Lasting effects of early life stress in mice: interaction of maternal environment and infant genes.

In the mouse, a powerful paradigm of early life stress, infant maternal separation (IMS), can trigger emotional and cognitive dysfunctions in adulthood similar to those found in humans with a history of childhood adversity. The magnitude of IMS effects differs among diverse inbred strains suggesting an interaction between the genetic background of pups and the maternal care they received. Here, we investigated this interaction with studies on reciprocal F1 hybrid mice of the stress-susceptible Balb/c and the resilient C57Bl/6 strains that were either raised by Balb/c mothers (low maternal care) or by C57Bl/6 mothers (higher maternal care) with or without IMS exposure. The ultrasonic vocalization response to isolation was recorded from infant F1 pups, and their emotional, executive cognitive and epigenetic phenotypes were assessed in adulthood. These studies showed that, regardless of the maternal care received, the emotional phenotype of F1 hybrids was not significantly affected by IMS exposure. However, F1 pups raised by Balb/c (but not C57Bl/6) mothers during IMS exposure exhibit deficits in working memory and attention-set-shifting in adulthood. They also exhibit reduced histone deacetylase 1 levels at promotors of brain-derived neurotrophic factor and early growth response 2 genes, and abnormally high induction of expression of these genes during cognitive testing. As one of affected genes was previously shown to associate with the Balb/c and the other with the C57Bl/6 genetic background, these findings indicate that both parental alleles interact with the maternal environment to modulate the cognitive and epigenetic phenotypes of F1 mice exposed to the IMS.

Effects of neonatal growth on adult blood pressures of borderline hypertensive rats.

We conducted this study to test the hypothesis that there are long-term effects of litter-size manipulations during the preweaning period on growth and adult blood pressure of rats. Litter size of genetically homogeneous borderline hypertensive rats, which were produced by cross-mating male Wistar-Kyoto rats with female spontaneously hypertensive rats, was manipulated from 10 to 16 days of age. In addition, a subset of males and females was castrated within the first 30 hours of life. Body weights were measured at several preweaning and postweaning ages, and tail-cuff blood pressures were recorded at 90 days of age. Intact and castrated pups of both sexes that were reared in small (n = 4) litters from 10 to 16 days of age gained nearly twice the weight of animals reared in large (n = 9 to 12) litters during this period. Intact males from small litters had significantly higher adult blood pressures than those from large litters. These long-term effects remained even in groups matched for adult weight and length. Neonatal castration of males completely blocked the consequences of litter-size manipulation on adult blood pressure, suggesting either an organizational or activational role for androgens. Neither intact nor neonatally castrated females exhibited differences in adult blood pressure as a function of litter-size manipulation.

Trigeminal and olfactory pathways mediating isolation distress and companion comfort responses in rat pups.

Isolation in a novel test cage elicits ultrasonic vocalization (USV) in 12-13-day-old rat pups, and the presence of an anesthetized littermate companion reduces USV while eliciting body contact. These responses were studied in pups with surgical lesions of trigeminal snout afferents and after olfactory denervation by nasal ZnSO4 perfusion. With lesions of only one system, body contact with the passive companion was reduced by 20%, and interpup orientation was differentially affected by the two types of lesions. However, USV rates were no different from littermate controls in the presence of the companion or when tested alone. Combined lesions of both systems reduced contact time 55% and eliminated any reduction in USV rates by the companion while leaving intact USV and locomotor responses to isolation. These results suggest that different sensory pathways may mediate isolation distress and companion comfort responses.

Independence of ultrasonic vocalization and thermogenic responses in infant rats.

Ultrasonic vocalizations (USV) normally accompany brown adipose tissue (BAT) thermogenesis in infant rats exposed to cold. BAT activation (measured by implanted thermistors) was pharmacologically blocked with hexamethonium (20 mg/kg ip) in 12-13-day-old pups, but they nevertheless showed normal USV responses to cold. Activation of BAT in warm pups by norepinephrine (800 micrograms/kg sc) failed to elicit USV. It is concluded that BAT activation is neither necessary nor sufficient for USV production. To evaluate how tightly the two responses may be coupled centrally, rat pups deprived of nutrients for 24 hr, in which sympathetic activation is known to be inhibited centrally (Bignall, Heggeness, & Palmer, 1975), were studied. These pups vocalized with the same latency in response to cold as normals but failed to show evidence of concurrent BAT activation. It is concluded that USV and BAT thermogenesis are normally elicited together by cold but are not tightly linked physiologically.

Ultrasonic vocalization, laryngeal braking, and thermogenesis in rat pups: a reappraisal.

In these experiments we tested the hypothesis that the respiratory events underlying rat pup ultrasonic vocalization (USV) facilitate brown adipose tissue (BAT) thermogenesis, as proposed by Blumberg and Alberts (1990). Laryngeal denervation or tracheostomy were used to reduce or eliminate laryngeal braking and prevent USV in 9- to 10-day-old pups during recovery from deep hypothermia and during cooling induced by isolation for 1 hr at room temperature. Both forms of devocalization slowed rewarming of hypothermic pups, but neither had any effects on cooling rate or BAT thermogenesis in normothermic pups that were allowed to cool from 35 degrees C to 29 degrees C. Physiological effects of laryngeal braking on thermogenesis appear to be limited to conditions, such as severe hypothermia, in which ventilatory and oxygen transport functions are compromised. The results have implications for understanding the regulation and evolution of USV.

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.

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.

Afferent control of pressor responses to feeding in young rats.

A series of experiments investigated the eliciting stimuli and physiological systems involved in triggering the blood pressure (BP) response to feeding in the young rat. The studies included 1) tongue cannula (TC) infusions of milk to nipple-attached and unattached pups; 2) TC infusions of saline, sucrose, and water to nipple-attached pups; 3) maternal milk ejections to 3-week-old pups that rarely have stretch responses; 4) gastric cannula infusions of milk to nipple-attached pups; and 5) maternal milk ejections and TC infusions to pups whose mouths were topically anesthetized. The results implicate the contact of fluid with oral and/or lingual surfaces as the critical stimulus primarily responsible for the BP rise. Although nipple attachment appears to be an important precondition for the typical pressor response, the co-occurrence of a behavioral stretch response is not essential.

Baroreceptor sensitivity in two-week-old rat pups: effects of nutrient deprivation and sino-aortic denervation.

Resting cardiac rates are reduced 40% by nutrient deprivation in two-week-old rats while arterial pressure is maintained at stable levels. Previous evidence implicated arterial baroreceptors and suggested the hypothesis that the cardiac rate changes result from increased baroreceptor sensitivity following nutrient deprivation. In order to test this hypothesis, cardiac reflex responses were elicited by graded doses of phenylephrine and sodium nitroprusside before and after nutrient deprivation. Although cardiac rate decelerations in response to phenylephrine were greater in fed pups (p less than 0.05) and acceleration in response to nitroprusside were greater in the deprived condition (p less than 0.01), these could be attributed to 'ceiling' and 'floor' effects of the resting cardiac rates characteristic of the two nutrient conditions. Sino-aortic denervation eliminated cardiac reflex responses, substantiating their dependence on baroreceptor afferents. Regression analysis of cardiac reflex responses to arterial pressure changes failed to show changes in baroreceptor sensitivity that would support the hypothesis. Alternate mechanisms mediating the nutrient effect on cardiac rate (e.g., involving neural and peptide hormonal pathways) should be pursued.

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.

Blood pressure responses to milk ejection in the young rat.

When young rats receive milk ejections from their mothers they exhibit abrupt 30-50% increases in blood pressure. In our first experiment, we recorded, via carotid cannulae, blood pressure and heart rate during natural nursing bouts. Blood pressure changes coincident with behaviorally defined milk ejections were twice as large as the changes associated with other behaviors observed. In addition, the increases in blood pressure during milk ejection were significantly greater when pups were separated from their mothers for 18-20 hours prior to testing. A second experiment showed that these responses were independent of the mother's overt behavior because virtually identical results were obtained when pups received milk from anesthetized dams stimulated to release milk with oxytocin. These results are discussed with regard to possible immediate functions of the response, and long term effects of repeated cardiovascular activation in early development.

Hypothermic vocalizations of rat pups (Rattus norvegicus) elicit and direct maternal search behavior.

Emissions of ultrasonic vocalizations (USVs) by rat pups (Rattus norvegicus) during hypothermia have consequences for recovery and warming. We investigated the effects on dam behavior of USVs emitted by 3- to 11-day-old pups during hypothermia at rectal temperatures between 18 and 22 degrees C. Rat dams were tested in a Y maze with the home cage as a start box. Dams were given, in one condition, a choice between a hypothermic pup emitting USVs or a hypothermic, silent (anesthetized) pup and, in the other, a choice between 2 hypothermic, silent pups. Although differing in some acoustic properties from normal isolation calls, USVs emitted by hypothermic pups both elicited maternal search behavior and acted as directional cues for dams, in comparisons with control dams exposed only to silent pups. Thus USVs of pups recovering from extreme hypothermia have communicative as well as physiological significance.

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.

Isolation distress in two-week-old rats: influence of home cage, social companions, and prior experience with littermates.

Behavioral and autonomic manifestations of acute isolation distress were studied in 2-week-old rats under novel and home-cage conditions. Levels of ultrasonic vocalization, heart rate, self-grooming, and rises were higher in novel surroundings, while locomotion and digging were more frequent in home cages. Next, we found that a group of 8 unanesthetized agemate pups was just as effective as an anesthetized dam in reducing (by 80-90%) all behaviors of isolated pups in novel surroundings. Finally, singletons reared without littermates during postnatal days 7-14 showed normal isolation responses, and these were as effectively alleviated by littermates as those of normally reared pups. Thus, isolation distress in rats this age is a complex response, modulated over time by aspects of the pups' environment and virtually prevented by the presence of cues common to both mother and littermates.

Effects of combined carotid sinus and aortic depressor nerve denervations in neonatal rat pups.

Denervation of the major peripheral chemo- and barosensory organs of neonatal mammals is known to produce age-dependent effects on mortality and on respiratory and cardiovascular functions. In the current series of experiments, these phenomena were more fully characterized in the young rat. 8- to 9-day-old rats showed few effects of combined carotid sinus and aortic depressor nerve (CSAD) denervation. During the days following surgery, these animals maintained normal body temperatures, had relatively normal weight gain, little cardiorespiratory disruption, and none died. Denervation at earlier ages (postnatal days 3-4 or 0-1) resulted in considerable mortality, 24 and 59%, respectively. In addition, body temperatures were lower, and the weight gain was impaired in the denervated pups as compared with controls, and these effects were more severe in the CSAD nonsurvivors than in the survivors. Also, nonsurvivors had much more severe disruptions of cardiorespiratory patterns measured 24-96 h after surgery than did CSAD survivors. Twenty-four hours after surgery, pups that later died could be identified based on low heart rates and long inspiratory times. CSAD survivors maintained a pattern of moderate cardiorespiratory disruption as compared with controls. Finally, our results indicate that the additional stress of subcutaneous electrocardiography lead implantation and/or daily maternal separation for cardiorespiratory recording increased the mortality in the 3- to 4-day-old CSAD pups from 11 to 40%.

Ultrasonic vocalization by rat pups during recovery from deep hypothermia.

Vocalization in the ultrasonic range (USV) has been reported to occur in young rodents in response to isolation, novelty, handling, and cold. Heretofore these calls have been known to occur only in alert, attentive, or emotionally aroused animals. These studies describe the emission of USV by comatose 9- to 10-day-old rat pups during recovery from deep hypothermia. Calling began at 15-18 degrees C core temperature while pups were virtually unresponsive to stimulation. Experimental results describe the patterns of call production in relation to respiration, cardiac function, colonic temperature, and brown adipose tissue thermogenesis. These vocalizations were 32-42 kHz in frequency, reached peak rates of 50/min at 23 degrees C, and were eliminated by laryngeal denervation, thus resembling isolation-induced vocalizations. However, contact with their dams failed to reduce call rates until pups had warmed above 25 degrees C. Newborn and weanling pups also emitted USV in deep hypothermia, but no USV were observed in pups recovering from general anesthesia. The possible functions and evolution of this behavior are discussed.

Potentiation of isolation-induced vocalization by brief exposure of rat pups to maternal cues.

Since their discovery in 1956, the highest rates of ultrasonic vocalization (USV) have been recorded from infant rats when first isolated in an unfamiliar place. We now report that peak USV rates can be doubled by allowing test pups a brief initial period of contact with their anesthetized dam (1-10 min) in the test chamber before isolating the pup by her removal. Potentiation of the isolation response was specific to the dam, for it failed to occur following initial contact with a group of 4 warm, anesthetized littermates. Control experiments showed that potentiation could not be attributed to thermal contrast, experimenter handling, general behavioral activation, novelty of maternal cues, or nursing deprivation. Furthermore, it did not occur when pups were taken for isolation testing directly from prolonged contact with their anesthetized dam in the home cage. Potentiation may be understood in terms of the communicative role of the pups' call and/or prior learning contingencies within the mother-infant interaction.

Isolation distress and maternal comfort responses of two-week-old rat pups reared in social isolation.

In 2-week-old rats, isolation from social companions in an unfamiliar place elicits ultrasonic vocalization rearing and locomotor behavior. These responses are prevented and close body contact is maintained if littermates or the dam are present. It has generally been believed that such isolation distress and comfort responses develop as a result of prior social experience. We reared pups in isolation from the third postnatal day and found essentially normal responses. A second experiment replicated and extended these results to pups reared in isolation from the day of birth. Isolation-reared pups did have higher levels of ultrasonic vocalization than their normally reared littermates, lower body weight, and lower post-test temperatures. We conclude that a lengthy period of close body contact and frequent social interactions with conspecifics are not necessary for the development of attachment responses in this species.

Nursing interaction of Wistar rats is modified by prior experience of altered nursing bout length.

We tested the capacity of 2 week postpartum Wistar rat litters and their dams to adapt to experimental shortening and lengthening of the time available for nursing while maintaining the frequency of bouts constant, using manual transfer between cage compartments. In 8-hr and 24-hr experiments, the short bout group (10 min/hr) gradually came to show normal litter weight gain, short latency milk ejections, and increased high arched nursing position, while the 40-min/hr group showed increased latency to attachment and longer latency to ME compared to the 20 min/hr control group. Pup body temperature did not vary between groups. Although pups in the 10-min group showed increased dam-directed behaviors in the first 8h, this difference vanished thereafter. In a twenty-fifth cycle, after dams and litters were exchanged, the prior experiences of the dams and not of the litters were found to determine the dynamics of milk exchange.

Feeding in infancy: short- and long-term effects on cardiovascular function.

Cardiovascular responses of adult organisms to feeding are well characterized and, in general, are understood as acute adaptations required for processing and distributing nutrients. Research over the past several years has shown that infants also have important cardiovascular responses to nutrient intake and that these are regulated by changes in autonomic activity to the heart and vasculature. Recent studies have provided results that suggest these responses in infancy may make an important contribution to the long-term development of cardiovascular function, in particular, adult blood pressure (BP). The purpose of this presentation will be to review the evidence that has led to this conclusion, offer ideas about how this potential early-life shaping of subsequent cardiovascular function may come about, and suggest further studies that will be required in order to characterize the mechanisms responsible for these effects.