Microglia regulate sleep through calcium-dependent modulation of norepinephrine transmission.

Sleep interacts reciprocally with immune system activity, but its specific relationship with microglia-the resident immune cells in the brain-remains poorly understood. Here, we show in mice that microglia can regulate sleep through a mechanism involving Gi-coupled GPCRs, intracellular Ca2+ signaling and suppression of norepinephrine transmission. Chemogenetic activation of microglia Gi signaling strongly promoted sleep, whereas pharmacological blockade of Gi-coupled P2Y12 receptors decreased sleep. Two-photon imaging in the cortex showed that P2Y12-Gi activation elevated microglia intracellular Ca2+, and blockade of this Ca2+ elevation largely abolished the Gi-induced sleep increase. Microglia Ca2+ level also increased at natural wake-to-sleep transitions, caused partly by reduced norepinephrine levels. Furthermore, imaging of norepinephrine with its biosensor in the cortex showed that microglia P2Y12-Gi activation significantly reduced norepinephrine levels, partly by increasing the adenosine concentration. These findings indicate that microglia can regulate sleep through reciprocal interactions with norepinephrine transmission.

Role of auditory feedback for vocal production learning in the Egyptian fruit-bat.

Some species have evolved the ability to use the sense of hearing to modify existing vocalizations, or even create new ones. This ability corresponds to various forms of vocal production learning that are all possessed by humans, and independently displayed by distantly related vertebrates. Among mammals, a few species, including the Egyptian fruit-bat, would possess such vocal production learning abilities. Yet the necessity of an intact auditory system for the development of the Egyptian fruit-bat typical vocal repertoire has not been tested. Furthermore, a systematic causal examination of learned and innate aspects of the entire repertoire has never been performed in any vocal learner. Here we addressed these gaps by eliminating pups' sense of hearing at birth and assessing its effects on vocal production in adulthood. The deafening treatment enabled us to both causally test these bats vocal learning ability and discern learned from innate aspects of their vocalizations. Leveraging wireless individual audio recordings from freely interacting adults, we show that a subset of the Egyptian fruit-bat vocal repertoire necessitates auditory feedback. Intriguingly, these affected vocalizations belong to different acoustic groups in the vocal repertoire of males and females. These findings open the possibilities for targeted studies of the mammalian neural circuits that enable sexually dimorphic forms of vocal learning.

Transcriptomic profiles of stress susceptibility and resilience in the amygdala and hippocampus.

A single, severe episode of stress can bring about myriad responses amongst individuals, ranging from cognitive enhancement to debilitating and persistent anxiety; however, the biological mechanisms that contribute to resilience versus susceptibility to stress are poorly understood. The dentate gyrus (DG) of the hippocampus and the basolateral nucleus of the amygdala (BLA) are key limbic regions that are susceptible to the neural and hormonal effects of stress. Previous work has also shown that these regions contribute to individual variability in stress responses; however, the molecular mechanisms underlying the role of these regions in susceptibility and resilience are unknown. In this study, we profiled the transcriptomic signatures of the DG and BLA of rats with divergent behavioral outcomes after a single, severe stressor. We subjected rats to three hours of immobilization with exposure to fox urine and conducted a behavioral battery one week after stress to identify animals that showed persistent, high anxiety-like behavior. We then conducted bulk RNA sequencing of the DG and BLA from susceptible, resilient, and unexposed control rats. Differential gene expression analyses revealed that the molecular signatures separating each of the three groups were distinct and non-overlapping between the DG and BLA. In the amygdala, key genes associated with insulin and hormonal signaling corresponded with vulnerability. Specifically, Inhbb, Rab31 , and Ncoa3 were upregulated in the amygdala of stress-susceptible animals compared to resilient animals. In the hippocampus, increased expression of Cartpt - which encodes a key neuropeptide involved in reward, reinforcement, and stress responses - was strongly correlated with vulnerability to anxiety-like behavior. However, few other genes distinguished stress-susceptible animals from control animals, while a larger number of genes separated stress-resilient animals from control and stress-susceptible animals. Of these, Rnf112, Tbx19 , and UBALD1 distinguished resilient animals from both control and susceptible animals and were downregulated in resilience, suggesting that an active molecular response in the hippocampus facilitates protection from the long-term consequences of severe stress. These results provide novel insight into the mechanisms that bring about individual variability in the behavioral responses to stress and provide new targets for the advancement of therapies for stress-induced neuropsychiatric disorders.

Gene Expression Signatures of Contact Lens-Induced Myopia in Guinea Pig Retinal Pigment Epithelium.

Purpose: To identify key retinal pigment epithelium (RPE) genes linked to the induction of myopia in guinea pigs. Methods: To induce myopia, two-week-old pigmented guinea pigs (New Zealand strain, n = 5) wore -10 diopter (D) rigid gas-permeable contact lenses (CLs), for one day; fellow eyes were left without CLs and served as controls. Spherical equivalent refractive errors (SE) and axial length (AL) were measured at baseline and one day after initiation of CL wear. RNA sequencing was applied to RPE collected from both treated and fellow (control) eyes after one day of CL-wear to identify related gene expression changes. Additional RPE-RNA samples from treated and fellow eyes were subjected to quantitative real-time PCR (qRT-PCR) analysis for validation purposes. Results: The CLs induced myopia. The change from baseline values in SE was significantly different (P = 0.016), whereas there was no significant difference in the change in AL (P = 0.10). RNA sequencing revealed significant interocular differences in the expression in RPE of 13 genes: eight genes were significantly upregulated in treated eyes relative to their fellows, and five genes, including bone morphogenetic protein 2 (Bmp2), were significantly downregulated. The latter result was also confirmed by qRT-PCR. Additional analysis of differentially expressed genes revealed significant enrichment for bone morphogenetic protein (BMP) and TGF-ß signaling pathways. Conclusions: The results of this RPE gene expression study provide further supporting evidence for an important role of BMP2 in eye growth regulation, here from a guinea pig myopia model.

Evidence for hormonal control of heart regenerative capacity during endothermy acquisition.

Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.

The Role of RFamide-Related Peptide-3 in Age-Related Reproductive Decline in Female Rats.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic-pituitary-gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system.

Moderate Stress-Induced Social Bonding and Oxytocin Signaling are Disrupted by Predator Odor in Male Rats.

In times of stress, social support can serve as a potent buffering mechanism that enhances resilience. In humans, stress can promote protective affiliative interactions and prosocial behavior. Yet, stress also precipitates psychopathologies characterized by social withdrawal such as post-traumatic stress disorder (PTSD) and depression. The factors that drive adaptive vs maladaptive social responses to stress are not yet clear. Rodent studies have focused on pair-bonded, opposite-sex mates and suggest that a variety of stressors can induce social support-like behaviors. However, between same-sex conspecifics-particularly males-stress effects on social bonding are less understood and often associated with aggression and social unrest. We thus sought to investigate if a moderate stressor-3 h of acute immobilization-impacts social-support behaviors differently when experienced in a neutral vs more innately threatening context (ie, paired with predator odor). We found that moderate stress increased social support-seeking behavior in rat cagemates and facilitated long-term sharing of a limited water resource, decreased aggression, and strongly defined dominance ranks (an indicator of home cage stability). In contrast, experiencing the same stressor in the presence of predator odor eliminated the positive behavioral effects of moderate stress. Importantly, hypothalamic oxytocin (OT) signaling increased coincident with stress in a neutral-but not a predator odor-context. Our results define a novel rodent model of divergent stress effects on social affiliation and OT signaling dependent on odor context with particularly strong relevance to stress-related disorders such as PTSD, which are characterized by a disrupted ability to seek and maintain social bonds.

Knockdown of hypothalamic RFRP3 prevents chronic stress-induced infertility and embryo resorption.

Whereas it is well established that chronic stress induces female reproductive dysfunction, whether stress negatively impacts fertility and fecundity when applied prior to mating and pregnancy has not been explored. In this study, we show that stress that concludes 4 days prior to mating results in persistent and marked reproductive dysfunction, with fewer successful copulation events, fewer pregnancies in those that successfully mated, and increased embryo resorption. Chronic stress exposure led to elevated expression of the hypothalamic inhibitory peptide, RFamide-related peptide-3 (RFRP3), in regularly cycling females. Remarkably, genetic silencing of RFRP3 during stress using an inducible-targeted shRNA completely alleviates stress-induced infertility in female rats, resulting in mating and pregnancy success rates indistinguishable from non-stress controls. We show that chronic stress has long-term effects on pregnancy success, even post-stressor, that are mediated by RFRP3. This points to RFRP3 as a potential clinically relevant single target for stress-induced infertility.

Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2.

Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain. DOI:http://dx.doi.org/10.7554/eLife.00362.001.