Simultaneous detection of tyrosine hydroxylase-immunoreactivity and vasopressin mRNA in neurons of the human paraventricular and supraoptic nucleus

Our purpose was to investigate the proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons expressing vasopressin (VP) mRNA in the human paraventricular and supraoptic nuclei by combining in situ hybridization with immunohistochemistry on the same tissue section. A variability in the proportion of TH-IR neurons synthesizing VP mRNA was observed in adults which was usually more than 50%. In neonates almost all the TH-IR neurons appeared to contain VP mRNA.     

Activation of vasopressin neurons in the human supraoptic and paraventricular nucleus in senescence and senile dementia

A recent study has shown that vasopressin (AVP) cells in the human supraoptic (SON) and paraventricular (PVN) nuclei increase in size after 60 years of age, suggesting that AVP production is increased in senescence. In the present study, the same brain material was used for the determination of nucleolar size in immunocytochemically identified AVP and oxytocin (OXT) neurons as an additional parameter for peptide production.A strong correlation was found between nucleolar size and cell size, both in AVP and OXT neurons. Nucleolar size of AVP but not of OXT neurons increased significantly in senescence. Observations in brains from patients with senile dementia of the Alzheimer type (SDAT) were commensurate with their ages. These results strongly support the hypothesis that AVP neurons in the SON and PVN are activated in old age.     

Neurofibrillary pathology in the human paraventricular and supraoptic nuclei

Severe neurofibrillary changes were identified in the paraventricular and supraoptic nuclei of elderly individuals using markers for Alzheimer’s disease-related intraneuronal pathology. This neurofibrillary pathology is remarkable in that the magnocellular paraventricular and supraoptic nuclei are particularly resistant to Alzheimer’s disease. Moreover, the changes were observed even in non-demented controls, indicating that they develop independently of Alzheimer’s disease. The alterations in the paraventricular and supraoptic nuclei were consistently accompanied by neurofibrillary changes in the mediobasal hypothalamus. Received: 29 October 1996 / Revised, accepted: 24 January 1997     

Specific expression of secretogranin II in magnocellular vasopressin neurons of the rat supraoptic and paraventricular nucleus in response to osmotic stimulation

As secretogranin II is considered to be a marker for the regulated secretory pathway, its distribution in the hypothalamo-neurohypophyseal system of salt-loaded Wistar rats was studied in detail by immunocytochemistry. Although after an osmotic challenge both vasopressin and oxytocin neurons are stimulated, secretogranin II was exclusively expressed in a subpopulation of vasopressinergic magnocellular neurons in the supraoptic and paraventricular nucleus of Wistar rats. Secretogranin II was only surely visualized after a combination of osmotic challenge and blockade of axonal transport by colchicine treatment. When these pre-treatments were not performed, only punctate fibers situated around the magnocellular neurons within the paraventricular and supraoptic nucleus were observed. Oxytocinergic magnocellular neurons never displayed any secretogranin II immunoreactivity, not even during lactation and after colchicine treatment. These findings suggest that secretogranin II is of functional importance during enhanced secretory activity within vasopressinergic neurons.     

The effects of osmotic stimulation and water availability on c-Fos and FosB staining in the supraoptic and paraventricular nuclei of the hypothalamus

We studied the effects of osmotic stimulation on the expression of FosB and c-Fos in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Adult male rats were divided into two groups that were injected with lidocaine (0.1-0.2 ml sc) followed by either 0.9% or 6% NaCl (1 ml/100 g bw sc). After the NaCl injections, the rats were anesthetized and perfused 2, 6, or 8 h after the injections. Their brains were prepared for immunocytochemistry and stained with FosB and c-Fos antibodies. The number of c-Fos-positive cells was significantly increased only at 2 h in the SON and PVN. In contrast, the number of FosB-positive cells was significantly increased at 6, and 8 h in both the SON and PVN. In a second experiment, the effect of water availability on FosB staining 8 h after injections of 6% NaCl was tested in 3 groups of rats: water ad libitum, rats that had no access to water, and rats that were given water 2 h prior to perfusion. FosB staining was significantly reduced in both the SON and the PVN of rats that had ad libitum water compared to the two water-restricted groups. In the third experiment, rats were injected with either 0.9% NaCl or 6% NaCl and were either given ad libitum access to water or water restricted for 6 h after the injections and perfused 24 h after the saline injections. FosB staining was not increased when water was available ad libitum. FosB staining was significantly increased at 24 h in the rats injected with 6% NaCl when water was restricted. Thus, FosB may continue to influence protein expression in the SON and PVN for at least 24 h following acute osmotic stimulation.     

Stress increases oxytocin release within the hypothalamic paraventricular nucleus

Evidence indicates that the hypothalamic paraventricular nucleus (PVN) and oxytocin (OT) neurons in particular play a role in the physiological response to stress. Microdialysis (MD) experiments were performed to determine whether OT is released into the PVN during shaker stress. Male rats were prepared with venous catheters and PVN guide cannulae. OT and vasopressin (VP) release into PVN and peripheral blood were measured under basal conditions and during and after shaker stress (10 min at 110 cycles/min). Stress produced a specific increase in PVN and plasma OT. Dialysate OT levels were 0.3±0.1, 2.8±1.2 and 1.3±0.6 pg/sample (control, stress and recovery, respectively). Plasma OT was significantly increased during stress (3.7±1.2 vs. 11.7±2.3 pg/ml, basal vs. stress, respectively). When MD probes were located outside the PVN, there was no increase in OT release, demonstrating site specificity. Stress produced no change in VP levels, either in dialysate or plasma. These results show that OT, but not VP, is released into the PVN and peripheral blood in response to shaker stress. The data raise the possibility that local release of OT into the PVN plays a role in the neuroendocrine stress cascade.     

Effect of enucleation on the expression of c-Fos protein in the supraoptic nucleus of the Japanese monkey (Macaca fuscata)

The aim of this study was to examine the effects of one eye enucleation on the expression of c-Fos protein in the hypothalamus of the Japanese monkey (Macaca fuscata). Compared with an intact monkey, significantly increased numbers of c-Fos positive neurons were observed in the supraoptic nuclei on both sides at 1 h after eye enucleation. This maximal c-Fos expression then started to decrease at 3 h after eye enucleation. Furthermore, by a dual-labeled immunocytochemical study, the c-Fos immunoreactivity was found mainly in the vasopressinergic but not in the oxytocinergic neurons within the supraoptic nucleus. These results suggest that vasopressinergic but not oxytocinergic neurons within the supraoptic nucleus may have critical roles in the stimulation of this nucleus in response to eye enucleation.     

Localization of κ opioid receptors in oxytocin magnocellular neurons in the paraventricular and supraoptic nuclei

It has been demonstrated previously that κ opioid receptor agonists, such as dynorphin, inhibit oxytocin secretion in the rat. To determine whether κ agonists act directly on oxytocin-containing magnocellular neurons to inhibit hormone secretion, we utilized immunofluorescence to examine the cellular localization of κ opioid receptors in the rat paraventricular and supraoptic nuclei. κ Opioid receptor immunoreactivity co-localized with oxytocin-containing cell bodies, their axons and axon terminals. Thus, our results suggest that κ opioid receptor agonists can exert direct inhibitory actions on oxytocin magnocellular neurons.     

Morphology of neurons in the anterior hypothalamic area and supraoptic hypothalamic nucleus of the adult human brain

Morphological features of neuronal cell types in the anterior hypothalamic area (AHA) and supraoptic hypothalamic nucleus (SON) of the adult human brain were analysed in Golgi impregnated preparations. Four neuronal cell types were described for the first time in these human nuclei. Type I neurons were found in both the AHA and SON, while the other three cell types (types II–IV) were found only in the SON. Type I neurons had elongated, triangular or multipolar somata, which emitted 2–5 sparsely branching primary dendrites with a moderate number of fine spines. Also many of type I neurons in the AHA had thin dendritic side-branches. Type II neurons had round or fusiform somata, and two occasionally branching primary dendrites. Type III neurons were multipolar neurons with 3–5 densely spined and sparsely branching dendrites. Their axons had collaterals. Type IV neurons had very small ovoid somata with one smooth and unbranched primary dendrite. The neurons in the human AHA and SON were similar to those observed in the same areas in other mammalian species, except for the very small neurons in the SON and the thin dendritic side-branches of type I neurons in the AHA, that had not been previously described.

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Sommario Sono state analizzate le caratteristiche morfologiche delle popolazioni cellulari impregnate con metodo di Golgi, nelle aree ipotalamiche anteriore (AHA) e sopraottica (SON) del cervello umano adulto. Per la prima volta vengono descritte quattro sottopopolazioni neuronali. I neuroni del tipo I sono stati osservati sia in AHA che in SON mentre gli altri tre sottotipi neuronali (tipi II – IV) sono stati trovati solo in SON. I neuroni di tipo I hanno soma allungato, triangolare o multipolare da cui emergono 2–5 dendriti primari con un numero ridotto di spine. Molti di questi neuroni in AHA hanno sottili ramificazioni dendritiche. I neuroni di tipo II presentano un soma tondeggiante o fusiforme e generalmente due ramificazioni dendritiche principali. Il tipo III è caratterizzato da neuroni multipolari con 3–5 ramificazioni dendritiche ricche di spine. I loro assoni presentano collaterali. Il tipo IV ha soma piccolo ed ovoidale con dendriti primari sottili e che non si ramificano. I neuroni osservati nei nuclei AHA e SON dell'uomo sono simili a quelli osservati nelle stesse aree di altre speci animali ad eccezione dei piccoli neuroni in SON e delle caratteristiche delle ramificazioni dendritiche dei neuroni di tipo I in AHA. Queste caratteristiche morfologiche non sono state precedentemente descritte.

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This study was supported by Jordan University of Science and Technology Grant No. 13/94.     

Tyrosine hydroxylase-immunoreactive neurons in paraventricular and supraoptic nuclei of the human brain demonstrated by a method adapted to prolonged formalin fixation.

We studied the distribution of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the adult human hypothalamus using a modification of the peroxidase-antiperoxidase immunohistochemical method which can be applied on autopsy brain material following prolonged formalin fixation. We observed that most of the TH-IR perikarya localized within the paraventricular (PVN) and supraoptic (SON) nuclei were large and showed homogeneous staining over the entire cytoplasm and processes. These results show that in the human brain a large population of neurons within the neurosecretory nuclei are able to synthesize a catecholamine.     

Cell loss in supraoptic and paraventricular nucleus in Alzheimer's disease

Previous studies have shown an activation of the hypothalamo-neurohypophyseal system (HNS) in normal aging and in senile dementia. Among other explanations, this activation might be secondary to cell loss in supraoptic (SO) and paraventricular (PV) nuclei. This study reports a 63% loss in the SO and a 56% loss in the PV in a group of Alzheimer disease (AD) patients. The remaining neurons undergo a compensatory hypertrophy that is more pronounced in the SO, affecting cell and nuclear size as well as nucleolar volume. The group of patients with a diagnosis of moderate dementia showed the greatest hypertrophy, as compared to the severely demented patients. Our results suggest that there is a compensatory capacity in the earlier stages of the dementia, that is lost in the final stages of Alzheimer's disease.     

Brain derived neurotrophic factor (BDNF) containing neurons in the hypothalamic paraventricular and supraoptic nuclei of juvenile and middle-aged rats after chronic stress

The type and duration of stress stimulation are postulated to affect the expression of the brain derived neurotrophic factor (BDNF) differentially during ontogenetic life. The aim of our study was to investigate the influence of two different stressors, i.e. chronic (15 min daily for 21 days) exposure to the forced swim (FS) test or the high light open field (HL-OF) test, on the BDNF contained in magnocellular (PVm) and parvocellular (PVp) neurons of the hypothalamic paraventricular (PV) and the supraoptic (SO) nuclei. The immunofluorescence (-ir) method was used to detect BDNF-ir cells. The research showed that only the PVp part of the PV in juvenile (P28; P-postnatal day) control rats had a significantly lower density of BDNF-ir neurons than that in middle-aged (P360) control subjects. After chronic FS, a significant decrease in BDNF-ir cells was observed in the studied hypothalamic nuclei of the juvenile rats, but no changes were noted in the middle-aged individuals. The PV (PVm, PVp) and the SO nuclei in juvenile rats showed a significantly lower density of BDNF-ir neurons than the corresponding area of the hypothalamus in middle-aged rats. However, following the HL-OF test, the density of BDNF-ir neurons remained unaltered both in the P28 and the P360 groups. The data suggest that the type of the stressor applied was the factor that differentiated the number of BDNF-ir cells in the PVm and the SO only in juvenile rats: chronic HL-OF was more severe than FS. The age of the animals was the main factor that conditioned the BDNF hypothalamic PV (PVm, PVp) and the SO response to FS stimulation. The different density of BDNF-ir containing cells in the PVp of juvenile versus middle-aged rats can be explained by a functional, age-related change in the demand of PVp neurons for BDNF.     

Inter- and intra-nuclear differences in galanin expression between the hypothalamic paraventricular and supraoptic nuclei in colchicine-untreated rats

Not much is known of the topography of galanin expression in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei neurons in colchicine (an axoplasmic inhibitor)-untreated animals. Insight into the biological implication(s) of galanin expression in the PVN and SON will depend, at least in part, on the nature of its distribution in colchicine-untreated animals. In this study therefore, the topographical distribution of galaninergic profiles was examined in the PVN and SON of colchicine-untreated rats. Staining in the parvocellular PVN (PVN(p)) was predominantly as varicose thin galanin fiber processes while the magnocellular PVN (PVN(m)) contained large cell soma and fiber processes. The relative fiber density was higher in the anterior, periventricular and medial PVN(p) than in the dorsal, lateral and posterior subdivisions. Large-sized cells and thick fibers were limited to the posterior PVN(m) while the anterior and medial PVN(m) contained varicose profiles. Light- and intensely-stained galanin-positive cells as well as large- and small-diameter (varicose or non-varicose) fibers were observed in the SON. The large and thin fibers exhibit preferential ventral and dorsal distribution, respectively. Together with the complexity of specific afferent and efferent connections within the PVN and SON, these observations underscore heterogeneous galanin expression and raise potential implications for understanding the biological role of galanin by physiologically challenging stimuli.     

Neurogenesis of oxytocin-containing neurons in the paraventricular nucleus (PVN) of the female pig in 3 reproductive states: puberty gilts, adult gilts and lactating sows

Evidence suggests that neurogenesis occurs in the adult hypothalamus, including centers containing oxytocin and vasopressin producing neurons. The present study was undertaken to look at one of these centers, the paraventricular nucleus (PVN), to describe its morphology, confirm the presence of neurogenesis and examine the effect of reproductive status on the incidence of neurogenesis. Serial sections of the paraffin-embedded hypothalamus were made from five puberty gilts, four adult gilts and four lactating sows. Specific sections were Nissl-stained for PVN morphology, while others were stained with an oxytocin (OT) primary antibody, which binds to the cytoplasm of oxytocin-containing neurons, and proliferating cell nuclear antigen (PCNA) primary antibody, which binds to PCNA, a protein expressed in the nucleus during cell division. Cells labeled with both OT and PCNA were considered to be oxytocin-containing neurons that had recently divided, signifying the recent synthesis of a mature neuron. The general morphology of the PVN was similar in all pigs, and three subnuclei were identified and named based on cytoarchitecture. Neurogenesis was consistently observed in OT-containing neurons of all pigs studied. However, a significantly greater number of double-labeled (OT + PCNA) cells occurred in the PVN of lactating sows and adult gilts, when compared to puberty gilts. These observations confirm the process of neurogenesis in the hypothalamus of the adult female pig and suggest that the up-regulation of OT-containing neurons is correlated to age and possibly driven by sexual maturation, but not necessarily lactation.     

Oxytocin and estrogen promote rapid formation of functional GABA synapses in the adult supraoptic nucleus

We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-beta estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions.     

Prolonged alcohol intake leads to irreversible loss of vasopressin and oxytocin neurons in the paraventricular nucleus of the hypothalamus

Previous data revealed that numerous neurons in the supraoptic nucleus degenerate after prolonged ethanol exposure, and that the surviving neurons increase their activity in order to prevent dramatic changes in water metabolism. Conversely, excess alcohol does not induce cell death in the suprachiasmatic nucleus, but leads to depression of neuropeptide synthesis that is further aggravated by withdrawal. The aim of the present study is to characterize the effects of prolonged ethanol exposure on the magnocellular neurons of the paraventricular nucleus (PVN) in order to establish whether or not magnocellular neurons display a common pattern of reaction to excess alcohol, irrespective of the hypothalamic cell group they belong. Using conventional histological techniques, immunohistochemistry and in situ hybridization, the structural organization and the synthesis and expression of vasopressin (VP) and oxytocin (OXT) in the magnocellular component of the PVN were studied under normal conditions and following chronic ethanol treatment (6 or 10 months) and withdrawal (4 months after 6 months of alcohol intake). After ethanol treatment, there was a marked decrease in the number of VP- and OXT-immunoreactive magnocellular neurons that was attributable to cell death. The surviving neurons were hypertrophied and the VP and OXT mRNA levels in the PVN unchanged. Withdrawal did not alter the number of VP- and OXT-producing neurons or the gene expression of these peptides. These results substantiate the view that after prolonged ethanol exposure numerous neurons of the hypothalamic magnocellular system degenerate, but the mRNA levels of VP and OXT are not decreased due to compensatory changes undergone by the surviving neurons.     

Survival and regeneration of neurons of the supraoptic nucleus following surgical transection of neurohypophysial axons depend on the existence of collateral projections of these neurons to the dorsolateral hypothalamus

The aim of the present study was to compare the postlesional responses of vasopressin-producing (VP) and oxytocin-producing (OT) neurons of the supraoptic nucleus (SON) to transection of neurohypophysial axons. At different times after sectioning the median eminence of adult rats, immunocytochemical staining of both types of neuronal cell bodies and axons indicated that: (1) the number of OT neurons detected within the SON was only slightly decreased as compared with controls (−20%), whereas the number of VP neurons was severely decreased (−60%); and (2) the large majority of axonal sprouts that regenerated into the external layer of the median eminence were OT neurohypophysial axons. The injection of a retrograde tracer into various areas surrounding the SON further showed that numerous SON neurons could be retrogradely labeled when the injection was centered in the lateral hypothalamus dorsal to the SON. The immunocytochemical identification of these retrogradely labeled neurons demonstrated that most of them were OT neurons. When animals were subjected to median eminence transection and to a unilateral surgical cut placed in the lateral hypothalamus above the SON, the survival of both OT and VP neurons was dramatically reduced in the SON ipsilateral to the hypothalamic lesion, as compared to the contralateral SON. Taken together, these data indicate that OT (and to a lesser extent VP) neurons of the SON display collateral projections towards the lateral hypothalamus that protect them from retrograde degeneration following the lesion of their neurohypophysial projections.     

Hypovolemia induces Fos-like immunoreactivity in neurons of the rat supraoptic and paraventricular nuclei.

Immunoreactivities to Fos proteins were detected in numerous neurons in the supraoptic, paraventricular and accessory neurosecretory nuclei 1 h following withdrawal of 4-5 cc of blood from the rat femoral arteries. Few or no positive cells were observed in the same nuclei in sham-operated or control animals. It is concluded that hypovolemia induces c-fos expression in hypothalamic neurons known to be associated with blood volume/pressure regulation.     

Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve

Sympathetic catecholaminergic fibers in the vagus nerve were immunohistochemically examined in formalin-fixed human cadavers using an antibody against the noradrenalin-synthetic enzyme tyrosine hydroxylase (TH). TH-positive fibers were extensively distributed in the vagal nerve components, including the superior and inferior ganglia, the main trunk and the branches (superior and recurrent laryngeal, superior and inferior cardiac, and pulmonary branches). The inferior ganglion and its continuous cervical main trunk contained numerous TH-positive fibers with focal or diffuse distribution patterns in each nerve bundle. From these findings, we conclude that sympathetic fibers are consistently included in the human vagus nerve, a main source of parasympathetic preganglionic fibers to the cervical, thoracic and abdominal visceral organs.