Alexandra Castillo-Ruiz

In search of a temporal niche: social interactions.

Circadian rhythms can be entrained to periodic cues in the environment including the solar day, food resources, and temperature. Work on a variety of organisms has suggested that social interactions within and between species may also influence circadian rhythmicity, but conceptual and technical difficulties relating to animal models, housing environments, rhythm assays, and experimental design have complicated mechanistic investigations in the laboratory. We review these issues and introduce the gregarious Nile grass rat, Arvicanthis niloticus, as a suitable model for research on this problem. Understanding social influences on temporal organization at this supra-organismal, community level is of considerable translational value, as its implications range from conservation biology to human health.

In search of a temporal niche

Circadian rhythms can be entrained to periodic cues in the environment including the solar day, food resources, and temperature. Work on a variety of organisms has suggested that social interactions within and between species may also influence circadian rhythmicity, but conceptual and technical difficulties relating to animal models, housing environments, rhythm assays, and experimental design have complicated mechanistic investigations in the laboratory. We review these issues and introduce the gregarious Nile grass rat, Arvicanthis niloticus, as a suitable model for research on this problem. Understanding social influences on temporal organization at this supra-organismal, community level is of considerable translational value, as its implications range from conservation biology to human health.

Cellular and molecular mechanisms of sexual differentiation in the mammalian nervous system

Neuroscientists are likely to discover new sex differences in the coming years, spurred by the National Institutes of Health initiative to include both sexes in preclinical studies. This review summarizes the current state of knowledge of the cellular and molecular mechanisms underlying sex differences in the mammalian nervous system, based primarily on work in rodents. Cellular mechanisms examined include neurogenesis, migration, the differentiation of neurochemical and morphological cell phenotype, and cell death. At the molecular level we discuss evolving roles for epigenetics, sex chromosome complement, the immune system, and newly identified cell signaling pathways. We review recent findings on the role of the environment, as well as genome-wide studies with some surprising results, causing us to re-think often-used models of sexual differentiation. We end by pointing to future directions, including an increased awareness of the important contributions of tissues outside of the nervous system to sexual differentiation of the brain.

NEURAL ACTIVATION IN AROUSAL AND REWARD AREAS OF THE BRAIN IN DAY-ACTIVE AND NIGHT-ACTIVE GRASS RATS

In the diurnal unstriped Nile grass rat (Arvicanthis niloticus) access to a running wheel can trigger a shift in active phase preference, with some individuals becoming night-active (NA), while others continue to be day-active (DA). To investigate the contributions of different neural systems to the support of this shift in locomotor activity, we investigated the association between chronotype and Fos expression during the day and night in three major nuclei in the basal forebrain (BF) cholinergic (ACh) arousal system - medial septum (MS), vertical and horizontal diagonal band of Broca (VDB and HDB respectively) -, and whether neural activation in these areas was related to neural activity in the orexinergic system. We also measured Fos expression in dopaminergic and non-dopaminergic cells of two components of the reward system that also participate in arousal - the ventral tegmental area (VTA) and supramammillary nucleus (SUM). NAs and DAs were compared to animals with no wheels. NAs had elevated Fos expression at night in ACh cells, but only in the HDB. In the non-cholinergic cells of the BF of NAs, enhanced nocturnal Fos expression was almost universally seen, but only associated with activation of the orexinergic system for the MS/VDB region. For some of the areas and cell types of the BF, the patterns of Fos expression of DAs appeared similar to those of NAs, but were never associated with activation of the orexinergic system. Also common to DAs and NAs was a general increase in Fos expression in non-dopaminergic cells of the SUM and anterior VTA. Thus, in this diurnal species, voluntary exercise and a shift to a nocturnal chronotype changes neural activity in arousal and reward areas of the brain known to regulate a broad range of neural functions and behaviors, which may be also affected in human shift workers.

Cholinergic projections to the suprachiasmatic nucleus and lower subparaventricular zone of diurnal and nocturnal rodents

In nocturnal species cholinergic agonists alter circadian rhythm phase when injected intraventricularly or directly into the suprachiasmatic nucleus (SCN), but the phase shifts obtained differ depending upon the site being injected. Cholinergic projections reach the SCN of nocturnal laboratory rats, however, nothing is known about these projections in diurnal rodents. The first objective of this study was to evaluate the hypothesis that cholinergic projections to the SCN are only present in nocturnal species. The second objective was to evaluate the hypothesis that the lower part of the subparaventricular zone (LSPV) is a candidate for being a site that mediates the phase shifts observed when cholinergic agonists are injected intraventricularly. These hypotheses were tested in the diurnal unstriped Nile grass rat (Arvicanthis niloticus) and the nocturnal laboratory rat. Additionally, we evaluated if the light-dark (LD) cycle had an effect on the expression of the vesicular acetylcholine transporter (VAChT) in the SCN, LSPV, and in two control areas. Animals were kept in a 12:12 LD cycle and perfused at six time points. VAChT immunoreactivity was observed in the SCN, LSPV, and in the control areas of both species. The SCN and LSPV showed a differential distribution and density of cholinergic projections between the two species, but similar temporal patterns of VAChT expression were found across species. These results provide evidence for a differential cholinergic stimulation of the SCN between grass rats and laboratory rats that may reflect a rewiring of neural projections brought about by the adoption of a diurnal activity profile.

Day-night differences in neural activation in histaminergic and serotonergic areas with putative projections to the cerebrospinal fluid in a diurnal brain.

In nocturnal rodents, brain areas that promote wakefulness have a circadian pattern of neural activation that mirrors the sleep/wake cycle, with more neural activation during the active phase than during the rest phase. To investigate whether differences in temporal patterns of neural activity in wake-promoting regions contribute to differences in daily patterns of wakefulness between nocturnal and diurnal species, we assessed Fos expression patterns in the tuberomammillary (TMM), supramammillary (SUM), and raphe nuclei of male grass rats maintained in a 12:12 h light-dark cycle. Day-night profiles of Fos expression were observed in the ventral and dorsal TMM, in the SUM, and in specific subpopulations of the raphe, including serotonergic cells, with higher Fos expression during the day than during the night. Next, to explore whether the cerebrospinal fluid is an avenue used by the TMM and raphe in the regulation of target areas, we injected the retrograde tracer cholera toxin subunit beta (CTB) into the ventricular system of male grass rats. While CTB labeling was scarce in the TMM and other hypothalamic areas including the suprachiasmatic nucleus, which contains the main circadian pacemaker, a dense cluster of CTB-positive neurons was evident in the caudal dorsal raphe, and the majority of these neurons appeared to be serotonergic. Since these findings are in agreement with reports for nocturnal rodents, our results suggest that the evolution of diurnality did not involve a change in the overall distribution of neuronal connections between systems that support wakefulness and their target areas, but produced a complete temporal reversal in the functioning of those systems.

The microbiota influences cell death and microglial colonization in the perinatal mouse brain

The mammalian fetus develops in a largely sterile environment, and direct exposure to a complex microbiota does not occur until birth. We took advantage of this to examine the effect of the microbiota on brain development during the first few days of life. The expression of anti- and pro-inflammatory cytokines, developmental cell death, and microglial colonization in the brain were compared between newborn conventionally colonized mice and mice born in sterile, germ-free (GF) conditions. Expression of the pro-inflammatory cytokines interleukin 1β and tumor necrosis factor α was markedly suppressed in GF newborns. GF mice also had altered cell death, with some regions exhibiting higher rates (paraventricular nucleus of the hypothalamus and the CA1 oriens layer of the hippocampus) and other regions exhibiting no change or lower rates (arcuate nucleus of the hypothalamus) of cell death. Microglial labeling was elevated in GF mice, due to an increase in both microglial cell size and number. The changes in cytokine expression, cell death and microglial labeling were evident on the day of birth, but were absent on embryonic day 18.5, approximately one-half day prior to expected delivery. Taken together, our results suggest that direct exposure to the microbiota at birth influences key neurodevelopmental events and does so within hours. These findings may help to explain some of the behavioral and neurochemical alterations previously seen in adult GF mice.

Time management in a co-housed social rodent species ( Arvicanthis niloticus )

Sociality has beneficial effects on fitness, and timing the activities of animals may be critical. Social cues could influence daily rhythmic activities via direct effects on the circadian clock or on processes that bypass it (masking), but these possibilities remain incompletely addressed. We investigated the effects of social cues on the circadian body temperature (Tb) rhythms in pairs of co-housed and isolated grass rats, Arvicanthis niloticus (a social species), in constant darkness (DD). Cohabitation did not induce synchronization of circadian Tb rhythms. However, socio-sexual history did affect circadian properties: accelerating the clock in sexually experienced males and females in DD and advancing rhythm phase in the females in a light-dark cycle. To address whether synchronization occurs at an ultradian scale, we analyzed Tb and activity rhythms in pairs of co-housed sisters or couples in DD. Regardless of pair type, co-housing doubled the percentage of time individuals were simultaneously active without increasing individual activity levels, suggesting that activity bouts were synchronized by redistribution over 24 h. Together, our laboratory findings show that social cues affect individual “time allocation” budgets via mechanisms at multiple levels of biological organization. We speculate that in natural settings these effects could be adaptive, especially for group-living animals.

Birth delivery mode alters perinatal cell death in the mouse brain

Significance The rate of cesarean section (C-section) delivery worldwide far exceeds the World Health Organization’s recommended rate. C-section delivery has been linked to behavioral effects in the offspring. This suggests effects on the brain, but human studies are confounded by the medical complications, altered birth timing, and maternal factors associated with C-section delivery. We addressed these limitations in a carefully controlled study in mice. Vaginally-born offspring exhibited an acute decrease in cell death across the brain that was absent in C-section–born mice. C-section delivery also was associated with softer vocalizations, as well as with a reduction in at least one neuronal cell type and increased body weight at weaning. Thus, birth mode has acute effects on neurodevelopment that may lead to lasting changes in the brain and behavior. Labor and a vaginal delivery trigger changes in peripheral organs that prepare the mammalian fetus to survive ex utero. Surprisingly little attention has been given to whether birth also influences the brain, and to how alterations in birth mode affect neonatal brain development. These are important questions, given the high rates of cesarean section (C-section) delivery worldwide, many of which are elective. We examined the effect of birth mode on neuronal cell death, a widespread developmental process that occurs primarily during the first postnatal week in mice. Timed-pregnant dams were randomly assigned to C-section deliveries that were yoked to vaginal births to carefully match gestation length and circadian time of parturition. Compared with rates of cell death just before birth, vaginally-born offspring had an abrupt, transient decrease in cell death in many brain regions, suggesting that a vaginal delivery is neuroprotective. In contrast, cell death was either unchanged or increased in C-section–born mice. Effects of delivery mode on cell death were greatest for the paraventricular nucleus of the hypothalamus (PVN), which is central to the stress response and brain–immune interactions. The greater cell death in the PVN of C-section–delivered newborns was associated with a reduction in the number of PVN neurons expressing vasopressin at weaning. C-section–delivered mice also showed altered vocalizations in a maternal separation test and greater body mass at weaning. Our results suggest that vaginal birth acutely impacts brain development, and that alterations in birth mode may have lasting consequences.

Defective daily temperature regulation in a mouse model of amyotrophic lateral sclerosis

&NA; Current understanding of the pathogenesis of the familial form of amyotrophic lateral sclerosis has been aided by the study of transgenic mice that over‐express mutated forms of the human CuZn‐superoxide dismutase (SOD1) gene. While mutant SOD1 in motor neurons determines disease onset, other non‐cell autonomous factors are critical for disease progression, and altered energy metabolism has been implicated as a contributing factor. Since most energy expended by laboratory mice is utilized to defend body temperature (Tb), we analyzed thermoregulation in transgenic mice carrying the G93A mutation of the human SOD1 gene, using implantable temperature data loggers to continuously record Tb for up to 85 days. At room (22 °C) ambient temperature, G93A mice exhibited a diminished amplitude of the daily Tb rhythm compared to C57BL/6J controls, secondary to decreased Tb values during the dark (behaviorally active) phase of the light‐dark cycle. The defect arose at 85–99 days of age, around the age of symptom onset (as assessed by grip strength), well before observable weakness and weight loss, and could not be accounted for by decreased levels of locomotor activity or food consumption. Housing under thermoneutral (29 °C) ambient temperature partially rescued the defect, but age‐dependently (only in animals >100 days of age), suggesting that the deficit in older mice was due in part to inadequate thermogenesis by “peripheral” thermogenic organs as the disease progressed. In younger mice, we found that cold‐induced thermogenesis and energy expenditure were intact, hinting that an initial “central” defect might localize to the subparaventricular zone, involving neural output pathways from the circadian clock in the hypothalamic suprachiasmatic nucleus to forebrain thermoregulatory circuitry.