Search

Comparison of the localization of immunoreactivities which were obtained with antibodies to ?-endorphin, ?-neoendorphin, Leu- and met-enk and dynorphin with those obtained earlier using antisera to ?-endorphin, somatostatin, CRF, GRF, ?-MSH, CCK/gastrin, VIP and substance P (Dhainaut-Courtois et al., 1985a,b) show the particular status of opiate-like substances in Nereis. Indeed, immunoreactivities for vertebrate opiates were observed quite exclusively in the brain, on paraffin embedded as well as on frozen tissue sections. Furthermore, immunoreactive neurones for dynorphin occur only in light green cells (C II cells) located in nuclei 20; axons may be followed to the infracerebral region (ICR), a neurohemal area where immunoreactive nerve fibres for Leu-enk and met-enk are also present. The question to be answered now is whether the compounds immunologically related to vertebrate opiates are authentic Nereis neurohormones released in the blood vessel or the coelom or if they act only as neuromodulators at the ICR level by controlling the activity of classical neurosecretory cells or infracerebral cells. At present, a coexistence in a single neurone of an uncharacterized Nereis neurohórmone and an opiate-like substance may also be postulated. In this study, colocalization of materials immunologically related to CCK/gastrin and GRF or CCK/gastrin and 5-HT were also demonstrated. In addition to this, the use of antibody to 5-HT allowed us to confirm previous results which were obtained with radioautography and histofluorescence methods (Dhainaut-Courtois et al. 1979a,b) and to specify the distribution of this biogenic amine in the whole CNS and in the peripheral nervous system.

Adrenocorticotropin and gonadotropin producing cells were localized in the adenohypothysis of normal Lerots by using anti-beta 1-24 ACTH, anti-LH, anti-LH beta, anti-PMSG antisera. In order to study their fine structure two techniques were employed: a superimposition technique which consists of detailed comparisons between the same cells in light, fluorescence and electron microscopic preparations and an immunocytochemical technique on ultra-thin sections using the peroxidase anti-peroxidase complex technique. The superimposistion technique allows an excellent description of cell ultrastructure of individually identified cells of each type. With this method we were able to describe the corticotropin secreting cells as lucent cells with electron dense granules ranging in size from 2500 to 3500 A. The gonodotropin secreting cells are darker and their granules are about 2000 A in diameter.

An ultrastructural study of enkephalin-immunoreactive perikarya forming a hypothalamic nucleus, the magnocellular dorsal nucleus (MDN), was carried out in colchicine-treated guinea pigs. These large cells usually contain a well-developed rough endoplasmic reticulum, a prominent Golgi complex and neurosecretory granules approximately 100 nm in diameter. These granules are not numerous and sometimes appear preferentially located in the periphery of the cytoplasm. Using the PAP method on ultrathin sections, the immunostaining obtained with anti-enkephalin antisera is essentially confined to the granules but also to some ribosomes linked to membranes of the rough endoplasmic reticulum. These images suggest that the cells of the MDN synthetize the enkephalin-related peptide. Around the perikarya many nerve terminals are observed and many of them make axosomatic synaptic contacts. The exact nature of these nerve endings as well as the projection of these enkephalin-containing cells remain to be established.

In mammalian brain, dopaminergic (DA) cell bodies located in the ventral mesencephalon give rise to meso-cortical, meso-limbic and meso-striatal systems. Among these, the meso-cortical DA pathway is particularly involved in the processing of emotional and cognitive responses. We demonstrate that the rat meso-cortical neurons specifically contain, in addition to DA, another transmitter, Neurotensin. If this co-localization exists in man, it may provide an anatomical substratum for the biological theory of schizophrenia as well as an indication that potential anti-psychotic drugs which act differentially on the DA ascending transmissions can be developed.

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the last case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected [3H]5-HT. At the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle whereas fibers only rarely penetrated toward its ventral portion. By the 9th postnatal day, extensive networks of supraependymal fibers became noticeable in the lateral ventricles and in the dorsal portion of the 3rd ventricle. In addition, a number of 5-HT fibers surrounded the infundibular and preoptic recesses and sometimes penetrated to the ventricular cavity. The functional significance of hypothalamic and ventricular 5-HT as a modulator of either the growth and differentiation of the developing brain or of some specific neuroendocrine functions is discussed.

CSF is the most recently isolated and synthetized hypothalamic releasing factor in vertebrates. The use of an antiserum raised against CRF made it possible to reveal, in an oligochaetous annelid, immunoreactive cells in the suboesophageal ganglion and in all ganglia of the ventral nerve cord. Ten cells were numbered in each ganglion and each presents a pattern which is identical from one somite to the next. It thus appears that in the case of CRF as well as other vertebrate neuropeptides, there exist immunologically related neurosecretory products in the central nervous systems of some invertebrates.

In the brains of colchicine-treated guinea pigs, antibodies directed against the C-terminal sequence of cholecystokinin octapeptide (CCK-8) and cross-reacting with gastrin, visualized a dense population of cell bodies in the arcuate nucleus, periportal nerve endings in the external layer of the median eminence and a few fibers in the neurohypophysis. In the adenohypophysis, at least the major part of corticomelanotrophs were labelled. Immunostainings are compared to those reported in the rat.

This study attempted to visualize serotonin (5-HT)-immunoreactive (IR) neurons in the hypothalamus of intact fetuses (E18) and neonatal rats (P9) as well as after their pretreatment with some drugs interfering with the 5-HT metabolism and uptake in the serotoninergic neurons (L-tryptophan, pargyline, 5-hydroxytryptophan, fluoxetine). The 5-HT-IR cells were not observed in the hypothalamus of normal, untreated fetuses and neonatal rats. However, two large accumulations of 5-HT-IR neurons appeared in the anterolateral hypothalamus and in the dorsomedial nucleus after the subsequent injections of the monoamine oxidase inhibitor, pargyline, and the amino acid precursor of the 5-HT synthesis, L-tryptophan. A significantly less intensive reaction was observed after injections either of the second precursor of the 5-HT synthesis, 5-hydroxytryptophan instead of L-tryptophan, or pargyline only. Immunostaining, provoked by the pargyline and L-tryptophan pretreatment, was completely blocked by the injection of the specific 5-HT uptake inhibitor, fluoxetine. It means that the 5-HT immunostaining of the hypothalamic neurons may be accounted for by their capacity to take up specifically 5-HT from the environment rather than by its intraneuronal synthesis from L-tryptophan. Nevertheless, the 5-HT synthesis from 5-hydroxytryptophan in these cells cannot be excluded. The uptake of extracellular 5-HT into catecholaminergic neurons can be excluded as nomifensine, the specific inhibitor of the uptake to these neurons, did not modify the immunostaining.

Several antisera specific to neuropeptides of vertebrates were applied to sections of the earthworm Eisenia fetida Sav. Some of them were able to reveal specific neurones of the supraoesophageal ganglion: h-GHRF, CRF, Leu- and Met-enkephalin, dynorphin, substance P, CCK-8, and CCK-8S. A map of different areas or nuclei was established in the brain, taking position of reactive cells into account. Four symmetrically paired nuclei and one mediodorsal nucleus were identified and plotted. There is generally no characteristic modification correlated with maturation of the worms. However, the reaction against anti-Leu-enkephalin differs significantly according to age of Eisenia in nucleus No. 1 and particularly in nuclei No. 2 where one cell group presents specific reactivity on and after puberty. Moreover, the successive application of two antisera to the same sections after elution showed no coexistence of substances in a single neurone. By comparison with these vertebrate peptide-like containing nerve cells, the assay to anti-5-HT showed only a few reactive aminergic neurones. An attempt of correlation of cellular types with reactive perikarya is started.

The antiovulatory effect of sulpiride is mediated via inhibition of the LH-RH release: when given during 10 days in guinea pigs, sulpiride is able to promote an increase of LH-RH immunoreactive substance in anterior hypothalamic and suprachiasmatic neurons. This storage probably related with inhibition of LH-RH release, is permitted by catecholamines, since metatyrosine administration prevent this sulpiride effect. On the other hand, when administered to hamsters sulpiride is able to promote stimulation of hypothalamic catecholaminergic neurons of tubero infundibular and anterior periventricularis nuclei. The hypothalamic aminergic action upon sulpiride antiovulatory effect is discussed.

The distributions of somatocrinin (GHRF) and neuropeptide Y (NPY) immunoreactivities were compared in the infundibular area of the human hypothalamus. About 40% of the arcuate GHRF-stained perikarya were found to costain for NPY, while in the peripheral neurovascular zone of the median eminence the two stainings appeared dissociated. Therefore, it is concluded that a wide population of hypothalamic neurons producing a GHRF-related peptide does not project to the pituitary portal plexus and is thus not involved in the direct neurohemal control of the pituitary somatotropic secretion.

By use of antisera directed against met-enkephalin, leu-enkephalin, dynorphin or α-neoendorphin, immunoreactive structures were visualized in the central nervous system and proboscis of the leech Theromyzon tessulatum. Their distribution in the various compartments of the supra- and subesophageal ganglia was mapped. No correspondence could be established between the neurons containing met- or leu-enkephalin-like substances and the different types of neurosecretory cells classically described in Hirudinea. Successive localization of leu- and met-enkephalin on the same section revealed that these two peptides occur in different neurons. Only one cell located in compartment 6 of the supraesophageal ganglion was both dynorphin- and leu-enkephalin-positive. The other dynorphinimmunoreactive cells were not stained with the anti-leuenkephalin serum. The α-neoendorphin-immunopositive cells were leu-enkephalin immunonegative and vice versa.

After treatment with CB 154 of a prolactin-secreting pituitary adenoma a morphological study reveales that many cells present a positive immunofluorescent reaction with anti-ovine prolactine antibody. At the ultra structural level, many granulations and vacuolated dense bodies can be observed.

Corticotropin releasing factor (CRF)-immunoreactive (IR) perikarya, visualized by the indirect immunoperoxidase method in colchicine-pretreated cats, were localized in many discrete regions of the medulla oblongata. They were found mainly in the dorsal aspect and midline of the medulla oblongata, and more rostrally in the ventrolateral portion. Our results also demonstrated CRF-IR neurons in the rostrocaudal extent of the inferior olive, probably projecting to the cerebellar cortex via thick axons visualized along the lateral edge of the medulla. CRF-IR olivary cells were also found in the pontine cat from which the forebrain was removed, but neither in hypophysectomized nor adrenalectomized cats.

The effect of the main psychoactive component of marihuana, delta-9-tetrahydrocannabinol (THC) was investigated on the onset of puberty and on the reproductive function in female rats up to the seventy-fifth to eightieth day of life. The drug was administered i.p. at a dose of 1 microgram/kg/day between the twenty-second postnatal day and the day of vaginal opening (V.O.). The administration of THC caused a 2-day delay in V. O., and the number of ova on the day of first estrus was significantly lower in treated rats than in controls. No differences were observed in serum gonadotropin and prolactin (PRL) levels on the day of V. O. After puberty, alterations occurred in the neuroendocrine functions of animals receiving THC that persisted until adulthood: estrous cycles were irregular, the number of ova in animals killed 35-40 days after V. O. was reduced, and serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were decreased (diminution of serum FSH content was less expressed). An increase in serum PRL concentration could be demonstrated only in animals killed on the day of estrus. From these results, it might be concluded that THC administered to prepubertal rats--even in a very low dose--causes long-term irreversible alterations in reproductive functions. The importance of the fight against drug abuse is emphasized.

An antiserum raised against the synthetic human pancreatic tumour GRF (hpGRF) made it possible to localize an immunoreactive material in 100 nm granules of nerve terminals distributed in the external layer of the median eminence in Guinea-Pig and Rat. Moreover, especially in colchicine-treated Guinea-Pigs, numerous stained perikarya were observed, exclusively located in the arcuate nucleus. These results suggest that hypothalamic GRF is very similar, if not identical, to hpGRF.

Using 19 antisera raised against neuropeptides, amines or enzymes of amine biosynthesis, an immunohistochemical characterization of the sheep suprachiasmatic nucleus was performed. The most distinguishing characteristic of the sheep suprachiasmatic nucleus was the low density of serotonin- and neuropeptide Y-immunoreactive fibres; their concentration was similar to that in surrounding areas. This is different from observations in rodents but similar to those in primates. Moreover, the sheep suprachiasmatic nucleus is also characterized by a dense plexus of methionine-enkephalin-immunoreactive fibres. This has not been observed in other species. As in other species, such as rodents, the sheep suprachiasmatic nucleus contains numerous neurophysin-immunoreactive neurons and a few tyrosine hydroxylase-immunoreactive neurons. After colchicine pretreatment, many intensely stained vasoactive intestinal peptide-, vasopressin- and somatostatin-immunoreactive perikarya appeared, and more neurophysin-immunoreactive cell bodies were observed. Thus, although similarities exist among species, there are distinct differences in the neuro-chemical organization of the suprachiasmatic nucleus in the sheep and other species.

The relationship between peptidergic dystrophic neurites and paired helical filament (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SPs) was studied using combined fluorescence and bright-field optics. Cryostat sections of AD hippocampi were first stained with thioflavine-S and immunolabelled with antisera raised against different neuropeptides: somatostatin-28(1-12), somatostatin-14, neuropeptide Y, cholecystokinin (CCK) and substance P. Secondly, using the elution-restaining procedure, sections were immunolabelled with anti-tau/PHF. In immature SPs, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained fewer PHFs than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SPs without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHFs. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore, this phenomenon seems to precede any formation of PHFs in SPs.

Methionine-enkephalin (M-enk) and leucine-enkephalin (L-enk) were detected by PAP immunohistochemical method in the corpus luteum but not in the follicles of the rat ovary.

Light and electron microscope methods which can be used to examine the possibilities of co-occurrence of peptides are reviewed. The results obtained using some of them are exposed and discussed, concerning i/the light microscope demonstration of the CRF/vasopressin coexistence in rat and guinea-pig. ii/the electron microscope demonstration of the granular colocalization of LH-RH/C-terminal ACTH and somatostatin/enkephalin in the guinea-pig median eminence.

The supraependymal serotoninergic axons are identified in the Rat by immunocytochemistry. A dense plexus of fibres presenting various calibers is visualized over all the ventricular walls when tangential sections are used. By place, in the lateral ventricles, fibres emerging from an ependymal hollowing are often clustered in small groups. As soon as they penetrate the ventricles, they ramify profusely in various branches.

An attempt has been made to reveal 5-HT immunopositive (IP) neurones in the hypothalamus of intact foetuses (18th day of gestation) and neonatal (9-day) rats under normal conditions and after their treatment with drugs involved into 5-HT metabolism or into regulation of its uptake by serotoninergic neurones. 5-HTIP cells were not observed in intact animals as well as after L-tryptophan treatment, whereas two large colonies of these neurones were found in the anterio-lateral hypothalamus and dorsomedial nucleus after subsequent injections of monoamine oxidase inhibitor, pargyline, and amino acid precursor of 5-HT synthesis, L-tryptophan. Significantly less intensive reaction was observed after injections of another precursor of 5-HT synthesis, 5-hydroxytryptophan, or pargyline only. Immunostaining evoked by pargyline or L-tryptophan can be prevented by preliminary injections of fluoxetine, a specific inhibitor of 5-HT uptake by serotoninergic neurones. These data suggest that the immunostaining of hypothalamic neurones is due to their capacity to take up specifically 5-HT from the environment rather than to its intraneuronal synthesis from L-tryptophan. However, 5-HT synthesis from 5-hydroxytryptophan in the same cells may also take place. The uptake of extracellular 5-HT by catecholaminergic neurones is absent, since nomifensine, a specific inhibitor of this uptake, does not affect immunostaining.

Perikarya and nerve fibers containing a substance immunologically related to CCK-8 were detected in the nervous system of Nereis, a marine annelid worm. The most noteworthy immunostaining was seen in cell bodies, localized at the periphery of the brain, within nuclei 5, 6, 7, 9, 10, 13, 14, 15, 17, 20, 23-24. Immunoreactive fibers were also found in the neuropile without any particular grouping. Numerous other "positive" perikarya occur in the medioventral portion of the ventral nerve cord, and in the ventral and dorsal parts of the suboesophageal ganglion. In addition to the cell bodies in the cerebral external layer, immunoreactive axons were abundantly observed in the connectives between the ganglia. Moreover, our results demonstrate CCK-like staining in neurons showing variations in size and shape, and in affinity for paraldehyde fuchsin. The present results support the hypothesis that this peptide may exert a role as neurotransmitter or neuromodulator in annelids.

By the use of antisera to met-enkephalin and leu-enkephalin, enkephalin-containing structures were visualized in the lateral septum of the guinea-pig brain. The present results do not reveal immunoreactive perikarya in this area. The immunostaining is exclusively located in numerous nerve fibers and endings mostly encompassing neuronal perikarya, which accounts for the fact that at the light-microscopic level cellular somata appear to be immunostained. The immunoreactive terminals and fibers contain granules approximately 110 nm in diameter and synaptic vesicles. The origin and the functional role of these numerous enkephalin terminals remain to be established.

The distributions of perikarya exhibiting human somatocrinin (hGHRF) and neuropeptide Y (NPY) like immunoreactivities (IR) were compared in the arcuate nucleus of colchicine-treated rats. The majority of hGHRF perikarya was found to exhibit simultaneously NPY IR, while virtually no NPY IR was detected in the external layer of the median eminence. It is suggested that the majority of the hGHRF neurons of the rat arcuate nucleus constitutes a tubero-extra-infundibularly projecting system.

By means of a preembedding immuno-electron-microscopic technique, a large number of nerve endings containing a substance related to human growth hormone-releasing factor (hGRF) have been demonstrated in the paraventricular nucleus of the guinea pig. They made synaptic contacts primarily with dendritic shafts: 80% of these contacts were symmetrical. The immunoprecipitate was located mainly in large granules and around small clear vesicles. These findings suggest that a peptide related to hGRF may play a role in neural communication in the paraventricular nucleus.

The development of the hypothalamic serotoninergic system has been studied in rats from the 14th fetal till the 9th postnatal day. Serotoninergic elements were detected with immunocytochemistry using antiserum to serotonin (5-HT) and radioautography following the injections of [3H]5-HT into the cerebral ventricles. Immunocytochemistry failed to recognize hypothalamic 5-HT neurons either in fetuses or in neonatal rats. Conversely, radioautography demonstrated sparse radioactively labeled cells, undifferentiated in appearance, at the 16th and 18th fetal days. Moreover, at the 18th fetal day an accumulation of radioactively labeled typical neurons appeared in the suprachiasmatic region. By the 9th postnatal day, practically all radioactively labeled cells looking like highly differentiated neurons were concentrated in the dorsomedial nucleus. As to nerve fibers, a small number of 5-HT-immunoreactive axons arising from the raphe nucleus were first detected in the hypothalamus at the 16th fetal day. Two days later, both 5-HT-immunoreactive and radioactively labeled fibers became widely distributed through the hypothalamus with especially high concentrations in and around the optic chiasma. By the 9th postnatal day, the frequency of 5-HT fibers increased considerably both in the anterior and middle hypothalamus. Some 5-HT fibers were apposed to unlabeled neurons and others abutted on the capillaries either in the organum vasculosum of the lamina terminalis or in the median eminence. The present morphologic study suggests that during the perinatal period the hypothalamic 5-HT system becomes developed enough to be involved in the regulation of some neuroendocrine functions.

Alzheimer's disease is characterized by the presence of two argentophilic histopathologic brain lesions: neurofibrillary tangles (NFT) and senile (neuritic) plaques (SP). NFT consist of large perikaryal masses of abnormal cytoplasmic fibers, most of which have the ultrastructural appearance of pairs of intermediate-sized (10 nm) filaments wound into a double helix and named Paired Helical Filaments (PHF). PHF also occur within the degenerating neurites of SP. The insolubility of PHF in strong detergents is turned to account for their isolation. We have isolated these structures from an Alzheimer brain and raised antibodies against PHF. The anti-PHF antibodies detected specifically NFT and SP on nervous tissue sections of Alzheimer brains, and also NFT in the hippocampus of normal aged brains. The stainings of NFT and SP by the anti-PHF or the classical Bodian silver staining technique were compared. The immunohistochemical method is more precise, more reproducible, more specific and will be of great interest for the quantification of these structures, specially when they are in minor quantities particularly in the atypical disease. Furthermore, the anti-PHF antibodies did not visualize neurofilament containing structures, and did not react with neurofilament protein subunits on immunoblots. These results are compared to those reported in the literature.

Antisera raised against the synthetic CRF allowed us to visualize by immunohistochemistry, the hypothalamic neurons which contain this peptide or a related substance. The perikarya are distributed in the paraventricular nucleus. They likely represent the origin of the numerous nerve terminals localized around the vessels of the hypophyseal portal plexus. In guinea-pig and rat after adrenalectomy and in the intact lerot, the pericapillary terminals reacting for CRF, also contain a substance related to bovine neurophysin. Moreover, in the guinea-pig, adrenalectomy appeared to induce a CRF-immunostaining in the LH-RH terminals.

Vasopressin- and oxytocin-immunoreactive cells have been demonstrated in the brain of the leech Theromyzon tessulatum. A mapping of their localization in the different compartments of the brain has been undertaken. The cells immunohistochemically identified have been compared to previously described cell types defined by classical staining methods for neurosecretory material. Preliminary results obtained with high performance liquid chromatography confirm the presence in brain homogenates of substances with chromatographic properties similar to that of vertebrate nonapeptides. The possible role of these vasopressin- and oxytocin-like substances in osmoregulation is discussed.

By using immunohistoenzymatic techniques, cells of 4 pituitary adenomas are revealed with beta MSH, alpha and beta endorphins antisera but do not react with 1--24 and 17--39 ACTH antisera. These data suggest that adenomatous cells are able to synthesize and LPH-like molecule without ACTH. The levels of plasmatic LPH and beta MSH, studied by RIA, stay normal. These results are discussed.

Immature female rats were induced to ovulate by an injection of pregnant mare serum: melatonin, intraperitonealy injected, induce an increase of LH-RH immunoreactive neurons in the hypothalamus. These neurons are localized in septo preoptic, infundibular and premammillary areas.

The relationship between peptidergic neurites and paired helical filaments (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SP) was studied using combined fluorescence and bright field optics. Cryostat sections of AD hippocampi were first stained by thioflavine-S and immunolabeled with antisera raised against different neuropeptides: somatostatin 28(1-12) (som 28(1-12)), somatostatin 14 (som 14), neuropeptide Y (NPY), cholecystokinin (CCK) and substance P (sP). Secondly, using the elution-restaining procedure, sections were immunolabeled with anti-tau/PHF. In immature SP, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained less PHF than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SP without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHF. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore this phenomenon seems to precede any formation of PHF in SP.

Morphological studies of 3 pituitary "chromophobe" adenoma in patients without hypercorticism show positive immunofluorescenct cells with anti alpha, 17-39 ACTH antibody which are not revealed with anti beta, 1-24 ACTH antibody. These cells observed at the ultrastructural level contain secretory granules of 1,000 to 1,500 A in diameter. The existence in these cells of a substance having an immunological relationship with ACTH without biological activity is discussed.