Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunocytochemical localization of vasotocin and mesotocin in the hypothalamus of lacertilian reptiles

Summary The hypothalamic magnocellular neurosecretory system of lizards was studied with the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level. It was shown that vasotocin and mesotocin are synthesized in separate neurons. The vasotocinergic as well as the mesotocinergic perikarya are of different sizes. Both cell types occur in close juxtaposition, but without a distinct pattern of distribution. The external zone of the lacertilian median eminence contains numerous immunoreactive vasotocinergic fibers and only few immunoreactive mesotocinergic fibers. The general organization of the hypothalamic magnocellular neurosecretory system of lizards, as revealed by immunocytochemistry, is essentially similar to that revealed with unspecific staining methods.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunohistochemical demonstration of alpha-1-antitrypsin in the islet cells of human pancreas

Summary Using the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level, it has been shown that, in the dipnoan preoptico-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. In the preoptic nuclei, the perikarya of these two types of neurosecretory neurons are not located preferentially. The two types of neurosecretory perikarya give rise to separate vasotocinergic and mesotocinergic axons, respectively. The dipnoan median eminence and neural lobe contain separate vasotocinergic and mesotocinergic nerve fibres, the general distribution of which is described. In the pars distalis and the pars intermedia of the hypophysis, neurohypophysial hormone-containing nerve fibres have not been found.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk OnderzoekThe authors are greatly indebted to Prof. Dr. Hyder, Department of Zoology, University of Nairobi, Kenya for kindly supplying us with the fixed material used in this study     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the median eminence of the frog hypophysis

Summary With the use of the unlabelled antibody peroxidase-antiperoxidase complex (PAP) technique at the electron microscopic level, it was shown that both the internal and the external regions of the median eminence of the frog contain separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of previous immunocytochemical studies at the light microscopic level. The mean size of the neurohypophysial hormone-containing granules in the external region of the median eminence is significantly smaller than that of the neurohypophysial hormone-containing granules in the internal region of the median eminence. No significant difference could be found between the mean granule size of the vasotocinergic and mesotocinergic fibres of either the internal or the external region of the frog median eminence.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The extravascular channel system in the rostral pituitary of Mugil cephalus (Teleostei) as revealed by use of horseradish peroxidase

Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was demonstrated that, in the amphibian magnocellular hypothalamo-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. A tendency to preferential location of the two kinds of neuronal perikarya is described. The neurosecretory perikarya are the origin of separate vasotocinergic and mesotocinergic axons. In the neural lobe, the pattern of distribution of the two types of axons is different. The coarse ventricular dendrites of both kinds of neurons are hormone-containing processes. Staining with anti-bovine neurophysin I serum suggested that the vasotocinergic and the mesotocinergic neurons synthesize different neurophysins.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Identification,in the external region of the rat median eminence,of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres

Summary Immuno-enzyme cytochemical investigations, using single and double staining techniques, showed that the external region of the rat median eminence contains separate neurophysin-vasopressin fibres and neurophysinoxytocin fibres. These neurophysin-hormone containing nerve fibres are influenced by bilateral adrenalectomy and by colchicine treatment. The external region of the median eminence of the homozygous Brattleboro rat contains neurophysin-oxytocin fibres. It does not contain immuno-reactive neurophysin-vasopressin fibres. Bilateral adrenalectomy also influences the neurophysin-vasopressin containing neurons of the suprachiasmatic nuclei. In the neurons of the parvicellular part of the rat hypothalamic paraventricular nuclei, staining for vasopressin and for oxytocin is completely absent.     

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold

Summary The distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.     

Immunocytochemical investigation of the hypothalamo-neurohypophysial system in birds

Summary The results of an immunohistochemical investigation of the hypothalamo-neurohypophysial system in several species of birds have shown that: (1) mesotocin and vasotocin are synthesized in separate neurons; (2) in all species investigated the distribution of mesotocinergic and vasotocinergic perikarya follows a common pattern; (3) the external zone of the avian anterior median eminence contains exclusively vasotocinergic nerve fibers, originating in supraoptic and ventral paraventricular regions; (4) the distribution of immunoreactive elements in the neural lobe shows a definite species-dependent pattern.     

The function of the hypophysis without preoptic neurosecretory control

Summary In a large number of adult female and male specimens of Rana temporaria, a total extirpation of the magnocellular neurosecretory preoptic nuclei was performed. Several months after operation, no regeneration of the neurosecretory nuclei had occurred. The results showed 1. that all A.F.-positive neurosecretory nerve fibres of the hypothalamo-hypophysial region originate from nerve cell bodies localized in the magnocellular preoptic nuclei; 2. that the magnocellular preoptic nuclei do not play an important part in the inhibitory control of the activity of the pars intermedia of Rana temporaria; 3. that the possible role of the magnocellular preoptic nuclei in the seasonal development of the gonads and of the secondary sexual characteristics of Rana, could be only of secondary importance; 4. that, in accordance with previous experiments, in Rana, ovulation is dependent on preoptic hypothalamic structures. Some results of the present and of other experiments can be interpreted as supporting the hypothesis of a possible dual higher control over the gonadotropic centre (Dierickx, 1965b, 1966, 1967) of the pars ventralis of the tuber cinereum.A part of these studies has been reported previously in abstract form (Dierickx, 1963a, 1963b, 1965a).     

The origin of the vasopressinergic and oxytocinergic fibres of the external region of the median eminence of the rat hypophysis

Summary The origin of the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence was investigated by means of hypothalamic lesions, adrenalectomy and immunocytochemistry. The results obtained in bilaterally adrenalectomized animals with complete, or incomplete, destruction of the suprachiasmatic nuclei showed that, at least, the great majority of the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence do not originate from the suprachiasmatic nuclei. From the observations obtained in bilaterally adrenalectomized animals with total or subtotal destruction of both paraventricular hypothalamic nuclei, it appears that the paraventricular nuclei must be the origin of (nearly) all the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence. The results strongly suggest that both types of fibres originate from all parts of the paraventricular nuclei.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk OnderzoekThe authors are much indebted to Prof. Dr. E. Kühn (Leuven) in whose laboratory the stereotactic operations were done     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Mesotocin and vasotocin in the brain of the lizard Gekko gecko

Summary The distribution of mesotocin and vasotocin was studied in the brain of the lizard Gekko gecko with antisera specific for either peptide. Both mesotocinergic and vasotocinergic perikarya are found in the paraventricular and supraoptic nuclei of the hypothalamus, whereas vasotocinergic neurons are exclusively present in the bed nucleus of the stria terminalis and in a cell group of the rhombencephalon. The distributional pattern of the mesotocinergic fibers corresponds closely to that of the vasotocinergic fibers. However, throughout the entire brain the mesotocinergic innervation is less dense than the vasotocinergic innervation. No sex differences are present in the mesotocinergic fiber system.Abbreviations acc nucleus accumbens - bst bed nucleus of the stria terminalis - bv blood vessel - dB diagonal band of Broca - dc dorsal cortex - dth dorsolateral thalamic nucleus - lc lateral cortex - me median eminence - oc optic chiasma - ot optic tract - pag periaqueductal grey - pvn paraventricular nucleus - rc rhombencephalic cell group - sep septum - son supraoptic nucleus - tect mesencephalic tectum - vth ventrolateral thalamus     

The nerve fibres controlling the gonadotropic activity of the hypophysis of Rana temporaria

Summary In 27 adult female specimens of Rana temporaria, the total interruption of the nervous pathways to the median eminence and the hypophysis was performed, without disturbing the normal blood supply of the hypophysis. In eight cases this complete interruption persisted three months after operation. In these animals there occurred no seasonal development of the eggs, ovaries and oviducts. In the 19 other operated animals a varying degree of regeneration of the interrupted nerve fibres occurred. In these animals a varying number of ripe eggs was present, but in many of them there existed a significant difference between the weight of their ovaries compared with that of the ovaries of the control animals. In all control animals the seasonal development of the ovaries and oviducts was normal. From these and from previous experiments it could be concluded that the A. F. negative nerve fibres of the median eminence, not originating from the magnocellular neurosecretory preoptic nuclei, are responsible for the normal seasonal development of the ovaries and oviducts in Rana temporaria. It must be these nerve fibres that release gonadotropic releasing factors into the blood capillaries of the median eminence. The A. F. positive neurosecretory fibres, the portal circulation of the pars distalis and the pituicytes of the median eminence play only a secondary part during this period of control of the gonadotropic activity of the hypophysis.     

Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

The topography of mesotocin and vasotocin systems in the brain of the domestic mallard and japanese quail: Immunocytochemical identification

Summary The neurosecretory systems producing mesotocin (MT) and vasotocin (VT) (the avian homologues of oxytocin and vasopressin, respectively) were characterized in the brains of the domestic mallard and Japanese quail by means of indirect immunofluorescence techniques using specific antisera. In the anterior preoptic region, including the organum vasculosum of the lamina terminalis, and at different levels of the supraoptic and paraventricular nuclei, separate mesotocin- and vasotocin-producing neurons were identified. Mesotocinergic and vasotocinergic neurons were also located in the tuberomammillary area, among the ectomammillary tract fibers. The supraoptico-neurohypophysial tract, formed by vasotocin- and mesotocincontaining axons, enters the internal zone of the median eminence and ends in the posterior lobe of the pituitary. The external zone of the rostral median eminence appears to contain vasotocin and mesotocin fibers, which terminate in close contact with the capillaries of the hypophysial portal system.With contributions by Dr. B. Kerdelhué, Laboratoire des Hormones Polypeptidiques du CNRS, 91190 Gif-sur-Yvette, France     

Further data on the origin of gonadotropin releasing hormone in the median eminence of the rat hypothalamus

Gonadotropin releasing hormone (GnRH) content of the two halves of the median eminence of the rat hypothalamus was determined by radioimmunoassay three weeks after three different unilateral knife cuts around the preoptic area. A unilateral cut in front or above the area caused a more than 25% decrease in the GnRH content of the two halves of the median eminence. A cut lateral to the preoptic region had only a slight effect similar to that observed after sham operations. The data suggest that probably more than 50% of the rat median eminence GnRH derives from outside the preoptic-suprachiasmatic region. The GnRH fibres projecting to the median eminence but arising from outside the preoptic region, probably mainly from GnRH perikarya in the limbs of the diagonal band of Broca and septum, enter this area partly from rostral and partly from above, but not from lateral direction. partly from rostral and partly from above, but not from lateral direction. Several of these fibres probably cross before terminating in the median eminence.     

Quantitative histochemical studies of the hypothalamus enzymes of acetylcholine metabolism.

The quantitative histochemical distribution of acetylcholinesterase and choline acetyltransferase activity has been measured in individual hypothalamic nuclei and median eminence, as well as in entire hypothalamic sections by a mapping technique. There was an 18-fold range of nuclear choline acetyltransferase activity with highest activities in the lateral preoptic nucleus and median eminence. There was a nine-fold range of nuclear acetylcholinesterase activity with highest activities in the lateral preoptic and magnocellular nuclei and lowest activity in the median eminence. The substantial gradients of choline acetyltransferase activity found in the hypothalamus indicate the importance of using a technique that provides an objective, permanent record of contiguous sample locations thereby allowing detailed analysis of tissue areas using, but not dependent on, anatomical boundaries.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Changes in metabolic activity of neurons in the anterior hypothalamus nuclei during hyperthermia, fever, and hypothermia

Differently directed changes in metabolic activity of anterior hypothalamic nuclei's neurons in rats during hyperthermia, fever, and hypothermia were revealed with histochemical methods. During hyperthermia, the activity of energy metabolism enzymes increased as well as RNA content in the neurons of supraoptic, paraventricular and median preoptic anterior hypothalamic nuclei. This is shown by an increase in the metabolic activity of neurons of these nuclei. Metabolic activity in neurons of median preoptic nuclei decreased and was not changed considerably in neurons of supraoptic and paraventricular nuclei during endotoxin-induced fever. The development of hypothermia was characterised by a decrease in metabolic activity of neurons of supraoptic, paraventricular and medium preoptic nuclei. It is supposed that differently directed metabolic activity changes in neurons of anterior hypothalamic nuclei during hyperthermia are connected with the mechanisms of body temperature regulation (median preoptic nuclei) and neurosecretory processes (supraoptic and paraventricular nuclei).