Hypothalamic tanycytes as mediators of maternally programmed seasonal plasticity.

In mammals, maternal photoperiodic programming (MPP) provides a means whereby juvenile development can be matched to forthcoming seasonal environmental conditions.1,2,3,4 This phenomenon is driven by in utero effects of maternal melatonin5,6,7 on the production of thyrotropin (TSH) in the fetal pars tuberalis (PT) and consequent TSH receptor-mediated effects on tanycytes lining the 3rd ventricle of the mediobasal hypothalamus (MBH).8,9,10 Here we use LASER capture microdissection and transcriptomic profiling to show that TSH-dependent MPP controls the attributes of the ependymal region of the MBH in juvenile animals. In Siberian hamster pups gestated and raised on a long photoperiod (LP) and thereby committed to a fast trajectory for growth and reproductive maturation, the ependymal region is enriched for tanycytes bearing sensory cilia and receptors implicated in metabolic sensing. Contrastingly, in pups gestated and raised on short photoperiod (SP) and therefore following an over-wintering developmental trajectory with delayed sexual maturation, the ependymal region has fewer sensory tanycytes. Post-weaning transfer of SP-gestated pups to an intermediate photoperiod (IP), which accelerates reproductive maturation, results in a pronounced shift toward a ciliated tanycytic profile and formation of tanycytic processes. We suggest that tanycytic plasticity constitutes a mechanism to tailor metabolic development for extended survival in variable overwintering environments.

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions. This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

An Intact Krüppel-like factor 9 Gene Is Required for Acute Liver Period 1 mRNA Response to Restraint Stress.

The clock protein period 1 (PER1) is a central component of the core transcription-translation feedback loop governing cell-autonomous circadian rhythms in animals. Transcription of Per1 is directly regulated by the glucocorticoid (GC) receptor (GR), and Per1 mRNA is induced by stressors or injection of GC. Circulating GCs may synchronize peripheral clocks with the central pacemaker located in the suprachiasmatic nucleus of the brain. Krüppel-like factor 9 (KLF9) is a zinc finger transcription factor that, like Per1, is directly regulated by liganded GR, and it associates in chromatin at clock and clock-output genes, including at Per1. We hypothesized that KLF9 modulates stressor-dependent Per1 transcription. We exposed wild-type (WT) and Klf9 null mice (Klf9-/-) of both sexes to 1 hour restraint stress, which caused similar 2- to 2.5-fold increases in plasma corticosterone (B) in each genotype and sex. Although WT mice of both sexes showed a 2-fold increase in liver Per1 mRNA level after restraint stress, this response was absent in Klf9-/- mice. However, injection of B in WT and Klf9-/- mice induced similar increases in Per1 mRNA. Our findings support that an intact Klf9 gene is required for liver Per1 mRNA responses to an acute stressor, but a possible role for GCs in this response requires further investigation.

Photoperiodic regulation in a wild-derived mouse strain.

MSM/Ms (MSM) is a mouse strain derived from Japanese wild mice, Mus musculus molossinus, that maintains the ability to synthesize melatonin in patterns reflecting the ambient photoperiod. The objective of this study was to characterize the effects of photoperiodic variation on metabolic and reproductive traits, and the related changes in pituitary-hypothalamic gene expression in MSM mice. MSM mice were kept in long (LP) or short photoperiod (SP) for 6 weeks. Our results demonstrate that MSM mice kept in LP, as compared with mice kept in SP, display higher expression of genes encoding thyrotropin (TSH) in the pars tuberalis, thyroid hormone deiodinase 2 (dio2) in the tanycytes and RFamide-related peptide (RFRP3) in the hypothalamus, and lower expression of dio3 in the tanycytes, along with larger body and reproductive organ mass. Additionally, to assess the effects of the gestational photoperiodic environment on the expression of these genes, we kept MSM mice in LP or SP from gestation and studied their offspring. We show that the gestational photoperiod affects the TSH/dio pathway in newborn MSM mice in a similar way to adults. This result indicates a transgenerational effect of photoperiod from the mother to the fetus in utero Overall, these results indicate that photoperiod can influence neuroendocrine regulation in a melatonin-proficient mouse strain, in a manner similar to that documented in other seasonal rodent species. MSM mice may therefore become a useful model for research into the molecular basis of photoperiodic regulation of seasonal biology.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

An integrative view of mammalian seasonal neuroendocrinology.

Seasonal neuroendocrine cycles that govern annual changes in reproductive activity, energy metabolism and hair growth are almost ubiquitous in mammals that have evolved at temperate and polar latitudes. Changes in nocturnal melatonin secretion regulating gene expression in the pars tuberalis (PT) of the pituitary stalk are a critical common feature in seasonal mammals. The PT sends signal(s) to the pars distalis of the pituitary to regulate prolactin secretion and thus the annual moult cycle. The PT also signals in a retrograde manner via thyroid-stimulating hormone to tanycytes, which line the ventral wall of the third ventricle in the hypothalamus. Tanycytes show seasonal plasticity in gene expression and play a pivotal role in regulating local thyroid hormone (TH) availability. Within the mediobasal hypothalamus, the cellular and molecular targets of TH remain elusive. However, two populations of hypothalamic neurones, which produce the RF-amide neuropeptides kisspeptin and RFRP3 (RF-amide related peptide 3), are plausible relays between TH and the gonadotrophin-releasing hormone-pituitary-gonadal axis. By contrast, the ways by which TH also impinges on hypothalamic systems regulating energy intake and expenditure remain unknown. Here, we review the neuroendocrine underpinnings of seasonality and identify several areas that warrant further research.

Maternal photoperiodic programming enlightens the internal regulation of thyroid-hormone deiodinases in tanycytes.

Seasonal rhythms in physiology are widespread among mammals living in temperate zones. These rhythms rely on the external photoperiodic signal being entrained to the seasons, although they persist under constant conditions, revealing their endogenous origin. Internal long-term timing (circannual cycles) can be revealed in the laboratory as photoperiodic history-dependent responses, comprising the ability to respond differently to similar photoperiodic cues based on prior photoperiodic experience. In juveniles, history-dependence relies on the photoperiod transmitted by the mother to the fetus in utero, a phenomenon known as "maternal photoperiodic programming" (MPP). The response to photoperiod in mammals involves the nocturnal pineal hormone melatonin, which regulates a neuroendocrine network including thyrotrophin in the pars tuberalis and deiodinases in tanycytes, resulting in changes in thyroid hormone in the mediobasal hypothalamus. This review addresses MPP and discusses the latest findings on its impact on the thyrotrophin/deiodinase network. Finally, commonalities between MPP and other instances of endogenous seasonal timing are considered, and a unifying scheme is suggested in which timing arises from a long-term communication between the pars tuberalis and the hypothalamus and resultant spontaneous changes in local thyroid hormone status, independently of the pineal melatonin signal.

Bulk Electroporation-Mediated Gene Transfer into Xenopus Tadpole Brain.

In vivo gene transfer is a powerful tool for investigating protein function and gene regulation in living organisms. Delivery of plasmid DNA to the brain of Xenopus tadpoles by bulk electroporation-mediated (EM) gene transfer can be used to study the effects of ectopic gene expression on development, physiology, and behavior. It can also be used to mark cells for lineage tracing, investigate the in vivo function of gene regulatory elements when linked to a reporter gene, and introduce mutations into the genome of transfected cells, among other applications. Bilateral EM gene transfer allows for transfection of both sides of the brain, whereas unilateral EM gene transfer enables analysis of the effects of forced gene expression on one side of the brain, with the other side serving as the control.

Melatonin-independent Photoperiodic Entrainment of the Circannual TSH Rhythm in the Pars Tuberalis of the European Hamster.

Adaptation of biological rhythms to a seasonal environment in circannual mammals is achieved via the synchronization of intrinsic circannual rhythms to the external year by photoperiod. In mammals, the photoperiodic information is integrated to seasonal physiology via the pineal hormone melatonin regulation of pars tuberalis (PT) TSHß expression and its downstream control of hypothalamic dio2 gene expression. In the circannual European hamster, however, photoperiodic entrainment of the circannual clock is possible in pinealectomized animals. The present study explores whether the TSHß expression in the PT and the downstream hypothalamic pathways are regulated by photoperiod in European hamsters in the absence of melatonin. All animals were kept on an accelerated photoperiodic regime, which compressed the natural year to a 6-month cycle. Sham-operated European hamsters and half of the pinealectomized European hamsters entrained their annual cycle in reproduction, body weight, and activity pattern to this cycle, whereas the other half of the pinealectomized animals followed only each second cycle. In all animals, PT TSHß and hypothalamic dio2 expressions were higher in hamsters displaying a summer physiological state than in those in winter state. Moreover, in agreement with their seasonal state, reproductive animals (summer state) showed higher expression of rfrp and lower expression of kiss1-genes encoding central regulators of the reproductive axis-than those animals in reproductive quiescence (winter state), indicating the hypothalamic integration of the photoperiodic signal even in pinealectomized animals. The appropriate occurrence of a well-characterized activity pattern indicative of a so-called sensitive phase to short photoperiod suggested that the SCN constructs the melatonin-independent photoperiodic message. This message is sufficient to entrain the circannual rhythm in TSHß expression in the PT and the downstream hypothalamic neuroendocrine pathway through a yet unknown pathway. These results reinforce the hypothesis that the PT is the site for the integration of circannual and photoperiodic information.

Maternal photoperiod programs hypothalamic thyroid status via the fetal pituitary gland.

In wild mammals, offspring development must anticipate forthcoming metabolic demands and opportunities. Within species, different developmental strategies may be used, dependent on when in the year conception takes place. This phenotypic flexibility is initiated before birth and is linked to the pattern of day length (photoperiod) exposure experienced by the mother during pregnancy. This programming depends on transplacental communication via the pineal hormone melatonin. Here, we show that, in the Siberian hamster (Phodopus sungorus), the programming effect of melatonin is mediated by the pars tuberalis (PT) of the fetal pituitary gland, before the fetal circadian system and autonomous melatonin production is established. Maternal melatonin acts on the fetal PT to control expression of thyroid hormone deiodinases in ependymal cells (tanycytes) of the fetal hypothalamus, and hence neuroendocrine output. This mechanism sets the trajectory of reproductive and metabolic development in pups and has a persistent effect on their subsequent sensitivity to the photoperiod. This programming effect depends on tanycyte sensitivity to thyroid stimulating hormone (TSH), which is dramatically and persistently increased by short photoperiod exposure in utero. Our results define the role of the fetal PT in developmental programming of brain function by maternal melatonin and establish TSH signal transduction as a key substrate for the encoding of internal calendar time from birth to puberty.

A circannual clock drives expression of genes central for seasonal reproduction.

Animals living in temperate zones anticipate seasonal environmental changes to adapt their biological functions, especially reproduction and metabolism. Two main physiological mechanisms have evolved for this adaptation: intrinsic long-term timing mechanisms with an oscillating period of approximately 1 year, driven by a circannual clock [1], and synchronization of biological rhythms to the sidereal year using day length (photoperiod) [2]. In mammals, the pineal hormone melatonin relays photoperiodic information to the hypothalamus to control seasonal physiology through well-defined mechanisms [3-6]. In contrast, little is known about how the circannual clock drives endogenous changes in seasonal functions. The aim of this study was to determine whether genes involved in photoperiodic time measurement (TSHß and Dio2) and central control of reproduction (Rfrp and Kiss1) display circannual rhythms in expression under constant conditions. Male European hamsters, deprived of seasonal time cues by pinealectomy and maintenance in constant photoperiod, were selected when expressing a subjective summer or subjective winter state in their circannual cycle of body weight, temperature, and testicular size. TSHß expression in the pars tuberalis (PT) displayed a robust circannual variation with highest level in the subjective summer state, which was positively correlated with hypothalamic Dio2 and Rfrp expression. The negative sex steroid feedback was found to act specifically on arcuate Kiss1 expression. Our findings reveal TSH as a circannual output of the PT, which in turn regulates hypothalamic neurons controlling reproductive activity. Therefore, both the circannual and the melatonin signals converge on PT TSHß expression to synchronize seasonal biological activity.