Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Cholinesterase activity of the hypothalamo-hypophysial neurosecretory system in rats during the ontogenetic development

Summary The hypothalamic region and the neural lobe of rats from the 16th foetal day to adult animals have been studied for acetylcholinesterase and cholinesterase activity after Karnovsky. The attention was focused on the magnocellular nuclei-supraoptic and paraventricular, the median eminence and the neural lobe. Acetylcholinesterase activity appears in the paraventricular nucleus on the 18th foetal day, i.e. prior to that in the supraoptic nucleus. Heterochronic development and heteromorphism of paraventricular neurosecretory cells have been noticed. The median eminence shows no clear acetylcholinesterase activity. There are acetylthiocholine and butyrylthiocholine positive structures in the posterior pituitary. These structures are especially pronounced in 30–47-day rats. The cholinergic mechanism of release of neurohormones from the neural lobe is suggested. The results are discussed in functional and phylogenetic aspects.     

Reaction of the hypothalamo-hypophyseal system of cats to stimulation of the cervical sympathetic nerve and the afferent fibers of the vagus nerve]

Data are presented on the functional morphology of the hypothalamo-hypophysial neurosecretory system of cats in stimulation of the cervical sympathetic nerve and of the afferent fibers of the vagus. Stimulation of the sympathetic nerve selectively activated the supraoptic nucleus and caused the discharge of the neurohormones from the posterior lobe of the hypophysis, whereas its infundibular portion contained much neurosecretory material. In response to the stimulation of the vagus all the portions of the neurohypophysis were released of the Gomori-positive substance; both the supraoptic and the paraventricular nucleus were activated.     

Hypothalamo-neurohypophysial complex in leukemia L 1210-bearing mice.

Mice of the DBA strain were inoculated with 4.5 x 10(6) L1210 ascites cells and the time response of the hypothalamo-neurohypophysial complex was studied. The amount of neurosecretory material was determined in the neuroendocrine cells of supraoptic and paraventricular nuclei, throughout the hypothalamo-neurohypophysial tract and in the neural lobe of the hypophysis. Three methods of response evaluation were employed: the amoount of neurosecretory material was estimated according to an arbitrary scale, by cytophotometry while for the hypothalamic cell-nuclei the karyometric method was used. The existence of a functional interrelation between the presence of leukemic cells and the activity of the hormonogenic sites in the hypothalamus is suggested. A clear response to the tumor inoculation was established both in the amount of stainable substance and in the nuclear volume of the secretory neurones. It is suggested that an accelerated synthesis and release of neurosecretory material in tumor recipient mice reduces renal excretion in order to compensate a dehydration caused by the production of ascitic fluid.     

Early secretory activity in the hypothalamic nuclei and neurohypophysis in the rat; determined by selective staining

Visualization of stainable material in the neural lobe of the rat provided the most reliable index of the age at which secretory activity can first be recognized, though preceded by both hypothalamic synthesis and axonal transportation. A problem of interpretation was encountered in the neural lobes of fetal and infant animals, due to different staining responses obtained during this age period, to the two methods of staining employed; chrome alum hematoxylin-phloxin and aldehyde fuchsin after oxidation by either acidified potassium permanganate or performic acid. With aldehyde fuchsin the material of the neural lobe is stainable selectively from the eighteenth day of fetal life to adulthood. With hematoxylin phloxine the first staining response also occurred in the posterior lobe but much later, at the end of the first postnatal week. The staining situation in the pars neuralis has its counterpart in the differentiating hypothalamic nuclei; complicated by the differentiation of the supraoptic nuclei some days in advance of the paraventricular nuclei. After aldehyde fuchsin staining, evidences of neurosecretory activity were present in the perikarya of the supraoptic nuclei at birth, but mature neurons were rarely seen in the paraventricularis until at least 24 hours later. Nuclei of fetal hypothalami were not studied, but the demonstration of stainable material in the fetal neural lobes constitutes circumstantial evidence of functional competence of some neurons of either one or both types of nucleus, most likely the supraoptic.     

Morphometric and electron microscopic studies of the secretory granules of neurosecretory cells of the supraoptic and paraventricular nuclei of white rats and mice]

The morphometrical and electron microscopic analysis of secretory granules in the perikaryons of neurosecretory cells of the supraoptic and paraventricular nuclei in male rats and mice has shown than in the cells of these nuclei in both species of animals there occur secretory granules of the same kind and size. Therefore this method fails to determine which of them contain oxytocin and which of them contain vasorpressin. The neurosecretory granules located in the Golgi apparatus zone are of a less size and have more osmiophilic cnetral material than the granules localized on the periphery which mainly have granular central material and are of a greater size. The distinctions in the size and type of secretory granules are associated with certain stages of their "maturation". Granular particles appear to be "swallen", more active forms of storing neurohormones. The presence of larger granular particles in the supraoptic nucleus of mice allows to suggest greater reactivity of this nucleus than in rats which is likely to be associated with a higher ability of mice, as compared with rats, to adaptate to disturbances in water-salt metabolism.     

Neurosecretory and microstructural changes in the hypothalamus of rats following administration of lithium chloride and 3H-thymidin

Adult male rats were intraperitoneally administered aqueous solution of lithium chloride (LiCl). Studies, including neurosecretory and microstructural changes within particular neurocytes in supraoptic (NSO) and paraventricular nuclei (NPV) were performed on hypothalamic sections. In the experimental rats the administered LiCl increased the level of GOMORI-positive neurosecretory material both in supraoptic and paraventricular nuclei. Great amounts of the neurosecretory material were markedly conspicuous in the above areas after 20 days of LiCl administration. Investigations carried out on cellular nuclei of particular neurocytes showed a significant enlargement of the nuclei, and statistical calculations revealed that, in comparison with the basic control, the difference was essentially significant (p less than 0.001). 3H-thymidin administration to the rats which had previously been on LiCl for 20 days demonstrated also that within supraoptic nuclei the incorporation of the isotope in cellular nuclei took a faster course than in control animals.     

Effect of fixation on the demonstration of neurophysin and "Gomori-positive" substances in neurosecretory granules of the rat hypothalamus.

Proteins reacting with neurophysin antibodies and "Gomori-positive" substances were demonstrated histochemically in hypothalamic neurosecretory material of normal and bilaterally adrenalectomized rats after two different fixations: a) picric acid-formalin (PAF) for 7 days at 37 degrees C; b) Bouin's fluid for 20 h at 4 degrees C. After PAF-fixation anti-neurophysin reactive neurosecretory granules are found in all parts of the supraoptico-hypophysial system and in the suprachiasmatic nucleus of normal and adrenalectomized animals. In the latter they can additionally be demonstrated in the outer layer of the median eminence. Amount and distribution of "Gomori-positive" substances correspond to those described for the immunoreactive material, except for the suprachiasmatic nucleus, in which the substances can not be detected; Following fixation in Bouin's fluid the immunohistochemical reactions are unchanged whereas the staining of "Gomori-positive" substances is remarkably impaired. The amounts of the substances demonstrable in the neural lobe are diminished and in the cells of the supraoptic and paraventricular nuclei as well as in both median eminence layers only traces of the substances are to be seen. The findings indicate that negative results in demonstrating "Gomori-positive" substances may be caused by inappropriate fixation and need to be controlled by immunohistochemical techniques.     

Radioautographic studies on the neurohypophysial projections of the supraoptic and paraventricular nuclei in the rat

Summary The distribution of labelled axonal pathways was studied after unilateral stereotaxic injection of ³H-leucine into either supraoptic (SON) or paraventricular nuclei (PVN). In addition to extrahypothalamic projections of both nuclei, the main efferents appeared to run towards the neurohypophysis, yet with a strikingly different pattern. At the neurohypophysial level, the SO-neurohypophysial tract crossed the inner layers of the median eminence (ME) before scattering in the neural lobe. The PV-neurohypophysial pathway, by contrast, provided an exclusive innervation to the external layer of the whole neurohypophysial organ, including the median eminence, infundibular stalk and neural lobe. The functional correlates of the clear-cut anatomical distinctness between the two magnocellular neurosecretory systems are discussed.     

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica

Summary An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and anti-mesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina terminalis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunore-active fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.This work was partially supported by a Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile to E.M. Rodriguez     

Ultrastructural aspects of the inhibition of neurosecretion by puromycin

Summary The supraoptic nuclei and the infundibular process of the neurohypophysis were studied under the electron microscope in rats fed on dried food for 1 to 3 days and in similarly dehydrated animals but injected subarachnoidally with puromycin 4 to 24 hours prior to fixation. With 4 to 8 hours of puromycin the intracisternal filamentous material found in the control is greatly diminished. After 24 hours this inhibitory effect disappears in the perikaryon but the neurotubular material of the neuronal processes within the supraoptic nucleus and the neurosecretion in the infundibular process are greatly reduced. These results are discussed taking into consideration the inhibition of protein synthesis caused by puromycin and the neurosecretory cycle which characterizes the supraoptic neurons.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (963–65).     

Functional changes in neurosecretory cells of the anterior hypothalamic nuclei after stimulation of the midbrain reticular formation

The functional activity of the neurosecretory cells in the supraoptic and paraventricular nuclei was studied in male Wistar albino rats at various intervals after electric stimulation of the midbrain reticular formation. These studies showed that such stimulation elicits higher functional activity of the neurosecretory cells in the anterior hypothalamic nuclei, characterized by increased secretory synthesis by these cells and rapid transport of the neurosecretion. These changes were most pronounced 1 h after stimulation of the reticular formation. The changes observed were unidirectional in both neurosecretory centers, but their manifestation was different: in the supraoptic nucleus the reaction was more intense but short lived, in the paraventricular it was less intense but lasted longer.A. A. Bogomolets Institute of Physiology, Academy of Sciences, Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 3, No. 4, pp. 394–400, July–August, 1971.     

Changes in oxytocin content in the magnocellular neurons of the rat hypothalamus following water deprivation or estrogen treatment

Summary The effect of water deprivation or estrogen treatment on the oxytocin content of rat hypothalamic cells was examined using a quantitative immunohistological technique. Oxytocin-containing cells were visualized using the immunoperoxidase technique of Sternberger and a primary antiserum directed against oxytocin. The optical density of the darkest 3.2 m diameter spot in the cytoplasm of a cell was used as a measure of the oxytocin content of that cell. Water deprivation produced a significant decrease in anti-oxytocin staining in the anterior commissural nucleus of males and females. There was a similar decrease in the paraventricular nucleus of males, but not in the paraventricular nucleus of females or the supraoptic nucleus of either males or females. Estrogen treatment of ovariectomized female rats produced a fall in anti-oxytocin staining in the anterior commissural, but not paraventricular or supraoptic nuclei.     

The development of the corticoliberin- and vasopressinergic elements of the rat hypothalamus during postnatal ontogeny

In 10--81-day and adult Wistar rats, neurosecretory cells were revealed which react with antisera to corticoliberin and vasopressin. Morphometric analysis of these cells in the supraoptic, paraventricular and anterior commissural nuclei shows that in the latter vasopressinergic cells develop somewhat later than in the supraoptic and paraventricular nuclei. Complete differentiation of neurosecretory cells in all the centres investigated is observed in 2-month animals. Studies were also made on the amount of corticoliberin- and vasopressinergic terminals in the external zone of the median eminence. Vasopressin-immunoreactive fibers are more numerous in young rats than in adult ones. Corticoliberin-positive neurosecretory fibers are more abundant in adult animals. Earlier development of vasopressinergic elements corresponds to a hypothesis of a more ancient origin of nonapeptidergic structures as compared to those producing liberins and statins.     

CHANGES IN THE HYPOTHALAMO-NEUROHYPOPHYSICAL NEURO-SECRETORY MATERIALS OF RATS DURING DIFFERENT PHASES OF MORPHINE ADMINISTRATION

Abstract—

• 1 Acute morphine treatment of rats increased the concentration of neuro-secretory material in the posterior pituitary. These changes were accompanied by the presence of more neurosecretory materials in the axons of the hypothalamic supraoptic and paraventricular neurons and in the hypothalamic capillaries and sinuses. In the perikarya of the hypothalamic supraoptic and paraventricular neurons, the neurosecretory material is in a dispersed state.
• 2 Following chronic morphine treatment, neurosecretory material was almost absent from the posterior pituitary, whereas in the perikarya of the supraoptic and paraventricular neurons, congestion of neurosecretory material is observed which is accompanied by the absence of the neurosecretory material from the axons arising from the supraoptic and paraventricular nuclei and from the hypothalamic capillaries and sinuses.
• 3 During nalorphine-induced abstinence, there was sudden reappearance of the neurosecretory material in the posterior pituitary along with the appearance of neurosecretory material in the hypothalamic neurosecretory neuronal tracts, blood capillaries and sinuses together with the dispersion of the neurosecretory materials from the hypothalamic neurosecretory neurons.

     

Hypothalamo-hypophysial Neurosecretory System in Normal and Hypophysectomised Catfish,Clarias Batrachus L

Two neurosecretory centers, nucleus preopticus (NPO) and nucleus lateralis tuberis (NLT), are distinguished in the hypothalamus of Clarias batrachus L. The cell bodies of NPO are grouped into nucleus preopticus magnocellularis (NPM) and nucleus preopticus parvocellularis (NPP). The NLT is recognised into three divisions: pars rostralis, pars medialis and pars ventrolateralis. The neurons of both the NLT and NPO are aldehyde-fuchsin (AF) positive. The axons from NPO run into four or five bundles ventrolaterally and caudally for some distance before all of them from either side join ventromedially into a single thick cord of neurosecretory fibers which finally enters the pars intermedia to form the neurointermediate lobe. The fibers from NLT, which are separate from the neurosecretory fibers of the NPO, anastomose in the region of the pars distalis. The neurosecretory material (NSM) of NPO is transported by axonal route whereas that of NLT is by axonal as well as ependymovascular pathways. Hypophysectomy results in the increase of AF-positive material in the neurons of NPO shortly after the operation, but later on, the material begins to deplete in them. The AF-positive material at the cut end of neurosecretory fibers, however, accumulates. The AF-positive material in the cell bodies of NLT is also depleted, but the nuclei increase in size, after hypophysectomy.     

Biorhythm of arginine-vasopressin in the paraventricular, supraoptic and suprachiasmatic nuclei of rats

The diurnal variations in content of arginine-vasopressin in the supraoptic nucleus, the paraventricular nucleus and the suprachiasmatic nucleus of rats were determined using radioimmunoassay. In the supraoptic nucleus and the paraventricular nucleus the arginine-vasopressin level was relatively constant during the light phase (the inactive phase). When it became dark, the level of arginine-vasopressin lowered during the early and middle dark phase and then increased to the highest level during the late dark phase. In the suprachiasmatic nucleus the level was stable during the light phase, while in the early and the late dark phase it was significantly higher than that in the middle dark phase.     

Retinohypothalamic projections and immunocytochemical analysis of the suprachiasmatic region of the desert iguana Dipsosaurus dorsalis

Two separate and distinct retinal projections to the hypothalamus in the iguanid lizard Dipsosaurus dorsalis were described using horseradish peroxidase and cobalt-filling techniques. Both of the projections were unilateral and completely crossed; one terminated in the supraoptic nucleus and the other in the suprachiasmatic nucleus. Immunocytochemical analysis showed that the supraoptic nucleus contained cell bodies and fibers that cross-react with antibodies raised against arginine vasopressin, while the suprachiasmatic nucleus contained arginine vasopressin-like immunoreactive fibers emanating from cells in the nearby paraventricular nucleus. The suprachasmatic nucleus contained a dense plexus of fibers that cross-reacted with neuropeptide-Y antibody. Antiserum against vasoactive intestinal polypeptide showed no reactivity in any part of the forebrain, while antiserum against serotonin showed sparse and uniform reactivity throughout the forebrain, including the suprachiasmatic nucleus. These results, together with other data, indicate that the suprachiasmatic nucleus of D. dorsalis is homologous to the suprachiasmatic nuclei of rodents, structures known to contain circadian pacemakers. We suggest that the suprachiasmatic nucleus may play a similar role in the circadian system of D. dorsalis.     

Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat

Immuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.     

The influence of the antimitotic drug CCNU on the neurosecretion of rat hypothalamo-hypophyseal system

The purpose of this investigation was to evaluate the influence of the antimitotic drug CCNU on the morphology of the hypothalamo-hypophyseal neurosecretory system of rat. Adult Wistar rats were treated intragastrically with 2.5 mg CCNU once a week during 3 consecutive weeks and 5 mg at the end of the 4th week. The brains and hypophyses were fixed in Zenker-formol solution. Paraffin slices were stained with chromhematoxylin to demonstrate neurosecretory material and with cresyl violet. PAS reaction was also performed. The experiment resulted in disturbances of the neurosecretory function of the hypothalamo-hypophyseal system in form of alteration in the content of neurosecretion in the neuronal cytoplasm and processes within supraoptic and paraventricular nuclei as well as in the neurohypophysis. The morphometric measurements showed enlargement of the cell nuclei and cytoplasm volumes in the nucleus supraopticus of hypothalamus.