Family Nurture Intervention in preterm infants alters frontal cortical functional connectivity assessed by EEG coherence.

AIM: To assess the impact of Family Nurture Intervention (FNI) on cortical function in preterm infants at term age. METHODS: Family Nurture Intervention is a NICU-based intervention designed to establish emotional connection between mothers and preterm infants. Infants born at 26-34 weeks postmenstrual age (PMA) were divided into two groups, standard care (SC, N = 49) and FNI (FNI, N = 56). Infants had EEG recordings of ~one hour duration with 124 lead nets between 37 and 44 weeks PMA. Coherence was measured between all pairs of electrodes in ten frequency bands. Data were summarised both within and between 12 regions during two sleep states (active, quiet). RESULTS: Coherence levels were negatively correlated with PMA age in both groups. As compared to SC infants, FNI infants showed significantly lower levels of EEG coherence (1-18 Hz) largely within and between frontal regions. CONCLUSION: Coherence in FNI infants was decreased in regions where we previously found robust increases in EEG power. As coherence decreases with age, results suggest that FNI may accelerate brain maturation particularly in frontal brain regions, which have been shown in research by others to be involved in regulation of attention, cognition and emotion regulation; domains deficient in preterm infants.

Unexplained infant crying: an evolutionary perspective.

UNLABELLED: The absence of adverse health outcomes later in development and the similarity of defining features of "colic" across cultures suggest that an evolutionary perspective may give us some insight into the nature of this puzzling condition. Evidence suggests that the larynx evolved first as a protective valve and later as a means to stabilize the upper thorax momentarily for precision upper arm movements. Recently, we found another physiological role for the larynx in regulating respiratory function to promote the recovery of young rats from severe hypothermia. In the process, bursts of calling were emitted by unconscious pups, which were nevertheless effective in eliciting maternal search and retrieval. These unexpected findings reveal how infant calling may have evolved as a communicative signal derived from more primitive physiological functions of the larynx. Repetitive calling in the normal young rat when isolated from its littermates and mother is regulated by multiple sensory cues present in the infant's social interactions, and in the paper it is described how this sensory input projects to central neuromodulatory systems known to be active in the control of anxiety behaviors in adult rats and humans. CONCLUSION: This broad range of functions for infant calling in other mammals suggests several new ways to approach the further study and treatment of unexplained crying in human infants.

Development of rats' maternally directed orienting behaviors from birth to day 2.

We previously observed 2- to 3-day-old (P2-3) rats perform maternally directed orienting behaviors (MDOBs)-locomotion, torso flexion, rotation to the supine orientation, and snout probing, accompanied by audible vocalizations (barking)-under surfaces having properties of the dam's ventrum, which result in close contact between the pup's ventrum and the surface. We now report on the development of MDOBs from birth through P2. Wistar rats (24 litters) were tested at one of four times: 1 hr after birth before the first nursing bout (P0pre), on the day of birth after the first nursing bout (P0post), on postnatal Day 1 (P1), or on postnatal Day 2 (P2). Pups were placed prone on a warmed platform inside a corral where, for 4 min, they encountered one of two motherlike surfaces at head height: (a) a soft cotton roof or (b) a patch of the pup's own anesthetized dam's ventrum. Control pups received no overhead stimulus. The behaviors we had identified as MDOBs and others, including limb behaviors, are easily recognized at P0pre, and, in aggregate, are more frequent then than at any later age. The differential responsiveness of most of the behaviors to the dam's ventrum compared to the cotton roof or open condition is relatively low at P0pre, but rises rapidly through P2. Behavioral structure develops quickly from a rudimentary principal component with two behaviors at P0pre to two principal components incorporating seven behavior classes by P2. MDOBs are discussed in relation to late-term fetal behaviors, early postnatal righting responses, and the oral nipple grasp, and as a model of the development of filial attachment in an altricial mammal.

Developmental effects of selective breeding for an infantile trait: the rat pup ultrasonic isolation call.

This article describes how continued selection for divergent levels of the 10-day-old infant rat's ultrasonic vocal (USV) response to isolation affects the time course of development of that and other possible co-selected traits from 3 to 21 days postnatally. Since selective breeding for an infantile trait has not been reported before, we collected from colleagues a number of predicted outcomes that reflect the wide range of current opinion on the relationship between microevolutionary and developmental processes. After 15 generations of selective breeding, we found widely divergent USV responses between 10-day-old High USV line (300 USV/2 min) and low USV line (15 USV/2 min) pups. The developmental trajectory of USV responses at 3 and 7 days of age also was markedly altered in both these lines in comparison to the randomly bred controls, but was much less affected in 14-, 18-, or 21-day-old pups, contrary to all predictions. The development of other behavioral responses to isolation generally remained unaffected by the continued selection as did physical traits, measures of temperature regulation and classic developmental milestones. Only two traits showed evidence of co-selection: High line pups showed more urination/defecation in response to isolation from 10 days on, and more rapid ear canal opening at 10 days. These and other findings are presented and discussed in relation to the developmental and evolutionary concepts on which the different predictions were based.

Selective breeding for infant vocal response: a role for postnatal maternal effects?

N:NIH rats were selectively bred on the basis of high or low rates of ultrasonic vocalization (USV) response to isolation at 10 days of age (Brunelli et al., 1997: Dev Psychobiol 31:255-265). To examine the possibility of postnatal maternal effects in the generational transmission of divergent traits, pups were cross-fostered shortly after birth between dams of the two lines (Low- and High-USV). Controls were fostered to dams of the same line (in-fostered). Additional (population) control data were obtained from the entire 13th generation of the selectively bred lines. USV rates of cross-fostered pups in each line were not significantly different from rates of in-fostered pups of the same line. High USV line pups cross-fostered to Low USV line dams weighed significantly less than in-fostered pups, on the day of testing. The results provide no evidence for a postnatal maternal contribution to the USV phenotype. Prenatal and/or perinatal maternal effects have not been ruled out.

Maternally directed orienting behaviors of newborn rats.

We observed an organized repertoire of behaviors performed by newborns of the laboratory rat when placed under suspended artificial surfaces simulating various properties of the awake dam's ventrum. Pups traveled and wriggled, turned on their sides and upside down, ventroflexed and probed into the surface while on side or upside down, and audibly barked, all in a state of heightened behavioral arousal. Within 48 to 72 hr after birth, these behaviors generally increased with the addition to the simulation of more maternal features, including the texture of the dam's ventral fur and odor, separately and in combination. In addition, the behaviors were intensified by 18 hours of maternal deprivation. The possible functional relations among the components of this repertoire, which we refer to as "maternally directed orienting and proximity-seeking behaviors," and between these behaviors and nipple attachment and suckling, are discussed.

Behavioral mechanisms for active maternal potentiation of isolation calling in rat pups.

The ultrasonic vocalization (USV) response of the isolated infant rat is a promising model for studying the neurobiology of an early anxiety state, and potentiation of the USV response after brief maternal encounters is a newly discovered behavioral regulator of this state. Using experimental variations in the contexts and patterns of maternal behavior during pup encounters and manual simulations of maternal behavior, we have identified several specific mother-pup interactions that cause potentiation. When one of these, pup carrying, was manually simulated, potentiation followed only if a characteristic postural transport response had been fully expressed by the pup. These behavioral mechanisms and other data suggest hypotheses for the development and adaptive role of maternal potentiation.

The influence of olfaction on potentiation and inhibition of ultrasonic vocalization of rat pups.

Twelve-day-old isolated rat pups reduce their rates of ultrasonic vocalization (USV) when an anesthetized adult is placed into the test cage, whether the adult is their dam or an unfamiliar male. USV rates remain very low even after removal of the male (inhibition). However, after removal of the dam, pups greatly increase their rates of USV over their first isolation period (potentiation). USV potentiation can be induced by either an awake, normally behaving dam or by one that is anesthetized. To test the role of olfaction in inhibition and potentiation, PND12 pups were rendered anosmic via intranasal infusion of 5% zinc sulphate (ZnSO4). Control pups were infused with normal saline. After overnight separation from the dam, the USV and other behaviors of pups were recorded during a 6-min test. Each pup was tested during an initial isolation period and a final isolation period. In the first experiment, an anesthetized adult (dam or unfamiliar male) or no companion was placed in contact with the pup during the middle minute. Anosmia prevented both potentiation and inhibition of USV by passive adult contact. Thus, it seems likely that pups use olfactory discrimination as the basis for these two highly differentiated vocal responses to social stimuli. Results from two additional experiments demonstrate that anosmia does not prevent potentiation when the adult dam is active and interacting with the pup on either postnatal day 12 or 8.

Effects of biparental rearing on ultrasonic vocalization (USV) responses of rat pups (Rattus norvegicus).

This study investigated the influence of social rearing on ultrasonic vocalization (USV) responses of 11- to 12-day-old rat (Rattus norvegicus) pups in isolation to the presence or removal of an anesthetized adult. Pups were reared with the dam or dam plus a virgin female (aunt), their biological sire, or a castrated male. All pups reduced rates of USV in contact with anesthetized adults. Pups' USV rates after the adult's removal depended on their rearing experience. After removal of the dam, pups increased USV rates over baseline (potentiation). Pups reared with aunts potentiated USV after contact with adult females but suppressed USV after contact with males. Pups reared with sires potentiated USV after contact with sires or strange males. Pups reared with castrated males did not suppress USV after contact with castrates but did after contact with unfamiliar intact males. Thus, pups' differential USV responses to contact with passive adults in isolation can be acquired during prior rearing experience.

The ontogeny of maternal potentiation of the infant rats' isolation call.

Previous studies have shown that preweanling rat pups double or triple their rates of ultrasonic vocalization (USV) when isolated immediately after brief periods of maternal interaction (potentiation). We studied the ontogenetic pattern of USV and other behavioral responses of pups to 3-min periods of isolation in a novel test chamber, from 5 to 25 days postnatal age, before, during, and after 1 min or 5 min of interaction with an anesthetized or an active dam. USV potentiation did not develop until 7-9 days postnatal, a week after the initial isolation and maternal contact quieting responses were well established. Potentiation reached a peak at 13 days, and then declined until all USV responses ceased after 21 days. Other behavioral responses to isolation were not enhanced by maternal interaction at any age. The distinct ontogenetic pattern of this unusual response to maternal separation has implications for understanding its mode of development and possible adaptive value.

Olfactory preference for mother over home nest shavings by newborn rats.

The developmental course and behavioral processes by which infant rats come to prefer one of two prominent natural odors within their rearing environment, those of their mother and home nest shavings, was studied. Pups as young as 4-5 days and as old as 9-10 days prefer their mother's odor that of their home nest shavings in a two-choice test chamber; pups deprived overnight prior to testing express that preference more strongly than nondeprived littermates when the mother is awake, but not when anesthetized. Encountering maternal and shavings odors in a two-choice paradigm engages a behavioral sequence that progresses from arousal and active sampling of both odors during the establishment of preference to increased mouthing after the choice has been made. A .5 degrees C thermal gradient augments the maternal preference response from 4-5 days of age on, and is necessary to it in 2-day-olds. One-day-old pups do not express a preference for their mothers in our test apparatus on the basis of odor even with the addition of a .5 degrees C thermal gradient, suggesting that preference for the odor of mother over home shavings may be acquired between 1 and 2 days of age.

Five generations of selective breeding for ultrasonic vocalization (USV) responses in N:NIH strain rats.

This article reports on early results from an ongoing selective breeding study in which rats were selected for different rates of ultrasonic vocalization (USV) in response to isolation. Using the N:NIH strain, all litters were screened at 10 (+/- 1) days of age in a 2-min isolation test, and those males and females with the highest (or lowest) rates in each litter were selected for later breeding with like breeders from unrelated litters. A Random line (unselected control) was also bred. In the first selected generation (S1), the Low line diverged from Random line controls, and has maintained significantly lower rates over all generations since. In the S3 generation, the High line diverged significantly from Random line controls, and has shown significantly higher USV rates in each succeeding generation. No line differences were found in other behaviors measured in isolation. Data from a small sample of S5 pups tested at postnatal Days 3, 10, 14, and 18 days showed that individual pups' rates of USV from Day 10 predicted those at Day 14, consistent with findings from an unselected generation. Ambient temperature, modulated by body weight, controlled USV at Day 3, whereas at Days 10 and 14 line accounted for most of the variance in USV. This is the first instance of laboratory selection occurring on the basis of an infantile trait.

Potentiation and inhibition of ultrasonic vocalization of rat pups: regulation by social cues.

Eight- and 11- to 12-day-old rat pups were tested in isolation and in the presence of an anesthetized adult under cold conditions. Pups of both ages reduced rates of ultrasonic vocalization (USV) when an adult was placed into the test cage, independent of whether the adult was their dam or an unrelated male. However, after removal of the dam, pups greatly increased their rates of USV over their first isolation period and in comparison with control pups. USV rates remained low after removal of the male. The temperature challenges faced by the pups in the two experimental conditions were the same. These phenomena are better explained by a hypothesis that postulates USV rate as being multiply determined, including by social cues, rather than a theory that considers thermal challenge only.

On the nature and consequences of early loss.

OBJECTIVE: To describe how an animal model system can be used to explore basic questions about the nature of loss and the effects of early loss on later vulnerability to disease. METHOD: The physiological and behavioral responses of infant rats to separation from their mothers are first described and then analyzed experimentally into component mechanisms. RESULTS: These studies have revealed an extensive layer of processes underlying the psychological constructs generally used to understand the response to loss. Hidden within the observable interactions of parent and offspring, we found a number of discrete sensorimotor, thermal, and nutrient-based events that have unexpected long-term regulatory effects on specific components of infant physiology and behavior. Release from all of these inhibitory and excitatory regulators together during maternal separation constitutes a novel mechanism by which the experience of loss can be translated into a complex patterned response. Evidence for early regulatory processes has also been found in monkey and human mother-infant interactions. Here they may well constitute the building blocks from which attachment and object representations develop. We and others have found long-term effects of loss, and of selective replacement of regulators, on behavioral development and on later vulnerability to disease. CONCLUSIONS: The results give us a new understanding of early attachment as a developmental force and of human grief as a risk to health.

Maternal interactions prior to separation potentiate isolation-induced calling in rat pups.

The vocal response rates of 12-13-day-old infant rats to isolation in a bare test box are markedly increased by brief (1-min) periods of contact with an anesthetized dam prior to isolation, without affecting other isolation-induced behaviors. No such potentiation followed brief contact with littermates, novel test conditions, or experimenter handling. Brief contact with the dam was equally effective in the test chamber or home cage and was not further enhanced by repeated contact-separation sequences. Passive contact became ineffective when prolonged to 30 min, and potentiation could not be restored by providing the additional reinforcing events of continuous suckling, periodic oxytocin-induced milk letdown, or bouts of simulated maternal licking. However, when pups engaged in active interaction with an awake dam, potentiation was significantly enhanced following 1-, 10-, and 30-min periods. A working hypothesis is outlined for the adaptive role of potentiation in the development of the rat pup.

Development of ultrasonic vocalization responses in genetically heterogeneous National Institute of Health (N:NIH) rats. I. Influence of age, testing experience, and associated factors.

The N:NIH strain of rats was developed by the National Institutes of Health to provide a maximally heterogeneous population as a base for selective breeding (Hansen & Spuhler, 1984). Using the N:NIH strain, this laboratory will selectively breed adult animals that exhibited extremes of high or low ultrasonic vocalization (USV) rates as infants. Because nothing was known about USV in N:NIH rats, we characterized the development of isolation-induced USV in the first generation of this strain born in our laboratory. In a longitudinal/cross-sectional study of pups tested at 3, 10, 15, and 18 days postnatally, N:NIH pups emitted their highest rates of USV at 3-4 days postnatally and calling remained high for 10 days before declining. USV rates were found to be a relatively environmentally stable behavioral trait in that repeated testing did not significantly affect the calling rates of either individuals or litters, and only at 3 days postnatal age did naturally occurring ambient temperature variations (6 degrees C range) significantly affect USV responses. Individual differences in USV responses emerged by 10 days of age that were not simply correlations of body weight or rectal temperature, and pups at that age showed isolation calling rates that were highly predictive of their response levels 5 days later.

Development of ultrasonic vocalization responses in genetically heterogeneous National Institute of Health (N:NIH) rats. II. Associations among variables and behaviors.

In this study, the first three generations of laboratory-reared, 10-day-old pups of the N:NIH strain were examined for ultrasonic vocalizations (USV) rates in response to 2 min of isolation. The purpose of the study was to determine baseline USV rates in these progenitor (PR1, PR2, PR3) generations prior to selective breeding for high and low rates of USV. In addition, this study aimed to detect sources of variation in USV between and within generations and sexes, and factors associated with USV during isolation across the generations. Data were collected from 532 10 day-old pups in 81 litters. Evaluation by principal component analyses revealed four factors corresponding roughly to constructs indicative of: thermoregulation; maturity and exploratory behavior; distress/anxiety, in which USV consistently appeared; and activity. Different component structures for each sex suggested that USV may represent different dimensions of anxiety for component structures for each sex suggested that USV may represent different dimensions of anxiety for the sexes, with some degree of overlap. Nonetheless, all variables measured during isolation testing accounted for only a small portion of the variance in individual pup USV (less than 9%) at 10 days of age, when the effects of intercorrelations among all variables were taken into account. This suggests that variables currently measured do not represent a comprehensive model for the control of USV.

Isolation alters striatal met-enkephalin immunoreactivity in rat pups.

Isolated preweanling rats emit ultrasonic vocalizations. Mu- and delta-opioid agonists quiet isolated pups; naltrexone, an opioid receptor blocker, prevents this quieting. A littermate companion is as effective as morphine in quieting vocalizations, and naltrexone also blocks companion quieting. We have now quantified methionine enkephalin (Met-ENK) immunoreactivity in the brains of 10-day-old Wistar rat pups taken directly from the home cage or kept either alone or with a companion for a brief or prolonged period. Met-ENK is an endogenous ligand that binds to the mu- and delta-opioid receptors. Striatal peptide levels were higher when pups were with a companion than when they were kept alone; the peptide level of pups in the home cage did not differ from either. Comparisons of pups in the brief (5 min) and prolonged (60 min) separation conditions showed significantly higher peptide levels following a brief period out of the nest than at the end of an hour. In hypothalamus, hippocampus, and frontal cortex neither social condition nor duration of separation significantly altered peptide quantity. Larger amounts of Met-ENK in pups provided with a companion could reflect an increase in posttranslational cleavage of the precursor molecule leading to stimulation of receptors that act to diminish USV. Reduced levels following 60 min out of the home cage might reflect depletion of the peptide following an initial release during the period when the pup's vocal response is most vociferous.

Elevated plus-maze behavior in adult offspring of selectively bred rats.

Animals sampled from the third generation (S3) of selective breeding of N:NIH strain rats based on ultrasonic vocalization (USV) rates in infancy (High, Low, and Random lines) were tested as adults on the elevated plus maze, a well-validated test of anxiety. Results showed that the Low line spent a greater percent of time in the open arms of the maze than High and Random (control) line animals, and a lower percent of time exploring from a relatively more protected position. There were no significant sex differences. The present study represents a probe into the processes at work during the early stages of selection for infant USV, and suggests that an adult measure of anxiety may be affected by selection for this infantile behavior.

The isolation and companion comfort responses of 7- and 3-day-old rat pups are modulated by drugs active at the opioid receptor.

Rat pups emit ultrasonic vocalizations (USVs) when isolated in a novel environment. In 10-day-olds, USV has been shown to be reduced by either the administration of 0.125 mg/kg morphine (MOR) or the presence of a littermate; these effects were both reversed by naltrexone (NLX), an opioid receptor blocker. The present study reports that the same dose of MOR produced NLX-antagonized quieting without sedation in 7- and 3-day-old pups; higher doses of MOR decreased USV but produced motor deficits as well. The 0.125 mg/kg dose of MOR is less effective in reducing USV in 3- and 7-day-olds; calling rates declined by no more than 42%, compared with 65% at 10 days of age. The presence of a companion also lowered the USV of 3- and 7-day-olds by a lesser amount (55-57%) than the 67% seen in 10-day-olds or the 90% decline when pups are 2 weeks old. This suggests that age-related changes in the opioid system may be relevant to the increased salience of a social companion that comes with maturity.