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Adult neural stem cells (NSCs) located in the two canonical neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ), express the glial fibrillary acidic protein (GFAP). Recently, proliferative activity has been described in the hypothalamus although the characterization of hypothalamic neural stem/progenitor cells (NSPCs) is still uncertain. We therefore investigated whether hypothalamic GFAP-positive cells, as in the SVZ and SGZ, also have neurogenic potential. We used a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GFAP promoter. GFAP-GFP expressing cells are localized in the ependymal layer as well as in the parenchyma of the mediobasal hypothalamus (MBH) and express Sox2, a marker for NSCs. Interestingly, no sexual dimorphism was observed in the numbers of GFP + and GFP-Sox2 + cells. After cells sorting, these cells were able to generate neurospheres in vitro and give rise to neurons, astrocytes and oligodendrocytes. Taken together, these results show that hypothalamic GFAP-expressing cells form a population of NSPCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Most organisms on earth, humans included, have developed strategies to cope with environmental day-night and seasonal cycles to survive. For most of them, their physiological and behavioral functions, including the reproductive function, are synchronized with the annual changes of day length, to ensure winter survival and subsequent reproductive success in the following spring. Sheep are sensitive to photoperiod, which also regulates natural adult neurogenesis in their hypothalamus. We postulate that the ovine model represents a good alternative to study the functional and metabolic changes occurring in response to photoperiodic changes in hypothalamic structures of the brain. Here, the impact of the photoperiod on the neurovascular coupling and the metabolism of the hypothalamic structures was investigated at 3T using BOLD fMRI, perfusion-MRI and proton magnetic resonance spectroscopy ( 1 H-MRS). A longitudinal study involving 8 ewes was conducted during long days (LD) and short days (SD) revealing significant BOLD, rCBV and metabolic changes in hypothalamic structures of the ewe brain between LD and SD. More specifically, the transition between LD and SD revealed negative BOLD responses to hypercapnia at the beginning of SD period followed by significant increases in BOLD, rCBV, Glx and tNAA concentrations towards the end of the SD period. These observations suggest longitudinal mechanisms promoting the proliferation and differentiation of neural stem cells within the hypothalamic niche of breeding ewes. We conclude that multiparametric MRI studies including 1 H-MRS could be promising non-invasive translational techniques to investigate the existence of natural adult neurogenesis in-vivo in gyrencephalic brains., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The ovine model could be an effective translational model but remains underexplored. Here, Blood Oxygen Level dependent functional MRI during visual stimulation and resting-state perfusion MRI were explored. We aimed at investigating the impact of isoflurane anesthesia during visual stimulation and evaluate resting cerebral blood flow and cerebral blood volume parameters in the lamb and adult sheep brain. BOLD fMRI and perfusion MRI after a bolus of DOTAREM were conducted in 4 lambs and 6 adult ewes at 3 T. A visual stimulation paradigm was delivered during fMRI at increasing isoflurane doses (1-3%). Robust but weak BOLD responses (0.21 ± 0.08%) were found in the lateral geniculate nucleus (LGN) up to 3% isoflurane anaesthesia. No significant differences were found beween BOLD responses in the range 1 to 3% ISO (p > 0.05). However, LGN cluster size decreased and functional localization became less reliable at high ISO doses (2.5-3% ISO). BOLD responses were weaker in adult sheep than in lambs (4.6 ± 1.5 versus 13.6 ± 8.5; p = 0.08). Relative cerebral blood volumes (rCBV) and relative cerebral blood flows (rCBF) were significantly higher (p < 0.0001) in lambs than in adult sheep for both gray and white matter. The impact of volatile anesthesia was explored for the first time on BOLD responses demonstrating increased reliability of functional localization of brain activity at low doses. Perfusion MRI was conducted for the first time in both lambs and adult ewes. Assessment of baseline cerebrovascular values are of interest for future studies of brain diseases allowing an improved interpretation of BOLD responses., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Unlike mammals, the role of adipokines and more particularly of chemerin in the regulation of food intake is totally unknown in avian species. Here we investigated the effect of chemerin on the food and water consumption and on the body weight in chicken. We studied the effects on the plasma glucose and insulin concentrations and the hypothalamic neuropeptides and AMPK signaling pathway. Female broiler chickens were intraperitoneally injected, daily for 13 days with either vehicle (saline; n = 25) or chemerin (8 μg/kg; n = 25 and 16 μg/kg; n = 25). Food and water intakes were recorded 24 h after each administration. Overnight fasted animals were sacrificed at day 13 (D13), 24 h after the last injection and hypothalamus and left cerebral hemispheres were collected. Chemerin and its receptors protein levels were determined by western-blot. Gene expression of neuropeptide Y (Npy), agouti-related peptide (Agrp), corticotrophin releasing hormone (Crh), pro-opiomelanocortin (Pomc), cocaine and amphetamine-regulated transcript (Cart) and Taste 1 Receptor Member 1 (Tas1r1) were evaluated by RT-qPCR. In chicken, we found that the protein amount of chemerin, CCRL2 and GPR1 was similar in left cerebral hemisphere and hypothalamus whereas CMKLR1 was higher in hypothalamus. Chemerin administration (8 and 16 μg/kg) decreased both food intake and body weight compared to vehicle without affecting water intake and the size or volume of different brain subdivisions as determined by magnetic resonance imaging. It also increased plasma insulin levels whereas glucose levels were decreased. These data were associated with an increase in Npy and Agrp expressions and a decrease in Crh, Tas1r1 mRNA expression within the hypothalamus. Furthermore, chemerin decreased hypothalamic CMKLR1 protein expression and AMPK activation. Taken together, these results support that chemerin could be a peripheral appetite-regulating signal through modulation of hypothalamic peptides expression in chicken., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To survive in temperate latitudes, species rely on the photoperiod to synchronize their physiological functions, including reproduction, with the predictable changes in the environment. In sheep, exposure to decreasing day length reactivates the hypothalamo-pituitary-gonadal axis, while during increasing day length, animals enter a period of sexual rest. Neural stem cells have been detected in the sheep hypothalamus and hypothalamic neurogenesis was found to respond to the photoperiod. However, the physiological relevance of this seasonal adult neurogenesis is still unexplored. This longitudinal study, therefore aimed to thoroughly characterize photoperiod-stimulated neurogenesis and to investigate whether the hypothalamic adult born-cells were involved in the seasonal timing of reproduction. Results showed that time course of cell proliferation reached a peak in the middle of the period of sexual activity, corresponding to decreasing day length period. This enhancement was suppressed when animals were deprived of seasonal time cues by pinealectomy, suggesting a role of melatonin in the seasonal regulation of cell proliferation. Furthermore, when the mitotic blocker cytosine-b-D-arabinofuranoside was administered centrally, the timing of seasonal reproduction was affected. Overall, our findings link the cyclic increase in hypothalamic neurogenesis to seasonal reproduction and suggest that photoperiod-regulated hypothalamic neurogenesis plays a substantial role in seasonal reproductive physiology.

Neurogenesis is the process by which new neurons are generated. This process, well established during development, persists in adulthood owing to the presence of neural stem cells (NSCs) localized in specific brain areas called neurogenic niches. Adult neurogenesis has recently been shown to occur in the hypothalamus, a structure involved in the neuroendocrine regulation of reproduction and metabolism, among others. In the adult sheep-a long-lived mammalian model-we have previously reported the existence of such a neurogenic niche located in the hypothalamic arcuate nucleus and the median eminence. In addition, in this seasonal species, the proliferation as well as neuroblasts production varies depending on the time of the year. In the present study, we provide a better characterization of the hypothalamic neurogenic niche by identifying the main components (NSCs, migrating cells, glial cells and blood vessels) using immunohistochemistry for validated markers. Then, we demonstrate the strong sensitivity of these various neurogenic niche components to the season, particularly in the arcuate nucleus. Further, using an electron microscopic approach, we reveal the cellular and cytoarchitectural reorganization of the arcuate nucleus niche following exposure to contrasting seasons. This study provides evidence that the arcuate nucleus and the median eminence contain two independent niches that react differently to the season. In addition, our results support the view that the cytoarchitectural organization of the sheep arcuate nucleus share comparable features with the structure of the subventricular zone in humans and non-human primates.

Sheep have many advantages to study neurogenesis in comparison to the well-known rodent models. Their development and life expectancy are relatively long and they possess a gyrencephalic brain. Sheep are also seasonal breeders, a characteristic that allows studying the involvement of hypothalamic neurogenesis in the control of seasonal reproduction. Sheep are also able to individually recognize their conspecifics and develop selective and lasting bonds. Adult olfactory neurogenesis could be adapted to social behavior by supporting recognition of conspecifics. The present review reveals the distinctive features of the hippocampal, olfactory, and hypothalamic neurogenesis in sheep. In particular, the organization of the subventricular zone and the dynamic of neuronal maturation differs from that of rodents. In addition, we show that various physiological conditions, such as seasonal reproduction, gestation, and lactation differently modulate these three neurogenic niches. Last, we discuss recent evidence indicating that hypothalamic neurogenesis acts as an important regulator of the seasonal control of reproduction and that olfactory neurogenesis could be involved in odor processing in the context of maternal behavior.

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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Wiley Periodicals, Inc.)

To adapt to seasonal variations in the environment, most mammalian species exhibit seasonal cycles in their physiology and behavior. Seasonal plasticity in the structure and function of the central nervous system contributes to the adaptation of this physiology in seasonal mammals. As part of these plasticity mechanisms, seasonal variations in proliferation rate and neuron production have been extensively studied in songbirds. In this report, we investigated whether this type of brain plasticity also occurs in sheep, a seasonal species, by assessing variations in cell proliferation in the sheep diencephalon. We administered the cell birth marker 5'-bromodeoxyuridine (BrdU) to adult female sheep in July and December, during long and short photoperiod, respectively. The BrdU incorporation was analyzed and quantified in the hypothalamus, a key center for neuroendocrine regulations, as well as in other structures involved in relaying neuroendocrine and sensory information, including the median eminence, the pars tuberalis of the pituitary gland, and the thalamus. In December, 2-fold and 6-fold increases in the number of BrdU+ nuclei were observed in the hypothalamus and thalamus, respectively, when compared with July. This variation is independent of the influence of peripheral gonadal estradiol variations. An inverse seasonal regulation of cell proliferation was observed in the pars tuberalis. In contrast, no seasonal variation in cell proliferation was seen in the subventricular zone of the lateral ventricle. Many of the newborn cells in the adult ovine hypothalamus and thalamus differentiate into neurons and glial cells, as assessed by the expression of neuronal (DCX, NeuN) and glial (GFAP, S100B) fate markers. In summary, we show that the estimated cell proliferation rates in the sheep hypothalamus, thalamus, and pars tuberalis are different between seasons. These variations are independent of the seasonal fluctuations of peripheral estradiol levels, unlike the results described in the brain nuclei involved in song control of avian species.

In adult mammalian brain, two main germinative regions located in the subventricular zone of the lateral ventricle and in the subgranular cell layer of the hippocampal dentate gyrus have been considerably documented and are still under intense scrutiny. However, new neuron formation has recently been reported in various other brain areas including the hypothalamus. This central structure, responsible for the control of many major neuroendocrine functions such as reproduction, expresses high levels of PSA-NCAM and nestin, both proteins being involved in structural and morphological plasticity mechanisms. Cell proliferation and new neuron production have been demonstrated in the adult hypothalamus of numerous species, although not hitherto described in non-human primates and humans. Similarly to the subventricular zone and in the subgranular cell layer, the adult hypothalamic neurogenesis process is subject to dynamic regulation by various physiological and pharmacological signals. Several pieces of evidence support the hypothesis that a stem cell niche-like architecture exist in the hypothalamus region lining the third ventricle thereby enabling adult neural stem cells to continuously generate neurons in vivo throughout life. Furthermore, recent data indicating that new hypothalamic neurons may become functionally implicated in sensory information processing endorse the assumption that the hypothalamus might be a neurogenic region., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The aim of the present study was to explore the putative effects of agonists and antagonists of the estradiol receptor on the early phase of GnRH-1 neuron development. To address this question we used an in vitro model of GnRH-1 neurons using cultured olfactory placode from sheep embryos on day 26 of gestation. Previous studies on this model have shown that in vitro the development of GnRH-1 neurons mimics in vivo development up to the start of pulsatile GnRH-1 secretion, To address the effects of modulating the estrogen receptor, cultures were treated with the endogenous and synthetic ligands of estradiol receptors: 17beta-estradiol, 17alpha-estradiol and tamoxifen. Neurogenesis was measured by incorporation of [(3)H]-thymidine. Morphometric parameters were evaluated by image analysis. The main results are that antagonism of estradiol receptors induced an important decrease in neurogenesis but had little effect on morphometric parameters, suggesting that during this early phase of development, maternal estrogens are important to achieve correct development of the GnRH-1 neuronal network.

Background: During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, i.e. morphological plasticity, we conducted experiments in intact ewes at 4 different times of the oestrous cycle (before the expected LH surge, during the LH surge, and on day 8 and day 15 of the subsequent luteal phase). The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia., Results: The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 +/- 7) than during the luteal phase (25 +/- 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group., Conclusions: These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge.

The aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization.

The distribution of histaminergic neurons in the sheep brain was studied by immunohistochemistry by using antibodies raised against histamine. For the first time in this species, the presence of histamine-immunoreactive neurons was described in the caudal diencephalon, around the mammillary bodies, and in the tuberomammillary area. The general pattern of distribution of these neurons was similar to that described previously in other species, i.e., rodents and humans. The distribution in the five neuronal groups described in rodents was not easy to demonstrate in sheep, because the boundaries between each group were not clear. The labeled neurons appeared to form a continuous cell system, as in humans. Numerous histamine-immunoreactive mast cells were found in the habenula and the thalamus. Histamine-immunoreactive fibers were found in almost all of the structures studied. The highest density of fibers was seen in the tuberomammillary area, from which dense bundles of fibers ran rostrally and dorsally along the third ventricle in a parasagittal plane. Numerous immunostained fibers were found close to the wall of the ventricles; some of them appeared to reach the cerebrospinal fluid through the ependymal cell layer. Some fibers were also observed in the optic tract, and the lowest density was found in the supraoptic and paraventricular nuclei. These results should be useful for developing further physiological studies on the role of histaminergic neuronal systems in sheep.

The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones. Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture. Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry. LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant. Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons. Cell morphology was very similar to that which has been described in the adult sheep brain. These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode. This mode may represent a useful tool to study LHRH neurones directly in the sheep.

In all vertebrate species studied, the main central population of GnRH neurones, which produces the final messages regulating reproduction, originates outside the brain. Early during fetal life, they appear in the olfactory placode epithelium and then migrate toward the base of the telencephalon in close association with the nervus terminalis, penetrate the brain within the nervus terminalis roots, reach their final locations and eventually grow axons toward their targets. Only part of this process is documented in ruminants. In the sheep fetus, the olfactory placode develops between day 22 and day 26 of gestation, but the first GnRH-immunoreactive neurones have been detected only at day 35, associated with the extracerebral part of the nervus terminalis. During the next 30-40 days, the GnRH neuronal systems progressively invade the brain. In both sexes, most of the development, in terms of distribution and morphology of the neurones, appears to be completed by the middle of gestation (term being on day 145). On day 85 GnRH-immunoreactive neuronal systems of male and female fetuses have also been reported to be very similar to GnRH neuronal systems of adult females. Attention should now be focused on the earliest developmental steps.

The preoptic area contains most of the luteinizing hormone releasing hormone immunoreactive neurons and numerous monoaminergic afferents whose cell origins are unknown in sheep. Using tract tracing methods with a specific retrograde fluorescent tracer, fluorogold, we examined the cells of origin of afferents to the medial preoptic area in sheep. Among the retrogradely labeled neurons, immunohistochemistry for tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and serotonin was used to characterize catecholamine and serotonin fluorogold labeled neurons. Most of the afferents came from the ipsilateral side to the injection site. It was observed that the medial preoptic area received major inputs from the diagonal band of Broca, the lateral septum, the thalamic paraventricular nucleus, the lateral hypothalamus, the area dorsolateral to the third ventricle, the perimamillary area, the amygdala, and the ventral part of the hippocampus. Other numerous, scattered, retrogradely labeled neurons were observed in the ventral part of the preoptic area, the vascular organ of the lamina terminalis, the ventromedial part of the hypothalamus, the periventricular area, the area lateral to the interpeduncular nucleus, and the dorsal vagal complex. Noradrenergic afferents came from the complex of the locus coeruleus (A6/A7 groups) and from the ventro-lateral medulla (group A1). However, dopaminergic and adrenergic neuronal groups retrogradely labeled with fluorogold were not observed. Serotoninergic fluorogold labeled neurons belonged to the medial raphe nucleus (B8, B5) and to the serotoninergic group situated lateral to the interpeduncular nucleus (S4). In the light of these anatomical data we hypothesize that these afferents have a role in the regulation of several functions of the preoptic area, particularly those related to reproduction. Accordingly these afferents could be involved in the control of luteinizing hormone releasing hormone (LHRH) pulsatility or of preovulatory LHRH surge.

The intracranial course of the terminal nerve was studied in the sheep. Luteinizing hormone-releasing hormone (LHRH) immunohistochemistry and acetylcholinesterase (AChE) activity detected histochemically revealed the existence of a major bundle of neural fibers coursing along the anterior cerebral artery, from the olfactory tubercle to the olfactory bulbs, at the surface of which the fibers spread out in a dense plexus, before reaching the cribiform plate. There were ganglia along the nerve, which contained at least two separate populations of identified cells: one possessing AChE activity and the other presenting LHRH immunoreactivity.

Immunohistochemical double-labellings of luteinizing hormone-releasing hormone and neuropeptide Y, serotonin, tyrosine hydroxylase and dopamine-beta-hydroxylase, were performed in the preoptic area at the level of the organum vasculosum laminae terminalis. The observed neuropeptide Y-, serotonin-, tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive fibres presented large varicosities when they were found within close proximity to luteinizing hormone-releasing hormone-containing perikarya. The fact that neuropeptide Y- and dopamine-beta-hydroxylase-immunoreactive fibres exhibited the same morphological characteristics and the comparison of the distribution of these two fibre populations raised the possibility of the co-localization of neuropeptide Y and dopamine-beta-hydroxylase in the same fibres. Dopamine-beta-hydroxylase- and tyrosine hydroxylase-immunoreactive fibres were morphologically different, suggesting that both dopaminergic and noradrenergic fibres could contact luteinizing hormone-releasing hormone-containing perikarya. Serotonin-immunoreactive fibres were also found close to the perikarya and to the proximal dendrite of the luteinizing hormone-releasing hormone-containing neurons. This study showed only putative sites of interactions between chemically identified fibres and luteinizing hormone-releasing hormone-containing neurons. Further ultrastructural immunocytochemical investigations are needed to ascertain the existence of synaptic contacts.