Primary culture of GnRH neurons derived from the sheep embryo olfactory placode.
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Animals↗
Biomedical subjects
M Caldani
Publications and source records attributed to M Caldani.
Localization and quantification of melatonin receptors in the diencephalon and posterior telencephalon of the sheep brain.
In an attempt to better understand the mechanisms by which melatonin controls neuroendocrine activity, we tried to define with accuracy the brain areas where the density of melatonin receptors is the highest in sheep and to establish their characteristics. The specific labelling of 125I-melatonin was first revealed by autoradiography on brain sections of the posterior telencephalon and diencephalon in three ewes. The extent and position of the five structures where the binding was found to be the highest (i.e., the pars verticalis and pars horizontalis of the nucleus tractus diagonalis, the septal area, the bed nucleus of the stria terminalis, and the ventromedial hypothalamic area) were then accurately defined by image analysis. In comparison to the landmarks given by image analysis, photographs of coronal sections of another ewe permitted the accurate definition of the limits of the structures to be punched in a second step. In six ewes, each of the five structures previously identified were punched from frozen coronal sections and binding of 125I-melatonin to membrane preparations was studied individually by Scatchard analysis. The correlation coefficient between the B/F ratio and binding (B) was in the range of 0.96-0.98, indicating that a precise quantification was possible in these different structures. The Bmax was the highest in the bed nucleus of the stria terminalis, the septal area, and the ventromedial hypothalamic area (1.38, 1.25, and 0.95 fmol/mg protein, respectively). All Kd values were less than 10 pM and the Hill coefficient was close to 1, indicating the presence of a single class of receptor to 125I-melatonin. These results indicate the reliability of a method used to measure with accuracy low concentrations of melatonin receptors in brain structures. In addition, the ventromedial hypothalamic area was found to be rich in melatonin receptors. This region is known to be involved in the central gonadotrope control in sheep.
Animals↗
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.
Animals↗
Relationship between the number of immunostaining gonadotropes and the plasma concentrations of gonadotrophins in ewes with and without the FecBB gene.
Booroola ewes possess a major gene, FecBB, that influences their ovulation rate (number of ovulations per oestrous cycle). Homozygous (BB) carriers of the FecBB gene have higher plasma concentrations of FSH and sometimes LH relative to the non-carriers (++). The aim of this study was to determine whether the plasma concentration differences in FSH or LH between the genotypes were due to a greater number of FSH beta- or LH beta-immunostaining cells in the anterior pituitary gland of BB ewes during the luteal phase of the oestrous cycle. No differences were found between the BB (n = 7) ewes and ++ (n = 8) ewes in total number of pituitary cells, pituitary volume, numbers or diameters of FSH beta- or LH beta-immunostaining cells, notwithstanding significantly higher concentrations of immunoreactive plasma FSH (P < 0.001) but not LH in BB compared with ++ animals. Significant linear relationships were found within each genotype between plasma FSH and number of FSH beta-immunostaining cells. No such relationship was found for plasma LH and number of LH beta cells. For the FSH relationship, the slopes of the regression lines were the same. It is hypothesized that the differences in plasma concentration of FSH between the genotypes is due to a greater output of FSH per pituitary cell in the BB animals.
Animals↗
Bilateral lesions of the suprachiasmatic nuclei alter the nocturnal melatonin secretion in sheep.
The hypothalamic suprachiasmatic nuclei (SCN) constitute both the biological clock of many circadian rhythms, and the first relay in the transmission of light cues from the retina to the pineal gland, which releases, via nocturnal melatonin secretion, an endocrine expression of the daylength. The aim of the present work was to investigate the precise role of the SCN in the entrainment of the nocturnal rhythm of melatonin (MEL) in sheep. Bilateral lesions of the SCN were performed via a transsinusal surgical approach in 10 adult rams submitted to a constant photoperiod (16:8D). Lesioned rams were compared to 4 sham and 2 control animals. Blood samples were collected 8 days before, 8 days after, and one month after surgery. Plasma MEL levels were estimated using direct radioimmunoassay. At the end of the experiment, histology and immunohistochemistry of the suprachiasmatic area were performed, and the extent of lesions was evaluated using a computerized image analysis system. Six rams exhibited a complete lesion of the SCN, and in the four remaining animals, the lesions were restricted to the anterior part of the SCN. For all animals, the nocturnal melatonin secretion was altered, but depending on the extent of the lesion, two types of results were observed: Eight days after surgery, in 3 of the 4 rams bearing anterior lesions of the SCN (SCNx-), a nocturnal increase in melatonin secretion still occurred at dusk, but the duration of this secretion extended beyond the end of the night. One month after surgery, melatonin profiles were once again normal, as compared to sham animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Animals↗
The suprachiasmatic nucleus in the sheep: retinal projections and cytoarchitectural organization.
The retinal innervation, cytoarchitectural, and immunohistochemical organization of the suprachiasmatic nucleus (SCN) was studied in the domestic sheep. The SCN is a large elongated nucleus extending rostrocaudally for roughly 3 mm in the hypothalamus. The morphology is unusual in that the rostral part of the nucleus extends out of the main mass of the hypothalamus onto the dorsal aspect of the optic chiasm. Following intraocular injection of wheat-germ agglutinin-horseradish peroxidase or tritiated amino acids, anterograde label is distributed throughout the SCN. Retinal innervation of the SCN is bilaterally symmetric or predominantly ipsilateral. Quantitative image analysis demonstrates that, although the amount of autoradiographic label is greatest in the ventral and central parts of the nucleus, density varies progressively between different regions. In addition to the SCN, retinal fibers are also seen in the medial preoptic area, the anterior and lateral hypothalamic area, the dorsomedial hypothalamus, the retrochiasmatic area, and the basal telencephalon. Whereas the SCN can be identified using several techniques, complete delineation of the nucleus requires combined tract tracing, cytoarchitectural, and histochemical criteria. Compared with the surrounding hypothalamic regions, the SCN contains smaller, more densely packed neurons, and is largely devoid of myelinated fibers. Cell soma sizes are smaller in the ventral SCN than in the dorsal or lateral parts, but an obvious regional transition is lacking. Using Nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining, the SCN can be clearly distinguished in the rostral and medial regions, but is less differentiated toward the caudal pole. Immunohistochemical demonstration of several neuropeptides shows that the neurochemical organization of the sheep SCN is heterogeneous, but that it lacks a distinct compartmental organization. Populations of different neuropeptide-containing cells are found throughout the nucleus, although perikarya positive for vasoactive intestinal polypeptide and fibers labeled for methionine-enkephalin are predominant ventrally; neurophysin-immunoreactive cells are more prominent in the dorsal region and toward the caudal pole. The results suggest that the intrinsic organization of the sheep SCN is characterized by gradual regional transitions between different zones.
Acetylcholinesterase↗
A new surgical approach to the ram suprachiasmatic nuclei.
In mammals, lesions of the suprachiasmatic nuclei (SCN) cause alterations in several biological rhythms. Investigation of the role of the ovine SCN requires complete bilateral lesions of the nuclei. However, the elongated shape, location and horizontal orientation of this hypothalamic structure prohibit a classical stereotaxic vertical approach. We present here a new trans-sinusal surgical technique to reach the SCN for lesion or other studies, which avoids damage to hypothalamic or other structures. The surgical procedure allows direct viewing of the SCN, located dorsal to the optic chiasm on the rostro-ventral surface of the brain. The orientation of the access tunnel is roughly aligned with the nearly horizontal main axis of the nuclei. An example of the use of such a surgical procedure is described: the bilateral destruction of the SCN. Following surgery, the extent of lesions was assessed by histology, showing complete destruction of the SCN. In order to demonstrate the physiological effects of the lesions, melatonin secretion profiles were followed over 48 h periods and compared before and after surgery. Bilateral SCN lesions induced alterations in the rhythm of melatonin secretion: levels were no longer correlated with light/dark cycles.
Animals↗
Release of chicken luteinising hormone-releasing hormone-I (cLHRH-I) by mediobasal hypothalamus in the cockerel: validation of an incubation system and effect of excitatory amino acids.
An in vitro system for the incubation of mediobasal hypothalami (MBH) of cockerels and a radioimmunoassay for chicken luteinising hormone-releasing hormone-I (cLHRH-I) were developed. The size of the hypothalamic fragment (MBH including the median eminence) and the incubation conditions used (40 degrees C, under constant shaking and gassing) preserved the physiological properties of the tissue. It was possible to maintain the MBH in vitro and to study the LHRH release for several hours. The assay proved sensitive enough (ED80 = 0.794 pmol/tube, ie 4.59 pg/ml) and sufficiently precise (within-assay coefficient of variation = 4.4% and between-assay coefficient of variation = 10.2%) to measure the amounts of peptide released in the incubation medium. The use of this incubation system provided the first evidence of the stimulating effect of the excitatory amino acids glutamate, NMDA and kainate on the secretion of cLHRH-I in birds. Our results suggest that the effect on the NMDA receptor is predominant.
Amino Acids↗
Differences in ketanserin binding in the ventromedial hypothalamus of ewes responsive or refractory to short days.
Participation of central 5HT receptors in the inhibition of LH pulsatility during refractoriness to short days (SD) in ewes has been suggested by previous in vivo studies using various 5HT-antagonist such as ketanserin. In the present study, binding of [3H]ketanserin in ewe brain sections was similar to that described in the brain of other species and could correspond with an interaction at 5HT2 receptors sites. Rosenthal analysis from the caudate nucleus was linear (Kd = 3 nM). The displacement studies from the cortex slices showed that the 5HT antagonists such as methysergide, ketanserin, cyproheptadine and spiperone competed with the labelled ligand at nanomolar concentrations whereas serotonin was less active. However, the first 3 drugs recognized different populations of binding sites. Prazosin, an alpha 1-adrenergic antagonist was inactive, but a slight inhibition of [3H]ketanserin binding was induced by pyrilamine, an H1 histaminic antagonist, within a nanomolar range. Methysergide (10(-6) M), which does not bind to H1 receptors, was therefore used to determine the nonspecific binding. Quantitative analysis of the binding of 3 nM [3H]ketanserin on sections of the ewe brain at the preopticohypothalamic level was then carried out by autoradiography. The highest binding densities were observed in the caudate nuclei (64.0 fmol/mg tissue Eq) and the mammillary bodies (52.7 fmol/mg tissue Eq) whereas intermediate or low densities were found in the other structures. The anatomical distribution of the labelling was similar to that described in other species for 5HT2 receptors. Ketanserin binding in these areas was compared between two groups of ovariectomized estradiol-treated Ile-de-France ewes, submitted to artificial short days (SD: 8L:16D), one group with a high LH pulsatility (responsive to SD) and the other one with a low LH pulsatility (photorefractory to SD). Binding densities were similar for each one of the studied regions between the two groups, except in the ventrolateral part of the mediobasal hypothalamus, where ewes exhibiting high LH pulsatility had a more than 2-fold higher binding density than those with a low LH pulsatility (mean +/- SEM, 14.6 +/- 1.4 vs. 5.7 +/- 1.0 fmol/mg tissue Eq, respectively; p < 0.0016). These results suggest that [3H]ketanserin binding sites in the ventromedial part of the mediobasal hypothalamus could be associated to the regulation of the photoperiodic inhibition of LH at the time of establishment of refractoriness to short days in the Ile-de-France ewe.
Adrenergic Antagonists↗
Role of hypothalamic catecholamines in the regulation of luteinizing hormone and prolactin secretion in the ewe during seasonal anestrus.
Separate studies with ewes have shown that catecholamines play an inhibitory role in the control of LH secretion during anestrus, and that there are structures in the lateral retrochiasmatic area (L-RCh), which could be involved in the regulation of gonadotrophin secretion. These observations have led to the hypothesis that the catecholaminergic structures in the L-RCh mediate the inhibition of pulsatile LH secretion by estradiol in the anestrous ewe. We tested this hypothesis by injecting 6-hydroxydopamine (6OH-DA) into the L-RCh of ovariectomized ewes during the anestrous season, and comparing the secretion of LH and prolactin in these animals with that in sham (injected with vehicle) and control (no injection) animals, in the presence and absence of exogenous estradiol. Finally, the effectiveness of the toxin was assessed by immunocytochemical techniques. When the ewes were treated with estradiol, LH pulse frequency was significantly lower in the controls (mean 1.1 pulses/4 h) and shams (0 pulses/4 h) than in the ewes treated with 6OH-DA (3.1 pulses/4 h). When the estradiol implants were removed, the frequencies increased to 5.1 pulses/4 h for the controls and 5.7 pulses/4 h in the ewes treated with 6OH-DA. These were not significantly different. Plasma prolactin levels were significantly reduced by 6OH-DA treatment. The 6OH-DA ewes recovered their response to estradiol by 14 weeks after the injection. The anatomical study at the end of the experiment revealed a difference between treated and control ewes of only 15% in the numbers of dopaminergic cells in the L-RCh.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Animals↗
Immunocytochemical identification of luteinizing hormone-releasing hormone-positive fibres and terminals in the olfactory system of the rat.
Luteinizing hormone-releasing hormone immunoreactivity was studied in the olfactory system of the rat in combination with acetylcholinesterase histochemistry. Neuronal perikarya containing luteinizing hormone-releasing hormone lie in the medial septal nucleus, the vertical limb of the diagonal band of Broca, the olfactory tubercule and the ganglionated plexus of the terminal nerve. Labelled fibres spread in the superficial layers of the main and accessory olfactory bulbs, some encompassing the strongly acetylcholinesterase-positive atypical glomeruli. Others are observed on the medial side of the bulb, running along the terminal nerve bundles and ganglia. These fibres join the vomeronasal nerve branches and proceed distally towards the nasal cavity. In the septal submucosa, immunoreactive fibres are partly associated with the terminal nerve network. Conspicuous endings filled with luteinizing hormone-releasing hormone are observed on blood vessels of the olfactory mucosa. Such well-differentiated terminals might be the neurosecretory afferents of a new neurohemal area. Immunoreactive terminals are also observed around the excretory ducts of the anterior medial glands. We have failed to observe any labelled fibres in the olfactory and vomeronasal epithelia. The results of the present study are discussed with respect to possible functional interpretations. It is suggested that significant amounts of luteinizing hormone-releasing hormone could be released in the submucosal capillaries in spite of the scarcity of immunoreactive fibres. Similar afferents could also modulate the secretory activity of some nasal glands. Synaptic events involving the neuropeptide might occur in the olfactory bulb, particularly in atypical glomerular areas previously characterized by their high acetylcholinesterase content. Finally, no anatomical support for a chemosensory function of fibres containing luteinizing hormone-releasing hormone has been brought out by our work.
Acetylcholinesterase↗
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.
Acetylcholinesterase↗
Absence of correlations between glutamine-synthetase activity and dysmyelination-associated modifications of astroglia in the brain of murine mutants.
Glutamine Synthetase (GS) activity was investigated in cerebellum (ce), cerebral cortex (cc), olfactory bulb (ob), and medulla oblongata (mo) of murine dysmyelinating mutants for correlations with modifications of astroglia associated with genetic dysmyelination. One of these mutants, jimpy, develops a strong gliosis throughout the CNS. The other three mutants: shiverer, mld, and quaking, exhibit various astrocytic responses to dysmyelination, but reduced gliosis if any. Comparison between CNS areas in control animals showed a higher GS activity in the olfactory bulb than in the cerebral cortex, medulla, and cerebellum. The developmental patterns of GS activity were similar in mutants and in controls in all four areas investigated. Data on Jimpy suggest that GS activity is not associated with reactive astrocytes.
Animals↗
Astroglial cells: glucocorticoid target cells in the brain.
Glutamine synthetase (GS), an enzyme localized in astroglial cells in the brain, is directly implicated in brain detoxification. An ontogenic study of GS activity was performed in homogenates from four distinct brain areas in comparison with the respective astrocytes obtained in primary cultures. GS was induced by hydrocortisone in the astrocytes of all brain areas studied; only cerebellum and cerebral hemisphere astroglial cells had a higher specific activity when compared with the corresponding homogenates. N6O2-Dibutyryl adenosine 3',5'-cyclic monophosphate (dBc AMP), insulin, soluble brain factors, and noradrenaline (NA) were also able to modulate GS activity. Brain factors as well as dBc AMP interfered with hydrocortisone induction of GS. Regulation by hydrocortisone paralleled the variation in its concentration in brain during development. We conclude that astroglial cells are target cells for glucocorticoids, which may modulate ammonia detoxification in these cells.
Animals↗
Glutamine synthetase activity during mouse brain development.
The specific activity of glutamine synthetase (GS) in mouse brain was 2-fold higher in the olfactory bulbs than in other regions. After birth, the specific activity of GS increased more rapidly in medulla oblongata and in olfactory bulbs, than in cerebral and cerebellar cortex. The activity of GS in primary cultures of brain hemispheres increased more slowly than in homogenates of whole brains. However, when astroblasts were treated in vitro with glucocorticoids or mouse brain extracts, GS activity reached 4 times the level measured in the homogenate of an adult mouse brain. We conclude that levels of GS activity may relate to the maturation of astrocytes, and propose that GS may be used as a marker of astrocytic maturation.
Animals↗
GnRH complementary peptide antibodies: outcome in GnRH receptor immunoanalysis.
The aim of this study was to obtain gonadoptropin-releasing hormone (GnRH) receptor antibodies of high affinity for receptor immunoanalysis. According to the complementary peptide theory, complementary nucleic acid segments encode for the hormone ligand and the receptor binding site, respectively. On this premise, we used as immunogen, GnRH complementary peptide [N-terminal]Ser-Arg-Ala-Gln-Ser-Ile-Gly-Pro-Val-Leu conjugated with a carrier protein. High antibody titers were obtained in rabbits, rats and mice. Our antisera recognized the hydrophobic middle part of the GnRH complementary peptide. A band of protein with a molecular weight similar to that of the GnRH receptor (60 kDa) was specifically detected by immunoblot of solubilized rat pituitary membranes with the highest titering rabbit antiserum. In bioassays on sheep pituitary cells in vitro, some antisera inhibit basal or GnRH-induced LH secretion. In order to elicit antibodies of high affinity, we used a selective receptor assay on rat brain and pituitary sections where the ligand was the labeled agonist Des-Gly10-D-Ala6 GnRH. None of the highest titering antisera prevented the binding of such a high affinity ligand. The complementary peptide approach thus appears not to be optimal for obtaining high affinity antibodies against the GnRH receptor binding site.
Amino Acid Sequence↗