Studies on the transport of secretory granules in the magnocellular hypothalamic neurons of the rat

Intrathecal administration of 20 mug of vincristine sulphate in the rat induced in vivo the formation of paracrystalline inclusions mainly in axonal processes. This is associated with an impairment in the migration of neurosecretory granules as shown by their accumulation in the perikarya of the magnocellular neurons. The granules are intermixed with numerous dense bodies of various shape, sometimes with a fibrillar content, and probably of lysosomal origin. In addition to the impairment of the flow of neurosecretory granules, there is also a striking accumulation of mitochondria and synaptic vesicles, and an apparent proliferation of the smooth endoplasmic reticulum. In the posterior lobe, the axonal endings contain a large number of neurosecretory granules, intermingled with bodies of varying shapes and electron density. Occasionally, a dense membrane surrounding a group of elementary granules is observed, reacting positively for acid phosphatase. This suggests an attempted crinophagia.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons

Summary The axonal flow of neurosecretory elementary granules has been studied in the paraventricular neurons of the rat (PVN), with the help of three techniques: light microscopy, radioautography after labelling with ³⁵S-L-cysteine, and electron microscopy.Colchicine treatment does not alter the uptake of ³⁵S cysteine in the PVN but the flow of labelled neurosecretory material towards the neurohypophysis is interrupted. Interruption of the axonal flow is also evidenced by the stagnation of neurosecretory granules at the periphery of the neuronal cytoplasm and by the presence of numerous axonal swellings, heavily loaded with neurosecretory granules and often containing abnormal elongated granules, surrounded by a single membrane, oriented more or less parallely to the long axis of the axons. Other cell organelles and neurotubules are not altered. The present experiments bring further evidence of the arrest by colchicine of the axonal flow of secretory granules without apparent changes of the neurotubules.This work was supported by a grant (1970/1971) from the Belgian National Fund for Scientific Research (J. F.-D), and by grant No 1120 from the Belgian National Fund for Medical Research (P. D).The authors wish to thank Mrs. A.-M. Hunninck-Couck for her devoted and skillful technical assistance, and are endebted to Dr. J. C. Heuson for kindly supplying the rats.     

Intranuclear coupling of hypothalamic magnocellular nuclei by glutamate synaptic circuits

Magnocellular neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) display bursting activity that is synchronized under certain conditions. They receive excitatory synaptic inputs from intrahypothalamic glutamate circuits, some of which are activated by norepinephrine. Ascending noradrenergic afferents and intrahypothalamic glutamate circuits may be responsible for the generation of synchronous bursting among oxytocin neurons and/or asynchronous bursting among vasopressin neurons located in the bilateral supraoptic and paraventricular nuclei. Here, we tested whether magnocellular neurons of the PVN receive excitatory synaptic input from the contralateral PVN and the region of the retrochiasmatic SON (SONrx) via norepinephrine-sensitive internuclear glutamate circuits. Whole cell patch-clamp recordings were performed in PVN magnocellular neurons in coronal hypothalamic slices from male rats, and the ipsilateral SONrx region and contralateral PVN were stimulated using electrical and chemical stimulation. Electrical and glutamate microdrop stimulation of the ipsilateral SONrx region or contralateral PVN elicited excitatory postsynaptic potentials/currents (EPSP/Cs) in PVN magnocellular neurons mediated by glutamate release, revealing internuclear glutamatergic circuits. Microdrop application of norepinephrine also elicited EPSP/Cs, suggesting that these circuits could be activated by activation of noradrenergic receptors. Repetitive electrical stimulation and drop application of norepinephrine, in some cases, elicited bursts of action potentials. Our data reveal glutamatergic synaptic circuits that interconnect the magnocellular nuclei and that can be activated by norepinephrine. These internuclear glutamatergic circuits may provide the functional architecture to support burst generation and/or burst synchronization in hypothalamic magnocellular neurons under conditions of activation.     

The development of connections of the magnocellular hypothalamic nuclei with the posterior hypophyseal lobe in rat prenatal ontogeny]

The development of projections of the hypothalamic nuclei into the posterior lobe of the pituitary was studied on the fixed brain of rat fetuses from day 15 until day 19 of embryogenesis using retrograde staining with the fluorescent carbocyanine dye DiI. The formation of connections of the supraoptic and retrochiasmatic nuclei of the hypothalamus with the posterior lobe of the pituitary takes place during prenatal development on days 15 and 16-17, respectively, while only an insignificant number of the paraventricular nucleus neurons send their axons to the posterior lobe of the pituitary in rat fetuses. These facts suggest different temporal involvement of the above nuclei in formation of the hypothalamic-hypophysial neurosecretory system in rat fetuses.     

Reactions of oxytocin- and vasopressinergic cells of the hypothalamic supraoptic and paraventricular nuclei in the rat to repeated administrations of thyroliberin

Male Wistar rats were injected with 1 microgram thyroliberin twice a day for 8 days. Vasopressinergic (VE) and oxytocinergic (OE) cells of the supraoptic (SON) and paraventricular nuclei (PVN) were detected immunohistochemically, their changes were estimated morphometrically. The blood level of the thyrostimulating hormone determined by radioimmunoassay was 220% of the control on the 6th day but declined by the 9th day. The thyroid hormone concentration was also diminished. The nucleoli of the VE and OE cells of the SON were reduced by the 9th day (by 74 and 82%, respectively; P less than 0.01). The nucleoli of the OE cells of the PVN were enlarged to 120% (P less than 0.01), hence production of the neurohormone by these cells was intensified. The VE cells of the PVN remained essentially unchanged. The data obtained suggest that disagreement between the PVN and SON cell reactions is caused by various reactions of the nerve centers conveying toward the PVN and SON. Moreover, the OE and VE cells of the PVN apparently differ in their input. The importance of the OE cells of the PVN for the thyrostimulating hormone level normalization is suggested.     

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.     

Distribution of osmoregulatory peptides and neuronal-glial configuration in the hypothalamic magnocellular nuclei of desert rodents

The desert rodents Psammomys obesus and Gerbillus tarabuli live under extreme conditions and overcome food and water shortage by modes of food and fluid intake specific to each species. Using immunohistochemistry and electron microscopy, we found that the hypothalamic magnocellular nuclei, and in particular, their vasopressinergic component, is highly and similarly developed in Psammomys and Gerbillus. In comparison to other rodents, the hypothalamus in both species contains more magnocellular VP neurons that, together with oxytocin neurons, accumulate in distinct and extensive nuclei. As in dehydrated rodents, many magnocellular neurons contained both neuropeptides. A striking feature of the hypothalamic magnocellular system of Psammomys and Gerbillus was its display of ultrastructural properties related to heightened neurosecretion, namely, a significant reduction in glial coverage of neuronal somata and dendrites in the hypothalamic nuclei. There were many neuronal elements whose surfaces were directly juxtaposed and shared the same synapses. Their magnocellular nuclei also showed a high level of sialylated isoform of the Neural Cell Adhesion Molecule (PSA-NCAM) that underlies their capacity for neuronal and glial plasticity. These species thus offer striking models of structural neuronal and glial plasticity linked to natural conditions of heightened neurosecretion.     

Functional difference between the magnocellular neurosecretory nuclei of the rat hypothalamus

The supraoptic, paraventricular, and postoptic nuclei (SON, PVN, and PON, respectively) of the hypothalamus were studied under conditions of 3 months training of rats to hypoxia (exposure for 6 h daily in a low pressure chamber under 7600m of simulated altitude). All the three nuclei were activated during the first 5 days, and then the state of the SON cells normalized. Functional activity of the PVN and PON decreased (the nucleolar volume of the neurosecretory cells diminished to 70--80%, the amount of the neurosecretory substance in the cells and the posterior lobe of the hypophysis was reduced). Such a decreased activity of the PVN and PON persisted till the end of the experiment. A positive correlation of the thyroid epithelium height and the nucleolar volume of the PVN and PON cells was established for both the PVN (r=0.81, P less than 0.05) and the PON (r=0.82; P less than 0.05); no significant correlation was revealed for the SON (r=0.51; P less than 0.05). Thus, functional similarity of the PVN and the PON, and some peculiarities in the SON reaction under conditions of the experiment described was demonstrated.     

Ultrastructural aspects of the paraventricular nuclei in the rat

Summary The ultrastructural aspects of the paraventricular nucleus and its neuropil are described in the normal rat.Two types of neurons can be distinguished morphologically. The first type contains numerous dense-core vesicles (mean diameter: 140 nm). The cisternae of the endoplasmic reticulum are arranged parallely at the periphery of the cell body.The second type of neuron contains a few dense-core vesicles (mean diameter: 75 nm) and the endoplasmic reticulum is randomly distributed in the cytoplasm. In the neuropil, two types of dense-core vesicles are observed in separated axons. The histogram of the distribution of their mean diameter clearly indicates a double population of vesicles.The signification of the second type of neuron in the paraventricular nucleus is discussed and its possible relation to TRF synthesis is evoked.This work was supported by a grant from the Belgian National Fund for Scientific Research.The author wish to thank Mrs. Hunninck-Couck for her devoted and skillful technical assistance.     

Plasticity in the hypothalamic magnocellular neurosecretory system

Over the past decade or so, plasticity has emerged as an important, quantifiable property of the mammalian hypothalamic magnocellular neurosecretory system. This plasticity has turned out to be genuinely related to normal function in the sense that it is a set of responses to physiological stimulation rather than only the sequelae of insult or injury, and it is generally completely reversible. This latter property, of course, distinguishes it further from the plasticity observed after injury. Four features of this magnocellular system that have been shown to display predictable and reversible intercellular plasticity are reviewed: the relationships between neurons and their associated astrocytic glia at various levels (dendritic somatic and terminal) of the magnocellular elements; the extent of terminal and glial contact with the basement lamina in the neurohypophysis; the type and possible efficacy of synaptic input, and the extent of electrotonic coupling among the magnocellular neurons.     

The hypothalamic magnocellular system in the domestic fowl

Summary In the rostral hypothalamus of the domestic fowl, the magnocellular neurosecretory nuclei show a peculiar differentiation. Golgi studies of the supraoptic and paraventricular nuclei of the fowl reveal at least two major cell types: 1) large multipolar neurons, and 2) small interneurons. Golgi impregnations provide a detailed cytoarchitectural picture of the large-sized cells; the latter may well correspond to the neurosecretory cells demonstrated in the same regions by selective staining, and immunocytochemical and electron microscopical techniques.Electron microscopically, neuronal perikarya are observed to contain variable amounts of neurosecretory granules (100–200 nm in diameter; mean diameter of 160 nm) scattered throughout the cytoplasm. The diameters of these granules do not differ statistically in the two principal nuclear areas examined. The perikarya of these neurons display only a few axosomatic synapses containing electron-lucent and dense-cored vesicles (70–90 nm in diameter). Numerous nerve terminals of this type also end on the dendritic ramifications in the surrounding neuropil.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

The spermatozoa of two closely related species of ophiuroids, Ophiocoma echinata and Ophiocoma wendti, were examined ultrastructurally. Morphologically, these spermatozoa resemble those of other non-echinoid echinoderms. The acrosomal complex, completely contained within an anterior fossa in the spherical nucleus, consists of a membrane-limited acrosomal vesicle and periacrosomal material. Events of the acrosomal reaction in O. echinata and O. wendti are presented. In both species, the reaction results in the establishment of an extracellular coat of acrosomal vesicle origin on the anterior surface of the spermatozoon. The possible role of this extracellular coat in the species-specific binding of sperm and ova is discusses. The origin of acrosomal tubule membrane is elucidated.     

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     

Cellular organization of paraventricular hypothalamic nuclei of the rat]

According to the data on reconstruction of the serial sections of the cerebral hypothalamic area, using morphometric and electron microscopical methods, the structure of the paraventricular nuclei (PVN) has been studied in white rats (Wistar strain) hypothalamus. Ten subnuclei have been revealed. Each of them has individual characteristics. Owing to the analysis of the groups revealed, it is possible to divide the subnuclei into 3 main groups: magnocellular, parvicellular and subnuclei consisting of middle size neurons. The coordinated interaction of these subnuclei are supposed to ensure possible participation of PVN in regulation of the vegetative nervous and endocrine systems.     

Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic–hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.     

Vasopressinergic axon collaterals and axon terminals in the magnocellular neurosecretory nuclei of the rat hypothalamus

Axon collaterals emerging from the vasopressinergic neurons of the supraoptic (SON) and paraventricular (PVN) nuclei and recurving back towards their respective nuclei have been previously reported. Since such axon collaterals can play a role in the neuromodulation of SON and PVN, these nuclei have been further investigated immunohistochemically under the light and electron microscope. The PAP technique, using a commercial antibody, was employed. Vasopressin-positive axon collaterals were seen to recurve towards their nuclei of origin. In the latter, vasopressinergic intrinsic neurons were also observed. Under the electron microscope, axon terminals containing vasopressin-immunoreactive neurosecretory granules were noted. Such terminals presumably arise from the vasopressin-positive recurrent axon collaterals or from the intrinsic neurons for the purpose of neuromodulation within the SON and PVN.