USV rates in isolation, consistent with other reports. PRX-00023 significantly reduced USV rates at the lowest doses (0.01-0.05 mg/kg). None of the PRX-00023 doses produced sedation, whereas all but the lowest dose of buspirone (0.1 mg/kg) produced sedation effects. The results suggest that this compound alleviates infantile anxiety-like behavior with great specificity in rats bred for high anxiety/depressive phenotypes by selectively targeting 5-HT1A receptors, possibly by both pre- and post-synaptic mechanisms.

Selective breeding for infant rat separation-induced ultrasonic vocalizations: developmental precursors of passive and active coping styles.

Human depression and anxiety disorders show inherited biases across generations, as do antisocial disorders characterized by aggression. Each condition is preceded in children by behavioral inhibition or aggressive behavior, respectively, and both are characterized by separation anxiety disorders. In affected families, adults and children exhibit different forms of altered autonomic nervous system regulation and hypothalamic-pituitary-adrenal activity in response to stress. Because it is difficult to determine mechanisms accounting for these associations, animal studies are useful for studying the fundamental relationships between biological and behavioral traits. Pharmacologic and behavioral studies suggest that infant rat ultrasonic vocalizations (USV) are a measure of an early anxiety-like state related to separation anxiety. However, it was not known whether or not early ultrasound emissions in infant rats are markers for genetic risk for anxiety states later in life. To address these questions, we selectively bred two lines of rats based on high and low rates of USV to isolation at postnatal (P) 10 days of age. To our knowledge, ours is the only laboratory that has ever selectively bred on the basis of an infantile trait related to anxiety. The High and Low USV lines show two distinct sets of patterns of behavior, physiology and neurochemistry from infancy through adulthood. As adults High line rats demonstrate "anxious"/"depressed" phenotypes in behavior and autonomic nervous system (ANS) regulation to standard laboratory tests. In Lows, on the other hand, behavior and autonomic regulation are consistent with an "aggressive" phenotype. The High and Low USV lines are the first genetic animal models implicating long-term associations of contrasting "coping styles" with early attachment responses. They thus present a potentially powerful model for examining gene-environment interactions in the development of life-long affective regulation.

The effects of selective breeding for infant ultrasonic vocalizations on play behavior in juvenile rats.

For over 25 generations, two lines of rats (High and Low USV lines) have been selectively bred for extreme rates of infantile (45 kHz) ultrasonic vocalizations (USV) in response to maternal separation at postnatal day (P)10. High and low line juveniles (P30-P40) were socially isolated and allowed to play in same-sex sibling pairs for 10 min per day over three days. Measures of play were nape contacts and pinning. Other social and nonsocial behaviors were also scored during the three sessions; two of these, 55 kHz USV and walk-overs, were statistically associated with play. Compared to the Random control line, both High and Low line juveniles showed deficits in play behavior. In the High line, play initiatory behavior (nape contacts) was reduced, but pinning, USV and walk-overs were relatively unchanged. In contrast, nape contacts, pinning, USV and walk-overs were all reduced in Low line juveniles compared to Random line controls. The results suggest that selection for extremes of infant USV rates has produced temperamental differences that are expressed in juvenile play in the High and Low USV lines.

Traumatized mothers can change their minds about their toddlers: Understanding how a novel use of videofeedback supports positive change of maternal attributions.

This study explored the use of a brief experimental intervention that integrates principles of infant-parent psychotherapy, videofeedback, controlled exposure to child distress in the context of parental posttraumatic stress disorder (PTSD), and stimulation of parental reflective functioning (RF). The Clinician Assisted Videofeedback Exposure Session (CAVES) was applied to 32 interpersonal violence-exposed mothers of very young children (8-50 months) with respect to change of maternal perception of her child. While we found no significant reduction over two videotaped assessment visits with a mental health professional, we did find a significant reduction in the degree of negativity of maternal attributions towards her child following the videotaped visit focused on the CAVES (p<.01). Maternal RF, a mother's capacity to think about mental states in herself and her child, accounted for 11% of the variance in reduction of maternal negativity after accounting for baseline levels of negativity. Clinician-assisted videofeedback appears to support emotional self-regulation of mothers with violence-related PTSD. Focusing with a therapist on videofeedback of child separation distress exposes mothers to avoided mental states of helplessness and perceived loss of protection. Negative maternal attributions may mark violent trauma-associated emotion dysregulation and projected self-representations of the maltreated mother.

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.

Development and evolution of hidden regulators: selective breeding for an infantile phenotype.

Mother-infant separation in the rat has been used as an analytical tool to reveal biosocial processes underlying infant physiology and behavior. The same strategy has guided a project in which selective breeding for an infantile behavior has provided insights into how biological systems become recruited and integrated as expressions of temperamental affective responses. Two lines of rats (High and Low USV lines) were selectively bred based on rates of USV emission to maternal separation and isolation at postnatal day (P) 10. After many generations of breeding, the High and Low lines show widespread and distinctly different profiles of physiology and behavior in the first 3 weeks of life. Insights gained from longitudinal studies suggest that selection may work by reorganizing developmental processes, not just a given trait, over the postnatal period. As animal models, the lines have the potential to provide valuable tools for understanding developmental mechanisms underlying genetic and developmental risk for depression/anxiety syndromes in children and adults.

Behavioral effects of toluene in rats selectively bred for infantile vocalization rate.

Glue sniffing is epidemic among children living in poverty in Latin America. Previous research has shown that abused inhalants such as toluene share pharmacological properties with anxiolytic drugs, and that personality factors such as degree of anxiety have been proposed to modulate the effects of these drugs. To study this interaction in an animal model, rats selectively bred for high (High) or low (Low) rates of distress calls after maternal separation (ultrasonic vocalizations, USVs) were used to investigate toluene's acute and long-term effects on two measures of anxiety behavior. At ten days of age, neonatal subjects were administered toluene (1 g/kg i.p.) and USVs were recorded. The subjects were retested as juveniles on an elevated plus maze to examine sequela of earlier toluene exposure. Acute toluene administration reduced USVs relative to control groups in neonates of both lines, indicating anxiolysis. As expected, Lows had reduced USVs relative to Highs. At 28 days of age, Highs spent more time in the open arms of the elevated plus maze than Lows. However, prior neonatal toluene exposure blocked this reversal of behavioral phenotype. This suggests that early toluene exposure compromised a compensatory process occurring during this developmental period, which may have been maternally mediated. These results have implications for the effects of early drug exposure on plasticity in the developing nervous system.

Differences in affective behaviors and hippocampal allopregnanolone levels in adult rats of lines selectively bred for infantile vocalizations.

Allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP), a progesterone metabolite, is an endogenous neurosteroid mediating affective behaviors via its positive modulation of GABA(A) receptors. In order to better understand the role of this neurosteroid in individual differences in affective behavior, we used an animal model based on selective breeding for an infantile affective trait, ultrasonic vocalizations (USV). Adult male and female (in either proestrus or diestrus) rats that had been bred for low (low line) or high (high line) rates of USV after maternal separation were tested in a series of affective behavioral tests: open field, emergence, social interaction, defensive freezing, and the Porsolt forced swim task. Concentrations of allopregnanolone in combined hippocampus and amygdala tissue were then measured. low line subjects showed significantly lower anxiety and depression responses in the emergence, open field, and Porsolt forced swim tasks than did high line subjects. Proestrus females exhibited less affective behaviors than diestrus females or males. Allopregnanolone levels in hippocampus/amygdala were significantly higher in low line subjects compared to high line subjects, and in proestrus females compared to diestrus females and males. These data indicate that: (1) affective behaviors in lines selectively bred for an infantile anxiety trait exhibit selection persistence into adulthood; and (2) levels of allopregnanolone in the limbic system parallel selected disparities in affective behavior, suggesting a selection for alterations in the neurosteroid/GABA(A) receptor system in these lines.

Selective breeding for an infant phenotype: rat pup ultrasonic vocalization (USV).

To examine processes underlying generational and developmental influences on anxiety, this laboratory produced two lines of (N:NIH strain) rats, selectively bred on the basis extreme rates of ultrasonic vocalization in 2 minutes of isolation at Postnatal Day 10. The research reviewed in this article focuses on: (1) establishment of the selectively bred lines; (2) defining infant behavioral and physiological phenotypes and (3) determining whether infantile USV phenotypes endure over development. The High and Low lines have diverged widely in their USV rates from each other and from the Random control line, which has maintained N:NIH strain rates overall from generation to generation. Beginning in the 11th generation, High USV pups have shown significantly higher frequencies of defecation and urination during isolation screening than the Low USV and random control line. Both lines show altered autonomic regulation of heart rates (HR) in response to stressors as juveniles and adults. These differences in HR responses in High and Low lines appear to be mediated by changes in the balance of sympathetic versus parasympathetic mechanisms. Other behavioral characteristics of the High line are consistent with an "anxious"/ "depressive" phenotype, such as vocalizations to touch in a novel environment, and performance in the Porsolt Swim, whereas Low line shows few differences in anxiety behavior. Future work will resolve the similarities and differences in the High and Low phenotypes and provide a developmental perspective to the growing body of information about affective regulation in humans and animals provided by selectively bred animal models.

Lack of evidence for mu-opioid regulation of a socially mediated separation response.

Maternal potentiation of ultrasonic vocalization (USV) occurs when, after a brief contact with the dam, reisolated infant rats produce USV at a rate as much as double or triple their calling rate in the initial isolation. Potentiation occurs most robustly when the dam is alert (active) but is also elicited by an anesthetized dam (passive). The possible role of mu-opioid receptors in control of both active and passive potentiation was tested using the mu-preferring receptor agonist morphine and antagonist naltrexone. While high levels of morphine did suppress USV rate in both the initial and the second isolation, neither agonist nor antagonist demonstrated an effect specific to potentiation. That is, no dose of morphine or naltrexone suppressed or enhanced the response to the active dam, and only the highest dose of morphine prevented potentiation with the passive dam. The results do not support the possibility that activation of mu-receptors by endogenous opioids regulates the increase in USV caused by maternal potentiation.

Brief maternal interaction increases number, amplitude, and bout size of isolation-induced ultrasonic vocalizations in infant rats (Rattus norvegicus).

The number, amplitude, duration, and bout structure of isolation-induced ultrasonic vocalization (USV) of infant rats (Rattus norvegicus) were measured on postnatal Day 10. Measurements were made before and after a brief, 1-min, active interaction with their mother or before and after a "pick-up" control procedure. Consistent with prior studies, the number of USVs emitted was significantly increased in the period following the maternal reunion but not after the control procedure. The average amplitude of USVs was also greater following maternal reunion. Finally, analyses characterizing the bout structure of USV production indicated that the average bout size (i.e. number of USVs/bout) was increased severalfold following the reunion with the mother, accounting for the greater rate of USV production during the second isolation period.

Psychobiological dysregulation in violence-exposed mothers: salivary cortisol of mothers with very young children pre- and post-separation stress.

UNLABELLED: To understand the determinants of frightening/frightened and other atypical maternal behavior, the authors studied a sample of 41 inner-city mothers of very young children (ages 8-50 months), the mothers of whom had lifetime histories of interpersonal violent trauma (i.e., physical or sexual abuse, and domestic violence) and related posttraumatic stress. METHOD: The authors measured (1) maternal salivary cortisol levels before and 30 minutes after a videotaped play paradigm with their children, involving two separations and reunions; and (2) cortisol reactivity 30 minutes after separation stress. Data were analyzed using Pearson bivariate correlations, ANOVA, and multiple linear regressions. RESULTS: Salivary cortisol "baseline" values were significantly negatively correlated with childhood interpersonal violent trauma severity (i.e., trauma severity prior to age 16). However, cortisol reactivity was not significantly correlated with interpersonal violent trauma severity at this level of analysis. Although baseline salivary cortisol values were not significantly correlated with current overall psychiatric or depressive symptoms, they were negatively correlated with severity of current posttraumatic stress symptoms (PTSS) and with dissociative symptoms. Neither dimensions of negativity nor distortion of maternal attributions showed any significant association with prestress or poststress salivary cortisol levels. Salivary cortisol baseline was negatively correlated with atypical maternal behavior via measurement of the level of disrupted communication, at a trend-level of significance. CONCLUSIONS: Violent trauma-associated dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis may be a marker for increased risk for intergenerational transmission via parenting behavior with young children. Low salivary cortisol prior to separation stress and blunted cortisol reactivity to separation may also be markers for posttraumatic stress.

Social, thermal, and temporal influences on isolation-induced and maternally potentiated ultrasonic vocalizations of rat pups.

Sensory and temporal factors have been demonstrated to be involved in the regulation of isolation-induced ultrasonic vocalizations (USV) of young rats. Sensory cues include thermal, olfactory, and tactile modalities. Temporal factors include the time spent in isolation. The goal of the present research was to examine the interaction of these factors in both isolation-induced and maternally potentiated USV. Maternal potentiation of USV occurs when a brief interaction with the dam, even a passive (anesthetized) dam, elicits an augmented vocal response to a subsequent isolation, with rates of USV in rat pups well above those emitted in standard isolation tests. We found that passive maternal potentiation of USV did occur under all conditions tested. Neither a 30-min prior isolation nor high ambient temperature prevented an increase in USV rate over the rate of the original isolation. After 30-min isolation at warm temperatures when the rate of USV had fallen to zero, the pups increased vocalization in the presence of the dam as well as in the subsequent isolation. Temporal and thermal factors also interacted significantly in regulating the level of the USV emitted by the pups during the first isolation, in the presence of the anesthetized dam, and during the second isolation.