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.

Mice display learning and behavioral deficits after a 30-day spaceflight on Bion-M1 satellite.

Profound effects of spaceflight on the physiology of humans and non-human animals are well-documented but incompletely explored. Current goals to undertake interplanetary missions increase the urgency to learn more about adaptation to prolonged spaceflight and readaptation to Earth-normal conditions, especially with the inclusion of radiation exposures greater than those confronted in traditional, orbital flights. The 30-day-long Bion M-1 biosatellite flight was conducted at a relatively high orbit, exposing the mice to greater doses of radiation in addition to microgravity, a combination of factors relevant to Mars missions. Results of the present studies with mice provide insights into the consequences on brain function of long-duration spaceflight. After landing, mice showed profound deficits in vestibular responses during aerial drop tests. Spaceflown mice displayed reduced grip strength, rotarod performance, and voluntary wheel running, each, which improved gradually but incompletely over the 7-days of post-flight testing. Continuous monitoring in the animals' home cage activity, in combination with open-field and other tests of motor performance, revealed indices of altered affect, expressed as hyperactivity, potentiated thigmotaxis, and avoidance of open areas which, together, presented a syndrome of persistent anxiety-like behavior. A learned, operant response acquired before spaceflight was retained, whereas the acquisition of a new task was impaired after the flight. We integrate these observations with other results from Bion-M1's program, identifying deficits in musculoskeletal and cardiovascular systems, as well as in the brain and spinal cord, including altered gene expression patterns and the accompanying neurochemical changes that could underlie our behavioral findings.

Physiologically-Based Pharmacokinetic Modeling of Atorvastatin Incorporating Delayed Gastric Emptying and Acid-to-Lactone Conversion.

The drug-drug interaction profile of atorvastatin confirms that disposition is determined by cytochrome P450 (CYP) 3A4 and organic anion transporting polypeptides (OATPs). Drugs that affect gastric emptying, including dulaglutide, also affect atorvastatin pharmacokinetics (PK). Atorvastatin is a carboxylic acid that exists in equilibrium with a lactone form in vivo. The purpose of this work was to assess gastric acid-mediated lactone equilibration of atorvastatin and incorporate this into a physiologically-based PK (PBPK) model to describe atorvastatin acid, lactone, and their major metabolites. In vitro acid-to-lactone conversion was assessed in simulated gastric fluid and included in the model. The PBPK model was verified with in vivo data including CYP3A4 and OATP inhibition studies. Altering the gastric acid-lactone equilibrium reproduced the change in atorvastatin PK observed with dulaglutide. The model emphasizes the need to include gastric acid-lactone conversion and all major atorvastatin-related species for the prediction of atorvastatin PK.

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.

Identification and Mitigation of Reactive Metabolites of 2-Aminoimidazole-Containing Microsomal Prostaglandin E Synthase-1 Inhibitors Terminated Due to Clinical Drug-Induced Liver Injury.

Two 2-aminoimidazole-based inhibitors, LY3031207 (1) and LY3023703 (2), of the microsomal prostaglandin E synthase-1 (mPGES-1) enzyme were found to cause drug-induced liver injury (DILI) in humans. We studied imidazole ring substitutions to successfully mitigate reactive metabolite (RM) formation. These studies support the conclusion that RM formation may play a role in the observations of DILI and the consideration of 2-aminoimidazoles as structure alerts, due to the high likelihood of bioactivation to generate RMs.

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.

Development of a rapid and sensitive multiple reaction monitoring proteomic approach for quantification of transporters in human liver tissue.

With increasing knowledge on the role of hepatic transporters in drug disposition, numerous efforts have been described to quantify the expression of human hepatic transporters. However, reported quantitative proteomic approaches often require long analysis times. Additionally, greater assay sensitivity is still necessary for less abundant transporters or limited quantity of samples (e.g. hepatocytes and liver tissue). In the present study, an LC-MS/MS method for rapid and simultaneous quantification of 12 hepatic transporters (BCRP, BSEP, MATE1, MRP2, MRP3, MRP4, NTCP, OATP1B1, 1B3, 2B1, OCT1, and P-gp) was developed. Using a high LC flow rate (1.5mL/min) and fast LC gradient (4min total cycle time), the run time was markedly reduced to 4min, which was much shorter than most previously published assays. Chromatographic separation was achieved using ACE UltraCore SuperC18 50mm×2.1mm 5-µm HPLC column. In addition, greater analytical sensitivity was achieved with both high LC flow rate/fast LC gradient and post-column infusion of ethylene glycol. The on-column LLOQ for signature peptides in this method ranged from 0.194 to 0.846 femtomoles. The impact of five protein solubilizers, including extraction buffer II of ProteoExtract Native Membrane Protein Extraction Kit, 3% (w/v) sodium deoxycholate, 20% (v/v) Invitrosol, 0.2% (w/v) RapiGest SF, and 10% (w/v) formamide on total membrane protein extraction and trypsin digestion was investigated. Sodium deoxycholate was chosen because of good total membrane protein extraction and trypsin digestion efficiency, as well as no significant MS interference. Good precision (within 15% coefficient of variation) and accuracy (within ±15% bias), and inter-day trypsin digestion efficiency (within 28% coefficient of variation) was observed for quality controls. This method can quantify human hepatic transporter expression in a high-throughput manner and due to the increased sensitivity can be used to investigate the down-regulation of hepatic transporter protein (e.g., different ethnic groups and liver disease patients).

Adaptation to a blood pressure telemetry system revealed by measures of activity, agility and operant learning in mice.

INTRODUCTION: Implantable telemetry enables continuous monitoring of physiological functions in freely moving animals and can greatly complement pharmacological research. Despite its miniaturization, a sensor/transmitter constitutes 5% or more of a mouse's bodyweight. The aim of the present study was to evaluate whether factors related to the presence of a probe/transmitter influence the ambulatory activity, strength, agility, or operant, motivated behaviors of this small rodent. METHODS: Adult male mice (C57BL/6N, 22-25g, 9-10weeks; implanted n=26, intact n=45) were evaluated during week-long tests, conducted three and eight weeks after surgical implantation of the PA-C10 blood pressure probe. An open field test, grip force measurement, Rotarod test were performed, followed by 7-day continuous monitoring of spontaneous wheel running activity and positively reinforced operant conditioning in an automated data collection system. RESULTS: An implanted blood pressure transmitter did not affect behavior of mice in the open field test, on the Rotarod or their grip force, compared to unoperated controls. Voluntary wheel running distance was reduced three, but not eight weeks after implantation. Three weeks after the surgery, performance in the positively reinforced operant conditioning in operated mice was slightly decreased compared to intact animals, while retention and acquisition of a 2nd, reversal-learning task eight weeks after the surgery were unaffected. DISCUSSION: We conclude that an implantable transmitter may have detectable effects in the first few weeks following implantation on some elements of mouse behavior. With sufficient recovery, mice perform comparably to unoperated controls in tests of strength, endurance, agility and learned operant behavior.

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

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5- dihydrothiazole in female mice.

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development,was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.

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.

Conformational positioning improves sleep in premature infants with feeding difficulties.

OBJECTIVE: To determine whether premature infants' sleep organization, total sleep time, and arousals may be modulated while on a conformational positioner that provides boundaries, customized positioning, and containment compared with standard positioning (standard crib mattress). STUDY DESIGN: A proof of concept trial using a within subject crossover design was conducted among 25 premature infants with feeding difficulties. Infants of 31.5 weeks gestational age served as their own control during overnight polysomnography at postconceptual age 38.4 weeks. Each baby received both interventions (order randomized), 1 for each one-half of the 10.5-hour study. RESULTS: Use of the conformational positioner resulted in higher sleep efficiency of 61% vs 54% for the standard mattress (P < .05). The interventions did not differ for percent active sleep, percent quiet sleep, percent indeterminate sleep, or spontaneous arousals. Sleep efficiency was higher on the conformational positioner than standard positioning for surgical subjects and for subjects with necrotizing enterocolitis or gastroschisis (n = 10). The surgical subjects (n = 9) had lower sleep efficiency, lower percentage of active sleep, and more spontaneous arousals compared with the nonsurgical group. CONCLUSIONS: The use of the conformational positioner improved sleep efficiency vs the standard mattress in premature infants with feeding difficulties. Infants requiring surgery or with gastrointestinal diagnoses may be more susceptible to environmental stress.

Mice in Bion-M 1 space mission: training and selection.

After a 16-year hiatus, Russia has resumed its program of biomedical research in space, with the successful 30-day flight of the Bion-M 1 biosatellite (April 19-May 19, 2013). The principal species for biomedical research in this project was the mouse. This paper presents an overview of the scientific goals, the experimental design and the mouse training/selection program. The aim of mice experiments in the Bion-M 1 project was to elucidate cellular and molecular mechanisms, underlying the adaptation of key physiological systems to long-term exposure in microgravity. The studies with mice combined in vivo measurements, both in flight and post-flight (including continuous blood pressure measurement), with extensive in vitro studies carried out shortly after return of the mice and in the end of recovery study. Male C57/BL6 mice group housed in space habitats were flown aboard the Bion-M 1 biosatellite, or remained on ground in the control experiment that replicated environmental and housing conditions in the spacecraft. Vivarium control groups were used to account for housing effects and possible seasonal differences. Mice training included the co-adaptation in housing groups and mice adaptation to paste food diet. The measures taken to co-adapt aggressive male mice in housing groups and the peculiarities of "space" paste food are described. The training program for mice designated for in vivo studies was broader and included behavioral/functional test battery and continuous behavioral measurements in the home-cage. The results of the preliminary tests were used for the selection of homogenous groups. After the flight, mice were in good condition for biomedical studies and displayed signs of pronounced disadaptation to Earth's gravity. The outcomes of the training program for the mice welfare are discussed. We conclude that our training program was effective and that male mice can be successfully employed in space biomedical research.

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.

Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure.

LY2090314 (3-[9-fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenous glycogen synthase kinase-3 inhibitor in oncology trials. Drug disposition was characterized after intravenous infusion of [(14)C]LY2090314 to rats and dogs, and was related to available clinical data. LY2090314 exhibited high clearance (approximating hepatic blood flow) and a moderate volume of distribution (∼1-2 l/kg) resulting in rapid elimination (half-life ∼0.4, 0.7, and 1.8-3.4 hours in rats, dogs, and humans, respectively). Scaled clearance from liver microsomes accurately predicted perfusion-limited clearance across species. LY2090314 was cleared by extensive metabolism, and the numerous metabolites were rapidly excreted into feces via bile (69-97% of dose; 62-93% within 0-24 hours); urinary recovery of drug-related material was low (≤3% of dose). Despite extensive metabolism, in rats and humans the parent compound was the sole identifiable drug-related moiety in plasma. Even in Mdr1a-, Bcrp-, and Mrp2-knockout rats, LY2090314 metabolites did not appear in circulation, and their urinary excretion was not enhanced, because the hypothesized impaired biliary excretion of metabolites in the absence of these canalicular transporters was not observed. Canine metabolite disposition was generally similar, with the notable exception of dog-unique LY2090314 glucuronide. This conjugate was formed in the dog liver and was preferentially excreted into the blood, where it accounted for the majority of circulating radioactivity at later times, and was predominantly recovered in urine (16% of dose). In conclusion, LY2090314 was rapidly cleared by extensive metabolism with negligible circulating metabolite exposures due to biliary excretion of metabolites into feces with no apparent intestinal reabsorption.

Evolution and development of dual ingestion systems in mammals: notes on a new thesis and its clinical implications.

Traditionally, the development of oral feeding is viewed as a continuous, unitary process in which reflex-dominated sucking behavior gives rise to a more varied and volitional feeding behavior. In contrast, we consider the thesis that the infant develops two separable ingestive systems, one for suckling and one for feeding. First, we apply an evolutionary perspective, recognizing that suckling-feeding is a universal, mammalian developmental sequence. We find that in mammalian evolution, feeding systems in offspring were established prior to the evolution of lactation, and therefore suckling is a separable feature that was added to feeding. We next review an experimental literature that characterizes suckling and feeding as separable in terms of their topography, sensory controls, physiological controls, neural substrates, and experience-based development. Together, these considerations constitute a view of "dual ingestive systems." The thesis, then, is that suckling is not a simple precursor of feeding but is a complete behavior that emerges, forms, and then undergoes a dissolution that overlaps with the emergence of independent feeding. This thesis guides us to focus differently on the challenges of properly managing and facilitating oral ingestion in infants, especially those born preterm, prior to the developmental onset of suckling.

The experience of being born: a natural context for learning to suckle.

Understanding the developmental origins of congenital capabilities such as sucking is fundamental knowledge that can contribute to improving the clinical management of early feeding and facilitate the onset of oral ingestion. We describe analyses in rats showing that sensory stimulation in utero and during birth establishes the newborn's sucking responses to maternal cues. We mimicked elements of labor and delivery (viz., compressions simulating labor contractions, stroking simulating postnatal maternal licking of the newborn, and postnatal thermal flux), and used them to induce postnatal respiration and nipple attachment in caesarian-delivered pups. We report herein new data showing that, by simulating a fetal rat's experience of being born, specific components of vaginal birth provide sufficient conditions for the odor learning that guides newborn's sucking responses. In contrast, the absence of in utero compressions was associated with poor sucking onset. Knowing how birth stimuli contribute to the first nipple attachment and constitute a context for learning to suckle is an important step toward better management of some early feeding problems. It can serve also as a foundation for understanding the challenges of facilitating sucking by babies born prematurely so that they do not experience the typical contingencies mediating onset of oral ingestion.