Martine Migaud

Biology of Mammalian Photoperiodism and the Critical Role of the Pineal Gland and Melatonin

In mammals, photoperiodic information is transformed into a melatonin secretory rhythm in the pineal gland (high levels at night, low levels during the day). Melatonin exerts its effects in discrete hypothalamic areas, most likely through MT1 melatonin receptors. Whether melatonin is brought to the hypothalamus from the cerebrospinal fluid or the blood is still unclear. The final action of this indoleamine at the level of the central nervous system is a modulation of GnRH secretion but it does not act directly on GnRH neurones; rather, its action involves a complex neural circuit of interneurones that includes at least dopaminergic, serotoninergic and aminoacidergic neurones. In addition, this network appears to undergo morphological changes between seasons.

Neuroendocrine interactions and seasonality.

Sheep in temperate latitudes are seasonal breeders. Of the different seasonal cues, photoperiod is the most reliable parameter and is used by animals as an indication of the time of the year to synchronize endogenous annual rhythms of reproduction and physiology. The photoperiodic information is transduced into neuroendocrine changes through variations in melatonin secretion from the pineal gland. Melatonin triggers variations in the secretion of luteinizing hormone-releasing hormone, luteinizing hormone and follicle stimulating hormone (LHRH/LH/FSH) responsible for seasonal changes in reproductive activity. In female sheep, the seasonal changes in the hormonal LH pattern mainly reflect an increase in the negative feedback exerted by estradiol under long days on the frequency of pulsatile LH secretion. The resulting seasonal inhibition of LH secretion involves the activation of monoaminergic and especially dopaminergic systems by estradiol. Other types of physiological regulation subject to seasonal changes such as voluntary food intake (VFI), fat metabolism, body mass and pelage growth also occur in sheep, goats or related wild species. Several neuroendocrine intermediates seem to be shared by these different systems and may participate in their synchronization, providing the advantage that this helps mammalian species to adapt to their environment.

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT1 melatonin receptor and directly regulates its Gi-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT1 carboxyl terminus. This high affinity interaction (Kd ∼ 4 nm), which is independent of MT1 activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT1/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT1, it destabilizes the interaction between MT1 and Gi and abolishes Gi-mediated signaling of MT1. Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

MTNR1A Melatonin Receptors in the Ovine Premammillary Hypothalamus: Day-Night Variation in the Expression of the Transcripts

Abstract Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

DCX-expressing cells in the vicinity of the hypothalamic neurogenic niche: A comparative study between mouse, sheep, and human tissues

Neural stem and precursor cells persist postnatally throughout adulthood and are capable of responding to numerous endogenous and exogenous signals by modifying their proliferation and differentiation. Whereas adult neurogenesis has been extensively studied in the dentate gyrus of the hippocampal formation and in the subventricular zone adjacent to the wall of the lateral ventricles, we and others have recently reported constitutive adult neurogenesis in other brain structures, including the hypothalamus. In this study, we used immunohistochemistry to study the expression of the neuroblast marker doublecortin (DCX), and compared its expression pattern in adult ovine, mouse, and human hypothalamic tissues. Our results indicate that DCX‐positive cells resembling immature and developing neurons occur in a wide range of hypothalamic nuclei in all three species, although with different distribution patterns. In addition, the morphology of DCX‐positive cells varied depending on their location. DCX‐positive cells near the third ventricle had the morphology of very immature neuroblasts, a round shape with no processes, whereas those located deeper in the parenchyma such as in the ventromedial nucleus were fusiform and showed a bipolar morphology. Extending this observation, we showed that among the cohort of immature neurons entering the ventromedial nucleus, some appeared to undergo maturation, as revealed by the partial colocalization of DCX with markers of more mature neurons, e.g., human neuronal protein C and D (HuC/D). This study provides further confirmation of the existence of an adult hypothalamic neurogenic niche and argues for the potential existence of a migratory path within the hypothalamus. J. Comp. Neurol. 522:1966–1985, 2014.

Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: focus on the sheep hypothalamus.

To cope with variations in the environment, most mammalian species exhibit seasonal cycles in physiology and behaviour. Seasonal plasticity during the lifetime contributes to seasonal physiology. Over the years, our ideas regarding adult brain plasticity and, more specifically, hypothalamic plasticity have greatly evolved. Along with the two main neurogenic regions, namely the hippocampal subgranular and lateral ventricle subventricular zones, the hypothalamus, which is the central homeostatic regulator of numerous physiological functions that comprise sexual behaviours, feeding and metabolism, also hosts neurogenic niches. Both endogenous and exogenous factors, including the photoperiod, modulate the hypothalamic neurogenic capacities. The present review describes the effects of season on adult morphological plasticity and neurogenesis in seasonal species, for which the photoperiod is a master environmental cue for the successful programming of seasonal functions. In addition, the potential functional significance of adult neurogenesis in the mediation of the seasonal control of reproduction and feeding is discussed.

Sensitivity to the photoperiod and potential migratory features of neuroblasts in the adult sheep hypothalamus.

Adult neurogenesis, a process that consists in the generation of new neurons from adult neural stem cells, represents a remarkable illustration of the brain structural plasticity abilities. The hypothalamus, a brain region that plays a key role in the neuroendocrine regulations including reproduction, metabolism or food intake, houses neural stem cells located within a hypothalamic neurogenic niche. In adult sheep, a seasonal mammalian species, previous recent studies have revealed photoperiod-dependent changes in the hypothalamic cell proliferation rate. In addition, doublecortin (DCX), a microtubule-associated protein expressed in immature migrating neurons, is highly present in the vicinity of the hypothalamic neurogenic niche. With the aim to further explore the mechanism underlying adult sheep hypothalamic neurogenesis, we first show that new neuron production is also seasonally regulated since the density of DCX-positive cells changes according to the photoperiodic conditions at various time points of the year. We then demonstrate that cyclin-dependant kinase-5 (Cdk5) and p35, two proteins involved in DCX phosphorylation and known to be critically involved in migration processes, are co-expressed with DCX in young hypothalamic neurons and are capable of in vivo interaction. Finally, to examine the migratory potential of these adult-born neurons, we reveal the rostro-caudal extent of DCX labeling on hypothalamic sagittal planes. DCX-positive cells are found in the most rostral nuclei of the hypothalamus, including the preoptic area many of which co-expressed estrogen receptor-α. Thus, beyond the confirmation of the high level of neuron production during short photoperiod in sheep, our results bring new and compelling elements in support of the existence of a hypothalamic migratory path that is responsive to seasonal stimuli.

Development of an in vivo adeno-associated virus-mediated siRNA approach to knockdown tyrosine hydroxylase in the lateral retrochiasmatic area of the ovine brain.

We developed a new technique of gene knockdown (KD) in a specific brain area of the ewe using an adeno-associated virus (AAV)-mediated short interfering RNA (siRNA) method to elucidate the importance of key factors of seasonal reproduction. Two 19-nucleotide sequences (TH1 or TH2) were chosen from the tyrosine hydroxylase (TH) gene. TH1, TH2 or a random sequence (TH3) was incorporated into an eGFP expressing AAV vector. Firstly, 5 microl of AAV-TH1 or AAV-TH2 solutions (8-9 x 10(11)Vg/ml) were stereotaxically injected into one A15 nucleus while the other received a control treatment. Ewes were killed after 15 or 75 days. The number of TH neurons was 49% and 36% lower on the AAV-TH1 treated side than on the control side 15 and 75 days post-injection, respectively. AAV-TH2 did not induce a significant variation in TH cell population. Finally, in order to increase the KD, two groups of ewes received 10 microl of AAV-TH1 either in a bolus injection or in two 5 microl inoculations carried out 2 weeks apart. Only ewes receiving a bolus injection showed a larger KD reaching 66% 2 months after inoculation. This method proved effective in reducing TH expression and will be further developed to understand cellular mechanisms driving seasonal functions.

Adult neurogenesis and reproductive functions in mammals

During adulthood, the mammalian brain retains the capacity to generate new cells and new neurons in particular. It is now well established that the birth of these new neurons occurs in well-described sites: the hippocampus and the subventricular zone of the lateral ventricle, as well as in other brain regions including the hypothalamus. In this review, we describe the canonical neurogenic niches and illustrate the functional relevance of adult-born neurons of each neurogenic niche in the reproductive physiology. More specifically, we highlight the effect of reproductive social stimuli on the neurogenic processes and conversely, the contributions of adult-born neurons to the reproductive physiology and behavior. We next review the recent discovery of a novel neurogenic niche located in the hypothalamus and the median eminence and the compelling evidence of the link existing between the new-born hypothalamic neurons and the regulation of metabolism. In addition, new perspectives on the possible involvement of hypothalamic neurogenesis in the control of photoperiodic reproductive physiology in seasonal mammals are discussed. Altogether, the studies highlighted in this review demonstrate the potential role of neurogenesis in reproductive function and emphasize the importance of increasing our knowledge on the regulation processes and the physiological relevance of these adult-born neurons. This constitutes a necessary step toward a potential manipulation of these plasticity mechanisms.

A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non‐human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co‐expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.