Maternal antibiotics disrupt microbiome, behavior, and temperature regulation in unexposed infant mice.

Maternal antibiotic (ABx) exposure can significantly perturb the transfer of microbiota from mother to offspring, resulting in dysbiosis of potential relevance to neurodevelopmental disorders such as autism spectrum disorder (ASD). Studies in rodent models have found long-term neurobehavioral effects in offspring of ABx-treated dams, but ASD-relevant behavior during the early preweaning period has thus far been neglected. Here, we exposed C57BL/6J mouse dams to ABx (5 mg/ml neomycin, 1.25 µg/ml pimaricin, .075% v/v acetic acid) dissolved in drinking water from gestational day 12 through offspring postnatal day 14. A number of ASD-relevant behaviors were assayed in offspring, including ultrasonic vocalization (USV) production during maternal separation, group huddling in response to cold challenge, and olfactory-guided home orientation. In addition, we obtained measures of thermoregulatory competence in pups during and following behavioral testing. We found a number of behavioral differences in offspring of ABx-treated dams (e.g., modulation of USVs by pup weight, activity while huddling) and provide evidence that some of these behavioral effects can be related to thermoregulatory deficiencies, particularly at younger ages. Our results suggest not only that ABx can disrupt microbiomes, thermoregulation, and behavior, but that metabolic effects may confound the interpretation of behavioral differences observed after early-life ABx exposure.

Oxytocin and the warm outer glow: Thermoregulatory deficits cause huddling abnormalities in oxytocin-deficient mouse pups.

Oxytocin is a social and reproductive hormone that also plays critical roles in a range of homeostatic processes, including thermoregulation. Here, we examine the role of oxytocin (OT) as a mediator of brown adipose tissue (BAT) thermogenesis, cold-induced huddling, and thermotaxis in eight-day-old (PD8) OT 'knock out' (OTKO) mouse pups. We tested OTKO and wildtype (WT) pups in single- and mixed-genotype groups of six, exposing these to a period of ambient warmth (~35°C) followed by a period of cold (~21.5°C). Whether huddling exclusively with other OTKO or alongside WT pups, OTKO pups showed reduced BAT thermogenesis and were significantly cooler when cold-challenged. Huddles of OTKO pups were also significantly less cohesive than WT huddles during cooling, suggesting that thermoregulatory deficits contribute to contact abnormalities in OTKO pups. To further explore this issue, we examined thermotaxis in individuals and groups of four OTKO or WT pups placed on the cool end of a thermocline and permitted to freely locomote for 2h. When tested individually, male OTKO pups displayed abnormal thermotaxis, taking significantly longer to move up the thermocline and settling upon significantly lower temperatures than WT pups during the 2h test. OTKO mouse pups thus appear to have deficits in both thermogenesis and thermotaxis-the latter deficit being specific to males. Our results add to a growing body of work indicating that OT plays critical roles in thermoregulation and also highlight the entanglement of social and thermoregulatory processes in small mammals such as mice.

Development of behavioral responses to thermal challenges.

Body temperature regulation involves the development of responses to cold and warm challenges. Matching our understanding of the development of body temperature regulation to warm challenges with that of cold challenges will enhance our understanding of the ontogeny of thermoregulation and reveal different adaptive specializations. Warm and cold thermoregulation are important processes, and they include direct thermal effects on offspring, as well as indirect effects on them, such as those imposed by thermally associated alterations of maternal behavior. The present paper is a selective review of the existing literature and a report of some new empirical data, aimed at processes of mammalian development, especially those affecting behavior. We briefly discuss the development of body temperature regulation in rats and mice, and thermal aspects of maternal behavior with emphasis on responses to high temperatures. The new data extend previous analyses of individual and group responses in developing rodents to warm and cool ambient temperatures. This literature not only reveals a variety of adaptive specializations during development, but it points to the earlier appearance in young mammals of abilities to combat heat loss, relative to protections from hyperthermia. These relative developmental delays in compensatory defenses to heating appear to render young mammals especially vulnerable to environmental warming. We describe cascading consequences of warming-effects that illustrate interactions across levels of physiological, neural, and behavioral development.

Oxidative Stress as Cause, Consequence, or Biomarker of Altered Female Reproduction and Development in the Space Environment.

Oxidative stress has been implicated in the pathophysiology of numerous terrestrial disease processes and associated with morbidity following spaceflight. Furthermore, oxidative stress has long been considered a causative agent in adverse reproductive outcomes. The purpose of this review is to summarize the pathogenesis of oxidative stress caused by cosmic radiation and microgravity, review the relationship between oxidative stress and reproductive outcomes in females, and explore what role spaceflight-induced oxidative damage may have on female reproductive and developmental outcomes.

Behavioral observation differentiates the effects of an intervention to promote sleep in premature infants: a pilot study.

PURPOSE: Sleep and ongoing cycling of sleep states are required for neurosensory processing, learning, and brain plasticity. Many aspects of neonatal intensive care environments such as handling for routine and invasive procedures, bright lighting, and noise can create stress, disrupt behavior, and interfere with sleep in prematurely born infants. The study empirically investigated whether a 30-minute observation of infant sleep states and behavior could differentiate an intervention to promote sleep in premature infants with feeding difficulties relative to conventional care (standard positioning, standard crib mattress [SP]). We included an intervention to determine the ability of the method to discriminate treatments and generate a benchmark for future improvements. The intervention, a conformational positioner (CP), is contoured around the infant to provide customized containment and boundaries. To more fully verify the 30-minute observational sleep results, standard polysomnography was conducted simultaneously and sleep outcomes for the 2 modalities were compared. SUBJECTS: In a randomized crossover clinical trial, 25 infants, 31.5 ± 0.6 weeks' gestational age and 38.4 ± 0.6 weeks at the study, with gastrointestinal conditions or general feeding difficulties used each intervention during an overnight neonatal intensive care unit sleep study. METHODS: Infant sleep states and behaviors were observed during two 30-minute periods--that is, on the positioner and mattress--using the naturalistic observation of newborn behavior. Two certified developmental care nurses assessed sleep state, self-regulatory, and stress behaviors during 2-minute intervals and summed over 30 minutes. Sleep characteristics from standard polysomnography were measured at the time of behavior observations. RESULTS: Infants on CP spent significantly less time in alert, active awake, or crying states by observation compared with SP. Surgical subjects spent more time awake, active awake, or crying and displayed a higher number of behavior state changes than the nonsurgical infants. The percentage of time in observed deep sleep and quiet sleep was correlated with both percentage sleep efficiency (r = 0.78) and fewer state shifts per hour (r = -0.65) from electroencephalogram (EEG). Sleep efficiency by EEG was greater on CP versus SP. CONCLUSIONS: The CP enabled sleep compared with the standard mattress (SP) over 30-minute observation periods. Sleep status from behavioral observation was verified by standard EEG-based sleep techniques. Behavioral observation of sleep states may be a useful strategy for measuring the effectiveness of strategies to facilitate sleep in premature infants. Surgical subjects may benefit from additional interventions to promote sleep.

Oxytocin mediates the acquisition of filial, odor-guided huddling for maternally-associated odor in preweanling rats.

The present study was designed to examine possible roles of oxytocin (OT) in the acquisition of a filial huddling preference in preweanling rats. We used a procedure in which a scented, foster mother can induce an odor-guided huddling preference in preweanling pups, following a single, 2-h-long co-habitation (Kojima and Alberts, 2009, 2011). This single, discrete period for preference learning enables us to observe the mother-pup interactions that establish the pups' preferences and to intervene with experimental manipulations. Four, 14-day-old littermates interacted with a scented foster mother that provided maternal care during a 2-h session. Two of the pups were pretreated with an intracerebroventricular injection of OT or an oxytocin antagonist (OTA), and the others received a vehicle injection. Filial preference for a maternally-paired odor was measured in a huddling test the next day. OT is necessary for acquisition of the filial preference: The preference learning was blocked in the pups treated with OTA, but not in their vehicle-treated littermates who experienced the same mother at the same time. Injection with exogenous OT did not augment the pups' preference. Manipulating pups' central OT also altered the contact interactions of the mother and pups. When some pups received OT, mother-litter aggregations formed as frequently and with similar combinations of bodies, but contact aggregations were significantly more cohesive than when some pups in the litter received OTA. We discuss dual, behavioral and neuroendocrine roles of OT in social learning by preweanling rats.

Warmth from skin-to-skin contact with mother is essential for the acquisition of filial huddling preference in preweanling rats.

During a single, 2-hr session with a scented foster dam, preweanling rat pups form an affiliative attraction to an odor associated with the maternal caregiver, manifest as a huddling preference. To identify maternal stimuli that induce this filial preference, we quantitatively examined behavioral interactions during odor conditioning. Bout duration of skin-to-skin (STS) contact was positively associated with the preference. In contrast, simple physical contact and anogenital licking were not significantly related to the preference. The frequency of nonanogenital licking was negatively associated with the preference as well as with bout duration of STS contact. When odor conditioning was conducted with a warm cylinder, ambient warmth, or stroking as the unconditioned stimulus, only pups exposed to the warm cylinder exhibited a preference for the conditioned odor. These results suggest a positive, affiliative effect of maternal STS contact on pup filial preference, which may be disrupted by maternal licking.

Maternal care can rapidly induce an odor-guided huddling preference in rat pups.

Olfactory-guided huddling is learned and expressed by postnatal day (PND) 15, when rat pups huddle preferentially with conspecifics or with targets bearing an odor previously associated with maternal care. Experiment 1 replicated this induction of an odor-guided huddling preference with a truncated regime of conditioning with a scented foster dam. Pups exposed to an odor in association with foster maternal care during five daily 2-hr sessions on PNDs 1-5, 5-9, or 10-14, but not pups merely exposed to the odor, displayed a huddling preference for the conditioned odor, but only when conditioning commenced after PND5. Experiment 2 demonstrated that a single, 2-hr exposure to a scented foster dam can induce a huddling preference in pups. Analysis of maternal behavior during the 2-hr conditioning sessions on PND14 revealed that frequency of maternal hovering over pups, but not licking/grooming or duration of contact, was associated with induction of the odor preference.

Orbital spaceflight during pregnancy shapes function of mammalian vestibular system.

Pregnant rats were flown on the NASA Space Shuttle during the early developmental period of their fetuses' vestibular apparatus and onset of vestibular function. The authors report that prenatal spaceflight exposure shapes vestibular-mediated behavior and central morphology. Postflight testing revealed (a) delayed onset of body righting responses, (b) cardiac deceleration (bradycardia) to 70 degrees head-up roll, (c) decreased branching of gravistatic afferent axons, but (d) no change in branching of angular acceleration receptor projections with comparable synaptogenesis of the medial vestibular nucleus in flight relative to control fetuses. Kinematic analyses of the dams' on-orbit behavior suggest that, although the fetal otolith organs are unloaded in microgravity, the fetus' semicircular canals receive high levels of stimulation during longitudinal rotations of the mother's weightless body. Behaviorally derived stimulation from maternal movements may be a significant factor in studies of vestibular sensory development. Taken together, these studies provide evidence that gravity and angular acceleration shape prenatal organization and function within the mammalian vestibular system.

Rat behavioral thermoregulation integrates with nonshivering thermogenesis during postnatal development.

Rat pups are capable of behavioral thermoregulation, both in the nest and on a thermocline, as early as the 1st week of postnatal life, and these pups can also produce heat metabolically without shivering. The rat pup's primary source of nonshivering thermogenesis is the sympathetically mediated metabolism of brown adipose tissue (BAT). BAT is well formed in newborns and functions shortly after birth. While infant behavioral thermoregulation and BAT thermogenesis have been extensively studied, little is known about the extent to which thermoregulatory behavior can be influenced by BAT thermogenesis. In the present study, 2-, 7-, and 14-day-old pups were observed on a thermal gradient following pharmacological stimulation or inhibition of BAT thermogenesis, and their thermal preferences were quantified. The authors found that 7- and 14-day-old pups treated with norepinephrine (NE), which increases BAT thermogenesis, preferred cooler portions of the gradient than saline-treated controls, whereas 2-day-olds failed to show a similar NE-induced behavioral adjustment. These findings indicate that the ability to adjust thermoregulatory behavior to compensate for enhanced metabolic thermogenesis develops during the 1st week of postnatal life.

Effects of labor contractions on catecholamine release and breathing frequency in newborn rats.

Plasma catecholamines in newborn rats (0-2 hr old) were analyzed following vaginal birth, cesarean section with simulated labor contractions, or cesarean section without labor contractions. Upon delivery, pups were exposed to key elements of the rat's natural birth process, that is, umbilical cord occlusion, tactile stimulation, and cooling. Only pups exposed to actual or simulated labor showed an immediate rise in norepinephrine and epinephrine. Initial postpartum respiratory frequencies were higher in vaginal than in cesarean delivered pups and, in all groups, inversely correlated with catecholamine titers, suggesting respiratory distress or transient tachypnea at lower catecholamine levels. These findings establish a rat model for analyzing effects of labor on neonatal adaptive response during the transition from prenatal to postnatal life.

Non-nutritive, thermotactile cues induce odor preference in infant mice (Mus musculus).

Mouse pups (Mus musculus) placed on the midline of a mesh floor suspended over an empty area bounded by 2 odor fields, 1 containing homecage bedding and the other clean bedding, preferentially selected the homecage area when tested on postnatal day (PD) 5, 10, or 12. PD5 pups given a choice of homecage bedding versus age-matched bedding from another litter showed no discrimination, whereas PD10/12 pups preferred own home odors. To test whether such home orientation can be shaped by experience, pups were placed for 2 hrs on PDs 8 and 9 with either a lactating dam, a nonlactating foster dam or a warm tube bearing 1 of 2 novel odors. Other pups were similarly exposed to scented gauze to test whether mere exposure (familiarization) to an odor could induce a preference. Pups naïve to both test odors and those familiar with 1 odor showed no preference for either odor on PD10. Pups placed with a lactating dam spent significantly more time over the conditioned odor. Moreover, pups placed with the nonlactating dams or the warm tube also preferred the conditioned odor, indicating that the preference can be attributed association with non-nutritive, thermotactile cues. (PsycINFO Database Record

Housing pregnant mice (Mus musculus) in small groups facilitates the development of odor-based homing in offspring.

Infant mice (Mus musculus) born to dams housed in isolation throughout pregnancy (IsoPreg) begin differentially approaching homenest bedding over clean bedding on Postnatal Day 6. Offspring of dams housed with 2 other potentially pregnant conspecifics (SocPreg) display such homing behavior on Day 4. Earlier onset of homing reflects facilitated olfactory responsiveness in SocPreg pups, rather than qualitative or quantitative differences in IsoPreg versus SocPreg nest odors, body growth, or motoric capabilities. Exposing pregnant IsoPreg dams to SocPreg bedding also accelerated homing onset in the offspring, though not to the same extent as the full social context. Thus, it appears that the facilitation of homing is mediated through the pregnant dam by a combination of chemical cues and other social stimuli.

The validity and utility of geotaxis in young rodents.

Negative geotaxis, an automatic, reliable, stimulus-bound, orientation and movement directionally against gravitational cues, is often used for behavioral assessments of infant rodents. We summarize historical and contemporary analyses and conclude that negative geotaxis does not exist in infant rats. Infant rodents placed on inclined surfaces (ranging from 15 degrees to 70 degrees in most tests) are posturally unstable and their compensatory responses have been misinterpreted as negative geotaxis. In fact, recent findings suggest that if infant rats display a geotaxis, they show positive geotaxis on shallow angles of inclination (e.g., 4 degrees and 8 degrees ). There may be utility in assessing postures and motoric responses of infant rats on relatively robust angles of inclination, but these are not tests of negative geotaxis.

Maternal responsiveness to infant Norway rat (Rattus norvegicus) ultrasonic vocalizations during the maternal behavior cycle and after steroid and experiential induction regimens.

When removed from the nest and placed in a cool environment, Norway rat (Rattus norvegicus) pups emit ultrasonic vocalizations that can elicit maternal search behavior. The authors examined the behavior of pregnant dams, mothers, and virgin females during exposure to a pup that was either warm and silent or cool and vocalizing. Results indicate potentiated maternal reactions to a vocalizing pup: Mothers approached and maintained proximal orientation to a vocalizing pup far more than did virgin females. Elevated levels of proximal orientation appeared within hours of birth, increased during the 1st week postpartum, and declined by the time of weaning. Estrogen plus progesterone administration facilitated virgin females' proximal orientation toward vocalizing pups, whereas prolonged exposure to pups in the absence of hormones was without effect, suggesting that the ontogeny of the maternal response is regulated, at least in part, by maternal hormones.

Stimulus control of maternal responsiveness to Norway rat (Rattus norvegicus) pup ultrasonic vocalizations.

Mother rats (Rattus norvegicus; 6 to 8 days postpartum) approach and maintain proximal orientation to a pup that is emitting ultrasonic vocalizations (USVs) far more than do virgin females (W. J. Farrell & J. R. Alberts, 2002). We used a playback regimen to examine the roles of acoustic and nonacoustic cues in regulating maternal proximal orientation toward vocalizing pups. When presented with recorded USVs, mothers of 6- to 8-day-old pups and nulliparous virgin females exhibited equivalent levels of proximal orientation toward the playback speaker. Mothers did show enhanced proximal orientation toward recorded USVs, however, if a silent pup was positioned below the speaker. Pup odors appear to be crucial for the maternal response to vocalizing pups, as peripherally induced anosmia attenuated maternal proximal orientation toward a vocalizing pup. Furthermore, spatial contiguity between olfactory and auditory stimuli was required for a maximal maternal response.

Positive geotaxis in infant rats (Rattus norvegicus): a natural behavior and a historical correction.

Infant rats (Rattus norvegicus) placed on a shallow incline (2 degrees, 4 degrees, or 8 degrees) oriented and moved downhill within 1 min; that is, they displayed positive geotaxis. Their downhill translocation increased with angle of inclination. A variety of possible behavioral elements (e.g., initial orientation, rotational movements, ambulatory velocities) were eliminated as explanations of the geotaxis. Wall contact was recognized as a determinative event: Pups on the inclines showed no orientation with respect to the geogravitational stimulus before contacting a wall. The event of wall contact, however, evoked reliable downhill orientation and more linear movements. Positive geotaxis was created by pups' orientation against walls and an associated increase in movement velocity. This is a distinct perspective on a behavioral response that replaces a traditionally misinterpreted phenomenon. The authors discuss the ecological validity and historical context of these findings.

Sex differences in thermogenesis structure behavior and contact within huddles of infant mice.

Brown adipose tissue (BAT) is a thermogenic effector abundant in most mammalian infants. For multiparous species such as rats and mice, the interscapular BAT deposit provides both an emergency "thermal blanket" and a target for nestmates seeking warmth, thereby increasing the cohesiveness of huddling groups. Sex differences in BAT regulation and thermogenesis have been documented in a number of species, including mice (Mus musculus)--with females generally exhibiting relative upregulation of BAT. It is nonetheless unknown whether this difference affects the behavioral dynamics occurring within huddles of infant rodents. We investigated sex differences in BAT thermogenesis and its relation to contact while huddling in eight-day-old C57BL/6 mouse pups using infrared thermography, scoring of contact, and causal modeling of the relation between interscapular temperature relative to other pups in the huddle (T IS (rel)) and contacts while huddling. We found that females were warmer than their male siblings during cold challenge, under conditions both in which pups were isolated and in which pups could actively huddle in groups of six (3 male, 3 female). This difference garnered females significantly more contacts from other pups than males during cold-induced huddling. Granger analyses revealed a significant negative feedback relationship between contacts with males and T IS (rel) for females, and positive feedback between contacts with females and T IS (rel) for males, indicating that male pups drained heat from female siblings while huddling. Significant sex assortment nonetheless occurred, such that females made more contacts with other females than expected by chance, apparently outcompeting males for access to each other. These results provide further evidence of enhanced BAT thermogenesis in female mice. Slight differences in BAT can significantly structure the behavioral dynamics occurring in huddles, resulting in differences in the quantity and quality of contacts obtained by the individuals therein, creating sex differences in behavioral interactions beginning in early infancy.

Observe, simplify, titrate, model, and synthesize: a paradigm for analyzing behavior.

Phenomena in behavior and their underlying neural mechanisms are exquisitely complex problems. Infrequently do we reflect on our basic strategies of investigation and analysis, or formally confront the actual challenges of achieving an understanding of the phenomena that inspire research. Philip Teitelbaum is distinct in his elegant approaches to understanding behavioral phenomena and their associated neural processes. He also articulated his views on effective approaches to scientific analyses of brain and behavior, his vision of how behavior and the nervous system are patterned, and what constitutes basic understanding. His rubrics involve careful observation and description of behavior, simplification of the complexity, analysis of elements, and re-integration through different forms of synthesis. Research on the development of huddling behavior by individual and groups of rats is reviewed in a context of Teitelbaum's rubrics of research, with the goal of appreciating his broad and positive influence on the scientific community.