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.

Depression and anxiety symptoms of mothers of preterm infants are decreased at 4 months corrected age with Family Nurture Intervention in the NICU.

Preterm delivery can precipitate maternal psychological morbidities. Family Nurture Intervention (FNI) was designed to minimize these by facilitating the emotional connection between mother and infant, beginning early in the infant's neonatal intensive care unit (NICU) stay. We examined depression and anxiety symptoms of mothers of preterm infants at 4 months infant corrected age (CA). One hundred fifteen mothers who delivered between 26 and 34 weeks gestational age were randomized to receive standard care (SC) or standard care plus FNI. Mothers' self-reported depressive symptoms (Center for Epidemiologic Studies Depression Scale: CES-D) and state anxiety (Spielberger State-Trait Anxiety Inventory: STAI) symptoms were assessed at enrollment, near to term age, and 4 months (CA). At 4 months CA, mean CES-D and STAI scores were significantly lower in FNI mothers compared to SC mothers. Effectiveness of FNI can only be evaluated as an integrated intervention strategy as it was not possible to control all aspects of FNI activities. Although there was considerable loss to follow-up, analyses suggest that resulting biases could have masked rather than inflated the measured effect size for depressive symptoms. FNI may be a feasible and practicable way to diminish the impact of premature delivery on maternal depressive and anxiety symptoms.

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.

Family nurture intervention improves the quality of maternal caregiving in the neonatal intensive care unit: evidence from a randomized controlled trial.

OBJECTIVE: This study assessed the impact of Family Nurture Intervention (FNI) on the quality of maternal caregiving behavior (MCB) while in the neonatal intensive care unit (NICU). FNI is a randomized controlled trial conducted in a high-acuity NICU to facilitate an emotional connection between mothers and their premature infants. FNI begins shortly after birth, continues until discharge, and involves mother/infant calming sessions that include scent cloth exchange, vocal soothing and emotion expression, eye contact, skin-to-skin and clothed holding, and family-based support sessions. METHODS: Maternal caregiving behavior was coded during a single holding and feeding session (∼30 min) in the NICU before discharge at approximately 36 weeks gestational age (GA). Sixty-five mothers and their premature infants (34 male, 31 female; 26-34 wk GA) were included in these analyses (FNI, n = 35; standard care [SC], n = 30). RESULTS: Relative to mothers in the SC condition, those in the FNI group showed significantly higher quality MCB, which remained significant when controlling for birth order, twin status, maternal depression, and maternal anxiety. CONCLUSION: This is the first study to demonstrate that in-unit MCB can be enhanced by a hospital-based intervention. FNI provides a new rationale for integrating nurture-based interventions into standard NICU care.

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.

Electroencephalographic activity of preterm infants is increased by Family Nurture Intervention: a randomized controlled trial in the NICU.

OBJECTIVE: To assess the impact of Family Nurture Intervention (FNI) on electroencephalogram (EEG) activity in preterm infants (26-34 weeks gestation). METHODS: Two groups were tested in a single, level IV neonatal intensive care unit (NICU; standard care or standard care plus FNI) using a randomized controlled trial design. The intervention consists of sessions designed to achieve mutual calm and promote communication of affect between infants and their mothers throughout the NICU stay. EEG recordings were obtained from 134 infants during sleep at ∼35 and ∼40 weeks postmenstrual age (PMA). Regional brain activity (power) was computed for 10 frequency bands between 1 and 48 Hz in each of 125 electrodes. RESULTS: Near to term age, compared to standard care infants, FNI infants showed robust increases in EEG power in the frontal polar region at frequencies 10 to 48 Hz (20% to 36% with p-values <0.0004). Effects were significant in both quiet and active sleep, regardless of gender, singleton-twin status, gestational age (26-30 or 30-35 weeks) or birth weight (<1500 or >1500 g). CONCLUSION: FNI leads to increased frontal brain activity during sleep, which other investigators find predictive of better neurobehavioral outcomes. SIGNIFICANCE: FNI may be a practicable means of improving outcomes in preterm infants.

Randomized controlled trial of Family Nurture Intervention in the NICU: assessments of length of stay, feasibility and safety.

BACKGROUND: While survival rates for preterm infants have increased, the risk for adverse long-term neurodevelopmental and behavioral outcomes remains very high. In response to the need for novel, evidence-based interventions that prevent such outcomes, we have assessed Family Nurture Intervention (FNI), a novel dual mother-infant intervention implemented while the infant is in the Neonatal Intensive Care Unit (NICU). Here, we report the first trial results, including the primary outcome measure, length of stay in the NICU and, the feasibility and safety of its implementation in a high acuity level IV NICU. METHODS: The FNI trial is a single center, parallel-group, randomized controlled trial at Morgan Stanley Children's Hospital for mothers and their singleton or twin infants of 26-34 weeks gestation. Families were randomized to standard care (SC) or (FNI). FNI was implemented by nurture specialists trained to facilitate affective communication between mother and infant during specified calming interactions. These interactions included scent cloth exchange, sustained touch, vocal soothing and eye contact, wrapped or skin-to-skin holding, plus family-based support interactions. RESULTS: A total of 826 infants born between 26 and 34 weeks during the 3.5 year study period were admitted to the NICU. After infant and mother screening plus exclusion due to circumstances that prevented the family from participating, 373 infants were eligible for the study. Of these, we were unable to schedule a consent meeting with 56, and consent was withheld by 165. Consent was obtained for 150 infants from 115 families. The infants were block randomized to groups of N = 78, FNI and N = 72, SC. Sixteen (9.6%) of the randomized infants did not complete the study to home discharge, 7% of those randomized to SC and 12% of FNI infants. Mothers in the intervention group engaged in 3 to 4 facilitated one- to two-hour sessions/week. Intent to treat analyses revealed no significant difference between groups in medical complications. The mean length of stay was not significantly affected by the intervention. CONCLUSION: There was no significant effect demonstrated with this intervention amount on the primary short-term outcome, length of stay. FNI can be safely and feasibly implemented within a level IV NICU. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01439269.

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.

An fMRI study of the brain responses of traumatized mothers to viewing their toddlers during separation and play.

This study tested whether mothers with interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) vs healthy controls (HC) would show greater limbic and less frontocortical activity when viewing young children during separation compared to quiet play. Mothers of 20 children (12-42 months) participated: 11 IPV-PTSD mothers and 9 HC with no PTSD. During fMRI, mothers watched epochs of play and separation from their own and unfamiliar children. The study focused on comparison of PTSD mothers vs HC viewing children in separation vs play, and viewing own vs unfamiliar children in separation. Both groups showed distinct patterns of brain activation in response to viewing children in separation vs play. PTSD mothers showed greater limbic and less frontocortical activity (BA10) than HC. PTSD mothers also reported feeling more stressed than HC when watching own and unfamiliar children during separation. Their self-reported stress was associated with greater limbic and less frontocortical activity. Both groups also showed distinct patterns of brain activation in response to viewing their own vs unfamiliar children during separation. PTSD mothers' may not have access to frontocortical regulation of limbic response upon seeing own and unfamiliar children in separation. This converges with previously reported associations of maternal IPV-PTSD and atypical caregiving behavior following separation.

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.

Evolutionary basis of adaptation in resilience and vulnerability: response to Cicchetti and Blender.

This masterful and wide-ranging paper gives us a clear picture of the need for interdisciplinary studies of resilience and vulnerability. We need to go beyond the interacting events at the different levels to see clearly that multiple levels of analysis and concept do not themselves interact, but instead give us different aspects of a biological, psychological, or behavioral event. Thus, no levels are reducible to any of the others. In the rest of my discussion, I raise the likelihood that the multiple level responses of "resilience" are no more adaptive in their evolutionary history than the very different responses we call "vulnerability." Keeping in mind the evolved functions of these responses, and the circumstances of their selection during evolution, will provide us with new ideas and approaches for understanding and intervention.

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.

Altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter-deficient mice.

BACKGROUND: As a key regulator of serotonergic activity and target of many antidepressant treatments, the serotonin transporter (SERT) represents a potential mediator of anxiety- and depression-related behaviors. Using mice lacking the SERT (SERT KO), we examined the role of SERT function in anxiety- and depression-related behaviors and serotonergic neuron function. METHODS: Serotonin transporter knockout mice were evaluated in paradigms designed to assess anxiety-, depression-, and stress-related behaviors. Dorsal raphe nucleus (DRN) function was assessed by quantitative serotonergic cell counting and extracellular electrical recording of neuronal firing properties. RESULTS: Serotonin transporter knockout mice showed an increase in latency to feed in a novel situation, more immobility in a forced swim, increased escape latency in a shock escape paradigm, and decreased immobility in tail suspension. No differences in anxiety-related behaviors were seen in the open field and the elevated plus maze. Serotonin transporter knockout mice exhibit a 50% reduction in serotonergic cell number and a fourfold decrease in firing rate in the DRN. CONCLUSIONS: Developmental loss of SERT produces altered behaviors in models of depression that are generally opposite to those produced by antidepressant treatment. The reduced serotonergic cell number and firing rate in the DRN of adult SERT KO mice suggest a mechanism for these altered behaviors.

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.

Effects of selective breeding for infant rat ultrasonic vocalization on cardiac responses to isolation.

Heart rates (HR) and autonomic control of heart period (HP) during maternal separation and isolation were measured at postnatal days (P) 10 and 18 in rat pups selectively bred for high, low, and random rates of infant ultrasonic vocalization. P10 pups displayed no significant line differences in HRs, strong tonic basal sympathetic control of HP and little sympathetic reactivity to isolation, and no evidence of tonic baseline or phasic parasympathetic responses to isolation. At P18, during isolation and recovery, Low HRs were significantly higher compared with Random and High pups. High HRs were significantly higher compared with the Random line in the last third of isolation and through recovery. The higher HRs of Low pups during stress were due to greater parasympathetic withdrawal, but greater sympathetic activation could not be ruled out. Higher HRs in High pups were due to increased sympathetic activation. Thus, increases in HR responses in the selected lines are due to different underlying autonomic processes.