Differential effects of oxytocin on olfactory, hippocampal and hypothalamic neurogenesis in adult sheep.

New neurons are continuously added in the dentate gyrus of the hippocampus, the olfactory bulb and the hypothalamus of mammalian brain. In sheep, while the control of adult neurogenesis by the social environment or the photoperiod has been the subject of several studies, its regulation by intrinsic factors, like hormones or neurotransmitters is less documented. We addressed this question by investigating the effects of central oxytocin administration on hippocampal, olfactory and hypothalamic neurogenesis. Endogenous markers, Ki67, Sox2 and DCX were used to assess cell proliferation, progenitor cells density and cell survival respectively in non-gestant ewes receiving a steroid treatment followed by intracerebroventricular injections of either oxytocin or saline. The results showed that oxytocin treatment significantly decreases the density of neuroblasts in the olfactory bulb, increases the density of neuroblasts in the ventromedian nucleus of the hypothalamus while no change is observed in both ventral and dorsal dentate gyrus. In addition, no change in the density of progenitor cells is found in the three neurogenic niches. These findings show for the first time that in females, oxytocin can regulate adult neurogenesis by acting on neuroblasts but not on progenitor cells and that this regulation is region specific.

Evidence that histaminergic neurons are devoid of estrogen receptor alpha in the ewe diencephalon during the breeding season.

In sheep as in rat, it has been highly suggested that neuronal histamine (HA) participates to the estradiol (E2)-induced GnRH and LH surges, through H1 receptor. With the aim of determining if E2 could act directly on HA neurons, we examined here whether HA neurons express estrogen receptor alpha (ERα) in the ewe diencephalon during the breeding season. We first produced a specific polyclonal antibody directed against recombinant ovine histidine decarboxylase (oHDC), the HA synthesizing enzyme. Using both this anti-oHDC antibody and an anti-ERα monoclonal antibody in double label immunohistochemistry, we showed that HA neurons do not express ERα in diencephalon of ewes with different hormonal status. This result diverges from those obtained in rat, in which around three quarters of HA neurons express ERα in their nucleus. This discrepancy between these two mammal species may reflect difference in their neuronal network.

Neuroanatomical distribution of the orphan GPR50 receptor in adult sheep and rodent brains.

GPR50, formerly known as melatonin-related receptor, is one of three subtypes of the melatonin receptor subfamily, together with the MT(1) and MT(2) receptors. By contrast to these two high-affinity receptor subtypes and despite its high identity with the melatonin receptor family, GPR50 does not bind melatonin or any other known ligand. Specific and reliable immunological tools are therefore needed to be able to elucidate the physiological functions of this orphan receptor that are still largely unknown. We have generated and validated a new specific GPR50 antibody against the ovine GPR50 and used it to analyse the neuroanatomical distribution of the GPR50 in sheep, rat and mouse whole brain. We demonstrated that GPR50-positive cells are widely distributed in various regions, including the hypothalamus and the pars tuberalis of the pituitary, in all the three species studied. GPR50 expressing cells are abundant in the dorsomedial nucleus of the hypothalamus, the periventricular nucleus and the median eminence. In rodents, immunohistochemical studies revealed a broader distribution pattern for the GPR50 protein. GPR50 immunoreactivity is found in the medial preoptic area (MPA), the lateral septum, the lateral hypothalamic area, the bed nucleus of the stria terminalis, the vascular organ of the laminae terminalis and several regions of the amygdala, including the medial nuclei of amygdala. Additionally, in the rat brain, GPR50 protein was localised in the CA1 pyramidal cell layer of the dorsal hippocampus. In mice, moderate to high numbers of GPR50-positive cells were also found in the subfornical organ. Taken together, these results provide an enlarged distribution of GPR50 protein, give further insight into the organisation of the melatoninergic system, and may lay the framework for future studies on the role of the GPR50 in the brain.

Functional characterization of polymorphic variants for ovine MT1 melatonin receptors: possible implication for seasonal reproduction in sheep.

In seasonal breeding species, the gene encoding for the melatonin MT(1) receptor (oMT(1)) is highly polymorphic and numerous data have reported the existence of an association between an allele of the receptor and a marked expression of the seasonality of reproduction in ewes. This allele called "m" (previously named "-" allele) carries a mutation leading to the absence of a MnlI restriction site as opposed to the "M" allele (previously named "+" allele) carrying the MnlI restriction site (previously "+" allele). This allows the determination of the three genotypes "M/M" (+/+), "M/m" (+/-) and "m/m" (-/-). This mutation is conservative and could therefore not be causal. However, it is associated with another mutation introducing the change of a valine to an isoleucine in the fifth transmembrane domain of the receptor. Homozygous "M/M" and "m/m" animals consequently express structurally different receptors respectively named oMT(1) Val(220) and oMT(1) Ile(220). The objective of this study was to test whether these polymorphic variants are functionally different. To achieve this goal, we characterized the binding properties and the transduction pathways associated with both variants of the receptors. Using a pharmacological approach, no variation in binding parameters between the two receptors when transiently expressed in COS-7. In stably transfected HEK293 cells, significant differences were detected in the inhibition of cAMP production whereas receptors internalization processes were not different. In conclusion, the possibility that subtle alterations induced by the non conservative mutation in "m/m" animals might modify the perception of the melatoninergic signal is discussed in the context of melatonin action.

Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants.

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.

Neuronal projections to the lateral retrochiasmatic area of sheep with special reference to catecholaminergic afferents: immunohistochemical and retrograde tract-tracing studies.

The retrochiasmatic area contains the A15 catecholaminergic group and numerous monoaminergic afferents whose discrete 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 retrochiasmatic area in sheep. The retrogradely labeled cells were seen by observation of the tracer by direct fluorescence or by immunohistochemistry with specific antibodies raised in rabbits or horses. Among the retrogradely labeled neurons, double immunohistochemistry for tyrosine hydroxylase, dopamine-beta-hydroxylase, and serotonin were used to characterize catecholamine and serotonin FG labeled neurons. The retrochiasmatic area, which included the A15 dopaminergic group and the accessory supraoptic nucleus (SON), received major inputs from the lateral septum (LS), the bed nucleus of the stria terminalis (BNST), the thalamic paraventricular nucleus, hypothalamic paraventricular and supraoptic nuclei, the perimamillary area, the amygdala, the ventral part of the hippocampus and the parabrachial nucleus (PBN). Further, numerous scattered retrogradely labeled neurons were observed in the preoptic area, the ventromedial part of the hypothalamus. the periventricular area, the periaqueductal central gray (CG), the ventrolateral medulla and the dorsal vagal complex. Most of the noradrenergic afferents came from the ventro-lateral medulla (Al group), and only a few from the locus coeruleus complex (A6/A7 groups). A few dopaminergic neurons retrogradely labeled with flurogold were observed in the periventricular area of the hypothalamus. Rare serotoninergic fluorogold labeled neurons belonged to the dorsal raphe nucleus. Most of these afferents came from both sides of the brain, except for hypothalamic supraoptic and paraventricular nuclei. In the light of these anatomical data, we compared our results with data obtained from rats, and we discussed the putative role of these afferents in sheep in the regulation of several specific functions in which the retrochiasmatic area may be involved, such as reproduction.

Histaminergic neurons in the sheep diencephalon.

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.

Primary cell culture of LHRH neurones from embryonic olfactory placode in the sheep (Ovis aries).

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.

Distribution of melanin-concentrating hormone (MCH)-like immunoreactivity in neurons of the diencephalon of sheep.

An immunohistochemical study with an antiserum raised against salmon melanin concentrating-hormone has demonstrated the presence of numerous melanin concentrating-hormone-immunoreactive neurons in the lateral hypothalamic areas of the sheep. The pattern of distribution of these perikarya is similar to that of rodents and primates. In sheep, however, melanin concentrating-hormone-immunoreactive neurons appeared to form two gatherings: the first is situated ventromedially to the internal capsule and the second in the dorsolateral hypothalamus. In these areas, numerous immunostained perikarya are observed. Compared to the rats, labelled neurons extended more caudally in the ventral tegmental area and more rostrally above the optic chiasma. Compared to primates, these neurons are less numerous in the periventricular area. In our study, dense networks of melanin concentrating-hormone-immunoreactive varicose fibers were observed in the supramamillary nucleus, the lateral hypothalamus, the nucleus medialis thalami and nucleus reuniens and in the bed nucleus of the stria terminalis.

Ontogeny of GnRH systems.

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.

Oestrogen receptors in the preoptico-hypothalamic continuum: immunohistochemical study of the distribution and cell density during induced oestrous cycle in ovariectomized ewe.

Oestrogen plays a key role in the regulation of the endocrine and behavioural events associated with the oestrous cycle. It is important, therefore, to know the location of neurones receptive to this steroid and to know whether their distribution varies with the oestrous cycle. We have undertaken experiments to identify the location of oestrogen receptors (ER) within the preoptico-hypothalamic continuum of ovariectomized ewes submitted to a variety of different hormone replacement regimes which mimic the different stages of the oestrous cycle. We used a monoclonal antibody to ER and detected receptors with immunohistological methods in the non-vascular part of the organum vasculosum of the lamina terminalis, the lateral septum, the medial preoptic area, the supraoptic, suprachiasmatic and arcuate (ARC) nuclei, the ventromedial hypothalamus (HVM) and in the region close to the mamillari recess. ER neurones were scarce or absent from the anterior hypothalamus and the paraventricular nucleus. The density of ER staining in the HVM, but in no other localization, was found to be higher, and in a more lateral position, during the induced luteal phase (progesterone treatment) than during the follicular phase (7 days of progesterone treatment followed by oestradiol) or in the ovariectomized female. In all areas studied, except for the ARC, the apparent surface area of the nucleus in ER immunoreactive cells varied with hormonal treatment. These data, and especially those in the HVM, contribute towards our understanding of how steroids may act in the ovine to control sexual behaviour.

Neuronal projections to the medial preoptic area of the sheep, with special reference to monoaminergic afferents: immunohistochemical and retrograde tract tracing studies.

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.

Immunohistochemical colocalization of tyrosine hydroxylase and estradiol receptors in the sheep arcuate nucleus.

In sheep, the arcuate nucleus contains numerous tyrosine hydroxylase (TH) and estradiol receptor (rE2) immunoreactive (IR) perikarya and it has been shown previously in this species that catecholaminergic neurons can mediate the gonadal steroid action on the reproductive function. In the present study, double immunohistochemical labelling with antibodies against TH and rE2 have been used to demonstrate the presence of rE2 in TH-IR neurons in the arcuate nucleus where the distribution of TH-IR and rE2-IR neurons overlap each other. Only less than 10% of all the rE2-IR perikarya presented TH immunoreactivity. It was therefore hypothesized that either such a low number of double labelled neurons can support the effects of estradiol in this area or that the effect of this steroid was indirect. In the latter case it might be first mediated by beta-endorphin neurons which have been previously described in this nucleus.

Presence of dopamine-immunoreactive cell bodies in the catecholaminergic group A15 of the sheep brain.

Antisera were raised in rabbits against dopamine or noradrenaline conjugated to thyroglobulin with glutaraldehyde. These antisera, tested in enzyme linked immunosorbent assay and immunohistochemistry specifically recognized their homologous antigens. With the aid of anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase, anti-dopamine-beta-hydroxylase, anti-dopamine, and anti-noradrenaline antisera, immunohistochemical reactions were performed on glutaraldehyde fixed sections of sheep diencephalon in order to determine the presence of dopamine in the catecholaminergic group A15. Perikarya of this nucleus were stained with anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase and anti-dopamine, but not with anti-dopamine-beta-hydroxylase or anti-noradrenaline. Both of these latter antisera stained fibers within this area. So as recently found in the rat, we could conclude that dopamine is present in group A15 of the sheep.

Anatomical relationships of monoaminergic and neuropeptide Y-containing fibres with luteinizing hormone-releasing hormone systems in the preoptic area of the sheep brain: immunohistochemical studies.

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.

LHRH-immunoreactive structures in the sheep brain.

Neural structures containing luteinizing hormone-releasing hormone (LHRH) are characterized in adult ewe and female lamb brains. Three anti-LHRH antisera are used in an immunofluorescent or immunoperoxidase method. On our preparations, all three gave the same results, expressed as number of labelled cells (about 2500 in a whole brain). It was found that 95% of the LHRH-immunoreactive cells are located in the preoptico-hypothalamic area, where cell bodies are localized mainly (50%) in the area surrounding the organum vasculosum of the lamina terminalis (OVLT); they are also found in a more anterior section of the medial part of the olfactory tubercle and the medial septum (14%), in a more posterior situation in the anterior and lateral hypothalamus (16%), and in the mediobasal hypothalamus (15%). Fibres originating in various part of the whole preoptico-hypothalamic group reach the OVLT and the median eminence. The remaining cells (5%) and fibres are found in various tel-, di-, and mesencephalic areas.

The sheep terminal nerve: coexistence of LHRH- and AChE-containing neurons.

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.