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Alzheimer's disease (AD) is a progressive neurodegenerative disorder, characterized by irreversible decline of mental faculties, emotional and behavioral changes, loss of motor skills, and dysfunction of autonomic nervous system and disruption of circadian rhythms (CRs). We attempted to describe the morphological findings of the hypothalamus in early cases of AD, focusing our study mostly on the suprachiasmatic nucleus (SCN), the supraoptic nucleus (SON), and the paraventricular nucleus (PVN). Samples were processed for electron microscopy and silver impregnation techniques. The hypothalamic nuclei demonstrated a substantial decrease in the neuronal population, which was particularly prominent in the SCN. Marked abbreviation of dendritic arborization, in association with spinal pathology, was also seen. The SON and PVN demonstrated a substantial number of dystrophic axons and abnormal spines. Alzheimer's pathology, such as deposits of amyloid-β peptide and neurofibrillary degeneration, was minimal. Electron microscopy revealed mitochondrial alterations in the cell body and the dendritic branches. The morphological alterations of the hypothalamic nuclei in early cases of AD may be related to the gradual alteration of CRs and the instability of autonomic regulation., (© The Author(s) 2014.)

The magnocellular neurones of the supraoptic nucleus (SON) and paraventricular nucleus release neuropeptide from their axon terminals and also from their dendrites. In the axon terminals, swellings known as Herring bodies are responsible for the degradation of aged, unreleased large dense-cored vesicles (LDCVs) by lysosomes. Dendrites of magnocellular neurones also contain a large number of LDCVs but specialised areas of vesicle degradation have yet to be discovered. Using immunofluorescence labelling for lysosomes in vasopressin-enhanced green fluorescent protein (vasopressin-eGFP) transgenic rats, we found that lysosomes are preferentially located in the centre of the dendrites where there was a high density of vasopressin-eGFP expression. These data suggest that there are local "hot spots", but not specific compartments for vesicle degradation in magnocellular dendrites., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Previous studies revealed that oxytocin release is increased by various forms of stress. Hypertonic saline injection, immobilization, and several other stressors elevated the blood level of oxytocin in rats. However, the mechanism of the stress-induced oxytocin release in human is not elucidated yet. Although numerous studies indicate that catecholamines play a pivotal role in modulating the release of oxytocin, there is a lack of data regarding the morphological substrate of this phenomenon. In order to reveal putative juxtapositions between tyrosine hydroxylase-immunoreactive (TH-IR) catecholaminergic and the oxytocinergic systems in the human hypothalamus, we utilized double-label immunohistochemistry in the present study. Numerous TH-IR axon varicosities abutted on oxytocin-IR neurons in the supraoptic and paraventricular nuclei, forming synapse-like associations. Close examination of these juxtapositions with high magnification failed to reveal any gaps between the contacting elements. In summary, the intimate associations between the TH-IR and oxytocin-IR elements may be functional synapses and may represent the morphological substrate of stress-influenced oxytocin release. The finding that several oxytocin-IR perikarya did not receive apparent TH innervation suggests that additional mechanisms may play significant roles in the oxytocin modulation by stressors.

The regulatory actions of estrogens on magnocellular oxytocin (OT) neurons of the paraventricular and supraoptic nuclei are well documented. Although the expression and distribution of nuclear estrogen receptor-beta, but not estrogen receptor-alpha, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently a G protein-coupled receptor (GPR) for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the paraventricular nucleus and supraoptic nucleus, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system.

Structures of the pancreas and magnocellular (supraoptic and paraventricular) nuclei of the hypothalamus of adult male rats were studied in conditions of acute pancreatitis at the light and electron microscopic levels. Histo-and organotypic changes in the parenchymatous and stromal elements of the pancreas were analyzed simultaneously with cytological assessment of the state of the nonapeptidergic neurosecretory centers of the hypothalamus. Blockade of the release of hypothalamic nonapeptides was found to occur at the level of axovasal complexes, and this aggravated the outcome of destructive and necrobiotic changes in the pathologically altered organ.

Light and electron microscopic methods were used to analyze changes in secretory neurons in the supraoptic (SON) and paraventricular (PVN) nuclei in the hypothalamus in 100 adult male rats at time points from the first minutes to 180 days after 48 hours of full-time exposure to bright light. At the early time points after exposure, the cellular formulae of the SON and PVN shifted towards functionally active neurons with minimal quantities of secretory granules, large nuclei and nucleoli, low RNA contents, small numbers of rough endoplasmic reticulum cisterns, vacuoles, and lysosomes in the perikarya. The number of cells depositing secretion was greater than in controls at 24 h in the SON and PVN and at 10 days in the SON. Normalization of the cellular formula and the structural organization of the protein-synthesizing apparatus of PVN neurons occurred at 10-30 days, with normalization in the SON at 30-180 days. These data provide evidence that the range of plasticity of neurons in the PVN on exposure to full-time bright light was more significant than that in the SON.

Changes of secreteory neurons of hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) were analyzed using light and electron microscopy in 100 adult male rats from the first minutes up to 180 days after their round-the-clock 48 hours-long exposure to bright light. At the early stages after the exposure, SON and PVN cellular formula was changed in favor of functionally active neurons containing few secretory granules, large nucleus and nucleoli, low RNA content, sparse complexes of rough endoplasmic reticulum, vacuoles and lysosomes in their perikarya. After 24 hours in both SON and PVN and after 10 days in SON the number of cells accumulating secretion was greater than that in control group. Normalization of the cellular formula and of the structural organization of protein-synthesis apparatus in PVN secretory neurons took place at days 10-30, while those ones in SON--at days 30-180. The results obtained suggest that the range of plasticity in PVN neurons in animals exposed to continuous bright light is larger than that in SON neurons.

The structures of pancreas and hypothalamic magnocellular nuclei (supraoptic and paraventricular) were studied using light and electron microscopes in acute pancreatitis induced experimentally in mature male rats. Histo- and organotypic transformations of the pancreatic parenchymal and stromal elements were analyzed with a simultaneous cytological assessment of the hypothalamic nonapeptidergic neurosecretory centers. A phenomenon of the inhibition of hypothalamic nonapeptide release at the level in axo-vasal complexes was established, that aggravated the outcome of the destructive and necrobiotic processes in pathologically changed organ.

Astrocytes clear synaptically released glutamate from the extracellular space through high-affinity transporters present on their plasma membrane. By controlling the extracellular level of the main excitatory transmitter in the central nervous system, astrocytes thus contribute prominently to the regulation of overall cellular excitability and synaptic information processing. We recently investigated the influence of the glial environment on glutamatergic and GABAergic neurotransmission in the supraoptic nucleus of the rat hypothalamus under physiological conditions such as lactation that significantly reduce astrocytic coverage of its neurons. By performing electrophysiological analyses on this unique model of dynamic neuronal-glial interactions, we have been able to show that the fine astrocytic processes normally enwrapping synapses serve two important functions. First, they govern the level of activation of presynaptic metabotropic glutamate receptors on glutamatergic terminals, thereby regulating synaptic efficacy at excitatory synapses. Second, they act as a physical and functional barrier to diffusion in the extracellular space, limiting spillover of glutamate and other neuroactive substances and therefore contributing to the regulation of heterosynaptic transmission and intercellular communication., (Copyright 2004 Wiley-Liss, Inc.)

The hypothalamic magnocellular neurons, synthesizing arginine vasopressin (AVP) and oxytocin, are well known to show structural plasticity during chronic physiological stimulation. We have previously reported that 6B4 phosphacan/receptor-type protein-tyrosine phosphatasebeta (RPTPbeta), a chondroitin sulfate proteoglycan is highly expressed in the supraoptic nucleus (SON) of adult hypothalamus. Here, we undertook to study the activity-dependent regulation of 6B4 phosphacan/RPTPbeta in this system. Double labeling confocal microscopy demonstrated in the SON that 6B4 phosphacan/RPTPbeta-immunoreactive perineuronal nets were seen around AVP-containing somata and dendrites and its distribution pattern was well coincided with that of TAG-1. Quantitative immunohistochemical and Western analyses showed that 1-week salt loading, known as the chronic physiological stimulation for inducing the structural changes such as synaptic remodeling and direct neuronal membrane apposition, decreased 6B4 phosphacan/RPTPbeta levels in the SON, but did not alter TAG-1 levels. The 6B4 phosphacan/RPTPbeta levels were returned to control basal values within 3 weeks after the cessation of the chronic stimulation. Activity-dependent decreases in 6B4 phosphacan/RPTPbeta levels of the SON were confirmed when Western and immunohistochemical samples were digested with chondroitinase ABC, indicating that the decrease in 6B4 phosphacan/RPTPbeta levels was due to disappearance of 6B4 phosphacan/RPTPbeta core protein rather than increase in chondroitin sulfate glycosaminoglycans. With electron microscopy, the electron-dense immunoproducts for 6B4 phosphacan/RPTPbeta were found on the membrane surface of axons and glial processes, but not at synaptic junctions in control SON, and its immunoreactivity was eliminated with the chronic salt loading. The present results indicate that the levels of 6B4 phosphacan/RPTPbeta are regulated with activity-dependent manner and may be concerned with the structural plasticity seen in the SON.

After commenting on some perceived reasons why our review may have been relatively frequently cited, a brief overview is presented that first summarizes what we knew 25 years ago about the dynamic neuronal-astroglial interactions that occur in response to changes in the physiological state of the animal. The brain system in which these dynamic interactions were studied was the magnocellular hypothalamo-neurohypophysial system (mHNS) of the rat. The mHNS developed as and continues to be the model system yielding the most coherent picture of dynamic morphological changes and insights into their functional consequences. Many other brain areas, however, have more recently come under scrutiny in the search for glial-neuronal dynamisms. Outlined next are some of the questions concerning this phenomenon that led to the research efforts immediately following the initial discoveries, along with the answers, both complete and incomplete, obtained to those research questions. The basis for this first wave of follow-up research can be characterized by the phrase "what we knew we didn't know at that time." The final section is an update and brief overview of highlights of both "what we know now" and "what we now know that we don't know" about dynamic neuronal-astroglial interactions in the mHNS.

We analyzed expression of osteopontin (OPN), a cytokine regulating tissue repair and inflammation, in astrocytes and microglia in response to systemic lipopolysaccharide (LPS) administration (250 microg/100 g). OPN mRNA expression appeared in subpial astrocytes as early as 6 h, and then spread over the brain parenchyma. The signal for OPN mRNA reached a peak at 24 h post-injection, and returned to basal levels after 48 h. Changes in OPN immunoreactivity in the LPS-injected rat mirrored OPN mRNA induction patterns. These results provide the first evidence of OPN induction in astrocytes and microglia following peripheral immune challenge, and suggest that OPN may play a key role in the pathogenesis of neuroinflammation.

Emotional stress inhibits vasopressin release from the pituitary but may facilitate its release from the dendrites in the hypothalamus. We examined effects of intermittently applied footshock upon the amount of vasopressin heteronuclear RNA in the hypothalamus. The footshock decreased plasma vasopressin concentration but increased its extracellular concentration within the supraoptic nucleus. The contents of the vasopressin heteronuclear RNA in the supraoptic nucleus were significantly decreased after the shock. These data suggest that intermittent footshock decreases not only vasopressin release from the axon terminals in the pituitary, but also vasopressin synthesis in the cell bodies in the hypothalamus while the stimulus facilitates vasopressin release from the dendrites in the hypothalamus. The data also suggest differential control of dendritic vasopressin release and synthesis in the hypothalamus.

The functional implications of intraneuronal coexistence of different neuropeptides depend on their respective targeting to release sites. In the rat hypothalamic magnocellular neurons, we investigated a possible differential routing of the coexpressed galanin and vasopressin. The respective location of proteins and messengers was assessed with double immunogold and in situ hybridization combining confocal and electron microscope analysis. The various populations of labelled granules were quantitatively compared in three subcellular compartments: perikarya, local processes and posthypophyseal nerve endings. Three subpopulations of granules were detected in all three compartments, but their respective amount showed significant differences. Galanin alone was immunolocalized in some secretory granules, vasopressin alone in others, and both peptides in a third subpopulation of granules. The major part of the granules containing vasopressin, either alone or in association with galanin, is found in neurohypophyseal nerve endings. In contrast, galanin single-labelled granules represent the most abundant population in dendritic processes, while double-labelled granules are more numerous in perikarya. This indicates a preferential distribution of the two peptides in the different compartments of magnocellular neurons. Furthermore, galanin and vasopressin messenger RNAs were detected at different domains of the endoplasmic reticulum, suggesting that translation might also occur at different locations, thus leading to partial segregation of galanin and vasopressin cargoes between two populations of secretory granules. The present study provides, for the first time in mammals, evidence suggesting that galanin and vasopressin are only partly copackaged and undergo a preferential targeting toward dendrites or neurohypophysis, suggesting different functions, autocrine/paracrine and endocrine, respectively.

In addition to its role as a potent vasodilator, adrenomedullin (ADM) affects an animal's physiological status through its effects in the brain. We have shown that circulating ADM activates neurons, including nitric oxide (NO)-producing neurons, in autonomic centers of the brain such as the hypothalamic paraventricular nucleus (PVN). Systemic ADM gains access to the brain through the area postrema (AP), a brainstem circumventricular organ, and the PVN is a major target of these ADM-sensitive AP neurons. Neurons expressing the preproADM (ppADM) gene are distributed throughout the brain, with high levels in autonomic centers. Lipopolysaccharide (LPS, immune stress), restraint (psychological stress), and 24 h dehydration all down-regulate ppADM gene expression in different subsets of autonomic centers. Receptor-activity-modifying protein (RAMP) 2 and RAMP3, ADM receptor subunits, are expressed in autonomic centers including the PVN and supraoptic nucleus. Intracerebroventricular injections of ADM increase arterial pressure, heart rate, tyrosine hydroxylase mRNA levels in the locus coeruleus, plasma levels of ACTH, and NO production in the hypothalamus. ADM excites putative GABAergic and cholinergic neurons in dissociated cells from a basal forebrain integrative center, the diagonal band of Broca. These results demonstrate that the signalling components necessary for ADM to influence physiological systems are present in the brain and that ADM is an important transmitter of brain autonomic pathways which are involved in regulating homeostatic balance.

The functional implications of intraneuronal coexistence of different neuropeptides depend on their respective targeting to release sites. In the rat hypothalamic magnocellular neurons, we investigated a possible differential routing of the coexpressed galanin and vasopressin. The respective location of proteins and messengers was assessed with double immunogold and in situ hybridization combining confocal and electron microscope analysis. The various populations of labelled granules were quantitatively compared in three subcellular compartments: perikarya, local processes and posthypophyseal nerve endings. Three subpopulations of granules were detected in all three compartments, but their respective amount showed significant differences. Galanin alone was immunolocalized in some secretory granules, vasopressin alone in others, and both peptides in a third subpopulation of granules. The major part of the granules containing vasopressin, either alone or in association with galanin, is found in neurohypophyseal nerve endings. In contrast, galanin single-labelled granules represent the most abundant population in dendritic processes, while double-labelled granules are more numerous in perikarya. This indicates a preferential distribution of the two peptides in the different compartments of magnocellular neurons. Furthermore, galanin and vasopressin messenger RNAs were detected at different domains of the endoplasmic reticulum, suggesting that translation might also occur at different locations, thus leading to partial segregation of galanin and vasopressin cargoes between two populations of secretory granules. The present study provides, for the first time in mammals, evidence suggesting that galanin and vasopressin are only partly copackaged and undergo a preferential targeting toward dendrites or neurohypophysis, suggesting different functions, autocrine/paracrine and endocrine, respectively.

The hypothalamo-neurohypophysial system synthesizes and releases arginine vasopressin (AVP) and oxytocin (OXT) with physiological stimulation. In the present study, we investigated localization of a chondroitin sulfate proteoglycan (CSPG), phosphacan/RPTPbeta, in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of adult rats at both the light and electron microscopic levels. Immunohistochemical analyses demonstrated stronger phosphacan/RPTPbeta immunoreactivity within the SON and PVN compared with adjacent hypothalamic areas. Double labeling experiments showed phosphacan/RPTPbeta immunoreactivity constituting punctate networks to surround the somata and dendrites of AVP- and OXT-secreting magnocellular neurons. Electron microscopic examination further revealed strong phosphacan/RPTPbeta immunoreactivity at extracellular membrane surface of some axons, somata, and dendrites of the SON, but not of synaptic junctions. Interestingly, phosphacan/RPTPbeta immunoreactivity was also observed at extracellular surface membrane between astrocytic processes and neurons rather than between magnocellular neurons. The present results indicate the high expression of the CSPG, phosphacan/RPTPbeta at the extracellular space in the hypothalamic AVP- and OXT-secreting magnocellular neurons., (Copyright 2002 Elsevier Science B.V.)

Agmatine, decarboxylated from arginine by arginine decarboxylase, is particularly prominent in the hypothalamus. The present study utilized the rat hypothalamo-neurohypophysial system to determine expression and "pre-synaptic" modulation of agmatine in the central nervous system (CNS). Under confocal-laser scanning, agmatine-like immunoreactivity (Agm-LI) was found enriched in arginine-vasopressin (AVP)-containing magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei (PVN). In addition, using electron microscopy, Agm-LI was found closely associated with large neurosecretory-like vesicles in neurohypophysial nerve terminals of posterior pituitary gland. Radioimmunoassay revealed that 10 and 30 microM agmatine concentration-dependently inhibited the depolarization-evoked AVP release from isolated neurohypophysial terminals. Using perforated patch-clamp, effects of agmatine on whole-terminal voltage-gated ion currents in the isolated neurohypophysial nerve terminals were examined. While it did not significantly affect either tetrodotoxin (TTX)-sensitive Na(+) or sustained Ca(2+)-activated K(+) channel currents, agmatine (1-40 microM) inhibited Ca(2+) channel currents in approximately 53% of the total nerve terminals investigated. The onset of inhibitory effect was immediate, and the inhibition was reversible and concentration-dependent with an IC(50)=4.6 microM. In the remaining (approximately 47%) neurohypophysial nerve terminals, only a higher (120 microM) concentration of agmatine could moderately inhibit Ca(2+) channel currents. The results suggest that: (1) endogenous agmatine is co-expressed in AVP-containing, hypothalamic magnocellular neurons of the SON/PVN and in neurohypophysial nerve terminals of posterior pituitary gland; (2) agmatine may serve as a physiological neuromodulator by regulating the voltage-gated Ca(2+) channel and, as a result, the release of AVP from neurohypophysial nerve terminals.

Developing oxytocin and vasopressin (OT/AVP) supraoptic nucleus (SON) neurons positively autocontrol their electrical activity via dendritic release of their respective peptide. The effects of this autocontrol are maximum during the second postnatal week (PW2), when the dendritic arbor transiently increases and glutamatergic postsynaptic potentials appear. Here, we studied the role and interaction of dendritic OT/AVP release and glutamate release in dendritic plasticity and synaptogenesis in SON. In vivo treatment with the peptides antagonists or with an NMDA antagonist suppressed the transient increase in dendritic arbor of SON neurons at the beginning of PW2. Incubation of acute slices with these compounds decreased the dendritic arbor on a short time scale (3-8 hr) in slices of postnatal day 7 (P7) to P9 rats. Conversely, application of OT/AVP or NMDA increased dendritic branches in slices of P3-P6 rats. Their effects were inhibited by blockade of electrical activity, voltage-gated Ca2+ channels, or intracellular Ca2+ mobilization. They were also interdependent because both OT/AVP and NMDA (but not AMPA) receptor activation were required for increasing the dendritic arbor. Part of this interdependence probably results from a retrograde action of the peptides facilitating glutamate release. Finally, blocking OT/AVP receptors by in vivo treatment with the peptides antagonists during development decreased spontaneous glutamatergic synaptic activity recorded in young adults. These results show that an interplay between postsynaptic dendritic peptide release and presynaptic glutamate release is involved in the transient increase in dendritic arbor of SON neurons and indicate that OT/AVP are required for normal synaptogenesis of glutamatergic inputs in SON.

It is generally believed that only L-amino acids have a physiological role in species other than bacteria. Recently, the existence of some D-amino acids, particularly D-aspartate, in various organs of several higher animals has been reported. Here we demonstrate that naturally occurring free D-aspartate is localized subcellularly to the heterochromatin in the nucleoli (but not in either the dendrites or axonal terminals) of magnocellular neurosecretory neurons in the rat hypothalamus, and also of microglia and pericytes in the posterior pituitary. Our results imply that naturally occurring free D-aspartate might have a physiological role in nuclear function in mammals. The findings provide new insight for the biological function of D-stereoisomers of amino acids as well as the organization of the nucleus of at least some eukaryotic cells.

Agmatine (decarboxylated l-arginine), an endogenous ligand of imidazoline and alpha(2) adrenoreceptors, is particularly enriched in the rat hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. The present study utilized light and electron microscopic immunocytochemical methods to determine the distribution and extent of colocalization of agmatine relative to subpopulations of vasopressin- (VP) and oxytocin- (OT) producing neurons in PVN and SON nuclei. By light microscopy, agmatine-immunoreactive perikarya were found in both the magnocellular and the parvocellular neuronal subdivisions of PVN and SON. Confocal and electron microscopy revealed that agmatine-immunoreactivity (I) within neuronal perikarya was associated with the nuclear membrane as well as mitochondria, Golgi complexes, endoplasmic reticula, and plasmalemma. Additionally, agmatine-I was identified in both axons and axonal terminals, which were enriched in large dense-core vesicles. Dual and triple immunocytochemical labeling experiments also demonstrated that agmatine coexists with VP or OT in most PVN and SON magnocellular neurons. Combinations of iontophoretic injections of Fluorogold into the dorsomedullary complex with immunocytochemical labeling revealed that many retrogradely labeled neurons in the parvocellular region of the PVN contained agmatine-I and either VP or OT. These findings provide evidence that agmatine may function as a modulator of both hypothalamically mediated neuroendocrine and autonomic responses., (Copyright 2001 Academic Press.)

The cell-specific expression of both the oxytocin (OT) and vasopressin (VP) genes in magnocellular neurons (MCNs) of the hypothalamus has been proposed to be under the control of cis-elements in an intergenic region downstream of the VP gene. We examined this hypothesis using transgenic mice containing mouse genomic DNA-derived constructs linked to chloramphenicol acetyltransferase (CAT) reporters. VP gene expression was studied using constructs containing 3.8 kbp of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to a CAT reporter. The two VP-transgene constructs differed by the lengths of their VP gene 3' flanking regions (2.1 versus 3.6 kbp). A similar construct for the oxytocin CAT transgene was used which contained the full-length (3.6 kbp) downstream intergenic region between the mouse genes. All three transgenic constructs produced cell-specific expression of the CAT-reporter in the magnocellular neurons as determined by CAT-immunoreactivity. Oxytocin transgene expression was restricted to OT cells in two founders, and the two VP transgenes to VP cells in five founders. Electron microscopic immunocytochemistry showed that the CAT fusion proteins produced from the OT- and VP-transgenes were efficiently trafficked through the regulated secretory pathways in their respective magnocellular neurons, packaged into large dense core vesicles, and transported to nerve terminals in the posterior pituitary., (Copyright 2001 Academic Press.)

The distribution of the purine receptor P2X(6) subtype was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were visualised with ExtrAvidin peroxidase conjugate and immunogold-silver pre-embedding immunocytochemistry using a polyclonal antibody against an intracellular domain of the receptor. Application of ExtrAvidin labelling revealed P2X(6) receptors in subpopulations of: (i) neurosecretory cell bodies, neurosecretory and non-neurosecretory axons and dendrites of neurones in the paraventricular and supraoptic nuclei; and (ii) pituicytes and neurosecretory axons of the neurohypophysis. Some of the neurosecretory granules observed in the supraoptic and paraventricular nuclei neurone cell bodies, dendrites and axons as well as those in neurohypophysial axons were also positive for the P2X(6) receptors. In the paraventricular nucleus, some axons and dendrites of non-neurosecretory neurones positive for P2X(6) receptors formed synapses between themselves. Using the immunogold-silver method, the electron-dense particles labelling P2X(6) receptors were found in neurosecretory cell bodies of the supraoptic and paraventricular nuclei, in relation to the cytoplasm, endoplasmic reticulum, Golgi complex and neurosecretory granules. The particles indicative of P2X(6) receptors were also located in neurosecretory and non-neurosecretory axons including axonal buttons making synapses with P2X(6)-negative dendrites. In the neurohypophysis, the electron-dense particles were localised in a subpopulation of pituicytes and neurosecretory axons. In neurohypophysial axons, particles were at times seen over the membrane of some neurosecretory granules (immunogold label) or microvesicles (immunoperoxidase label). We speculate that the P2X(6) receptors at the neurohypophysial level may be implicated not only in hormone release from the axon terminals, but also in membrane recycling of the granular vesicles and microvesicles.

The human supraoptic nucleus (SON) is the main production site of plasma arginine-vasopressin (AVP). The present study aimed to define the subpopulation of patients in which multinucleated SON neurons (MNN) are present. We determined the proportion of MNN in immunocytochemically defined SON AVP population and compared AVP mRNA levels in the SON of 29 patients with and without MNN. Interestingly, MNN appeared to be present in a high proportion in patients with pneumonia and other pulmonary pathologies (Pul P). The percentage of MNN in the SON of Pul P patients turned out to be age- and sex-dependent. In young women with Pul P their proportion was 10 times higher than in women without such a pathology and in young men with Pul P their frequency was 22 times higher than in other men. In those patients with the highest proportion of MNN, i.e. young females and males with Pul P, AVP mRNA expression in the SON was the lowest. In addition, young women (less than or = 50 years old) had lower AVP mRNA levels than young men and than elderly women (>50 years old). In conclusion, our study suggests that multinucleated neurons are a hallmark of Pul P in the human SON and that this phenomenon may be accompanied by lower AVP production in young subjects.

Previous studies suggest that cholinergic neurons in the diagonal band of Broca (DBB) participate in the baroreceptor-mediated inhibition of phasic vasopressin neurons in the supraoptic nucleus (SON). To test this hypothesis, extracellular recordings were obtained from putative vasopressin SON neurons of anesthetized rats injected with the cholinergic immunotoxin 192 IgG-saporin (0.8 microg/microl) in the DBB. Baroreceptor sensitivity of neurons was tested with brief phenylephrine-induced (10 microg/10 microl iv) increases in blood pressure of at least 40 mmHg. In rats injected with vehicle or unconjugated saporin, 19 of 21 and 18 of 20 phasic neurons, respectively, were inhibited by increased blood pressure. In rats injected with 192 IgG-saporin, which significantly reduced the number of choline acetyltransferase (ChAT)-positive DBB neurons, 33 of 36 phasic neurons were inhibited. Normal rats and rats with DBB saporin injections received rhodamine bead injections into the perinuclear zone (PNZ) to retrogradely label DBB neurons, and their brains were stained for ChAT. ChAT-positive DBB neurons were not retrogradely labeled from the PNZ. Together, these results indicate that the pathway relaying baroreceptor information to the SON involves noncholinergic DBB neurons.

The relationship between the cloned kappa opioid receptor, dynorphin, and the neurohypophysial hormones vasopressin and oxytocin was analysed in the guinea-pig hypothalamic magnocellular neurosecretory neurons. This analysis was performed in order to understand better which population of neuroendocrine neurons in the guinea-pig is modulated by kappa opioid receptors and its endogenous ligand dynorphin. Extensive co-localization was observed between kappa opioid receptor immunoreactivity and preprodynorphin immunoreactivity in neuronal cell bodies in the paraventricular and supraoptic nuclei. Cells positive for either the kappa opioid receptor or both the kappa opioid receptor and preprodynorphin were restricted to the vasopressin expressing neuronal population and not found in the oxytocin expressing neuronal population. The kappa opioid receptor and dynorphin were examined in the posterior pituitary and both were found to be extensively distributed. Staining for the kappa opioid receptor and dynorphin B co-localized in posterior pituitary. In addition, immunogold electron microscopy confirmed that kappa opioid receptor and dynorphin B immunoreactivity were found in the same nerve terminals. Ultrastructural analysis also revealed that kappa opioid receptor immunoreactivity was associated with both nerve terminals and pituicytes. Within nerve terminals, kappa opioid receptor immunoreactivity was often associated with large secretory vesicles and rarely associated with the plasma membrane. Our data suggest that the cloned kappa opioid receptor may directly modulate the release of vasopressin but not oxytocin in guinea-pig hypothalamic magnocellular neurosecretory neurons and posterior pituitary. Furthermore, we propose that this receptor is an autoreceptor in this system because our results demonstrate a high degree of co-localization between kappa opioid receptor and dynorphin peptide immunoreactivity in magnocellular nerve terminals.

Ultrastructure of axosomatic (AS) and axodendritic (AD) synapses in the supraoptic nucleus (NSO) was investigated in a group of normothermic cats and compared with another group of cats after short-term induced hypothermia. Quantitative analysis demonstrated a significant decrease of number of AS synapses, smaller size of AD synaptic knobs, shorter length of the synaptic contact and an increase of the active zone. Evaluation of the shape of the synaptic cleft demonstrated an increase of both positive (P) and negative (N) types in hypothermic animals. The observed morphological changes can be ascribed to the decreased synaptic activity in a greater part of the synaptic population and to the increased activity in a smaller portion of synapses.

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.

Inorganic mercury (HgCl2) was administered to adult mice in drinking water (20 mg/l). Animals were sacrificed after one or two years and fixed by whole-body perfusion. Sections of the hypothalamus and neurohypophysis were subjected to silver acetate autometallography for visualization of mercury at light and electron microscopy levels. Mercury deposits, which can be seen by light microscopy as black granules, were found to accumulate within neuronal perykaria of the supraoptic and paraventricular nuclei. Electron microscopy demonstrated that mercury deposits in neurosecretory neurons were detected exclusively within lysosomes. Mercury was also present in small vesicles, 40-70 nm in diameter, and in endocytic vacuoles within the axon terminals of the neurohypophysis. No mercury could be seen in sections obtained from control animals that had been drinking uncontaminated water. Mechanisms involved in uptake and transport of mercury to neuronal bodies are discussed.

The distribution of the P2X(2) subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X(2) receptors was localised in: (i) paraventricular and supraoptic nuclei-in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis-in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X(2)-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X(2)-positive axons on P2X(2)-positive endocrine cell bodies as well as on P2X(2)-negative somata were also observed. The functional significance of these findings is discussed.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Strong immunoreactivity for neuronal nicotinic acetylcholine receptor alpha4 subunit was detected in neurons of the supraoptic nucleus (SON). At the ultrastructural level, immunoreactivity for alpha4 was detected in the post-synaptic membranes as well as in the cytoplasmic matrices in the magnocellular neurons. Nicotine (1-10 microM) increased cytosolic Ca2+ concentrations ([Ca2+]i) in isolated arginine-vasopressin (AVP)-containing neurons in the rat SON. Nicotine (10 microM) was less potent in increasing [Ca2+]i in AVP-containing neurons than noradrenaline (1 microM), a known neurotransmitter in the SON. The nicotine-induced [Ca2+]i increase in AVP-containing neurons was markedly reduced when pre-treated with a protein kinase A (PKA) blocker, H89 (40 microM). These findings suggest that nicotine, a known neurotransmitter in the SON, activates AVP-containing neurons via nicotinic acetylcholine receptor which is linked to stimulation of cAMP-PKA-regulated Ca2+ signaling pathway.

The disector, an unbiased stereological method for evaluation of synaptic densities, was used to analyse putative GABA and glutamate innervations of the supraoptic nucleus of virgin and lactating rats. The analysis was performed on ultrathin sections labelled for either of the amino acids with a postembedding immunogold technique. Our observations showed that the volume of the nucleus increased by 40% in lactating animals, an increase due to a significant enlargement of dendritic and somatic, but not vascular, volumes. Nevertheless, values of overall synaptic densities in the whole nucleus remained as high as those in virgin rats (37-40 x l0(6) synapses/mm3). About 45% of all synapses were immunoreactive for GABA and 25% for glutamate; there were twice as many GABA- and glutamate-positive synapses on dendrites as on somata. When we estimated synaptic densities in relation to the neuropil (by subtracting the proportion of sampled areas occupied by somatic profiles), we found a significant increase in synaptic density in lactating animals. This affected axodendritic as well as axosomatic synapses, immunopositive and immunonegative for GABA or glutamate. The disector also allowed us to determine that the number of synapses from terminals making contacts on several somata and/or dendrites simultaneously constituted about 9% of all synapses in virgin rats, a proportion which more than doubled in lactating rats. About 50% were immunopositive for GABA and 30% for glutamate. Our data offer further evidence of physiologically-linked structural synaptic plasticity in the supraoptic nucleus and clearly demonstrate that it affects both inhibitory and excitatory inputs on dendrites, as well as on somata, throughout the nucleus.

Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.

Despite several studies showing that the rat supraoptic (SON) and paraventricular (PVN) nuclei are innervated by noradrenergic afferents, the respective contribution of these inputs to the oxytocinergic and vasopressinergic neuronal populations remains to be clearly defined. In the present study, we used the unbiased disector method to estimate the numerical density of noradrenergic varicosities on identified oxytocinergic and vasopressinergic somata in the rat SON and PVN. The analysis was carried out on semithin (1 micron) plastic sections cut from vibratome slices (50 microns) of the SON and PVN which had been double-labelled for noradrenaline (NA) and oxytocin- or vasopressin-related neurophysin. These preparations displayed many noradrenergic varicosities which electron microscopy showed to represent, in the main, synaptic boutons. Our quantitative analysis revealed that noradrenergic varicosities contacted oxytocinergic and vasopressinergic somata to a similar extent in male and female rats, under basal conditions of hormone secretion. The incidence of these axo-somatic contacts was similar in the SON and PVN. In contrast, in lactating rats, in which oxytocin secretion is enhanced, there was a significant increase in the density of noradrenergic varicosities apposed to oxytocinergic somata, in both nuclei. Our observations indicate that, in male and female rats under normal conditions, noradrenergic afferents innervate each type of neurosecretory somata, in both magnocellular nuclei, in a similar fashion. They reveal, moreover, that noradrenergic afferents participate in lactation-induced structural plasticity of synapses impinging on oxytocinergic somata.

The median preoptic nucleus (MnPo) is critical for normal fluid balance, mediating osmotically evoked drinking and neurohypophysial hormone secretion. The influence of the MnPo on vasopressin and oxytocin release is in part through direct connections to the supraoptic and paraventricular nucleus. In the present investigation the synaptic contacts between the MnPo and supraoptic neurons were investigated in rats by ultrastructural examination of terminals labeled anterogradely with the tracers Phaseolus vulgaris-leucoagglutinin or biotinylated dextran. At the light microscopic level, labeled fibers within the supraoptic nucleus branched frequently, were punctuated by varicosities, and were distributed throughout the nucleus without preference for the known distributions of oxytocin and vasopressin neurons. At the ultrastructural level, synapses were associated with many of these varicosities. The ratio of labeled axodendritic to axosomatic synapses encountered was roughly consistent with a uniform innervation of dendrites and somata. The great majority of synapses were characterized by symmetrical contacts. Similar results were found for a few injections made in the organum vasculosum of the lamina terminalis, just rostral to the MnPo, and in the immediately adjacent periventricular preoptic area. Coupled with other recent anatomical and electrophysiological evidence, these results suggest there is a strong monosynaptic pathway from structures along the ventral lamina terminalis to the supraoptic nucleus.

Activating direct olfactory (glutamatergic) inputs to supraoptic nucleus (SON) neurons increases interneuronal coupling in slices from lactating but from not virgin or male rats. Studied here were influences on coupling of another monosynaptic input to SON, the histaminergic tuberomammillary nucleus (TM) projection, activation of which selectively excites phasically firing (putative vasopressin) cells. Effects of TM stimulation and its possible downstream consequences on Lucifer yellow (LY) dye coupling among putative vasopressin cells were determined in male rat SONs. In unstimulated slices, 12 LY injections (1 cell/SON) yielded eight single and four pairs of coupled neurons. In slices in which TM was stimulated for 10 min at 10 Hz, 13 injections yielded 4 single and 28 coupled cells, with groups of 2 to 4 cells coupled to the injected neuron, a threefold increase in the number of coupled cells per injection (p < 0.02). Bathing slices in medium containing 10 microM pyrilamine (H1 antagonist) blocked this stimulation-induced coupling increase, suggesting mediation by activation of guanylate cyclase-cGMP to which H1 receptors often are linked . Bathing slices in medium containing 0.5-1 mM 8-bromo-cGMP yielded results similar to those of TM stimulation, a 2.5-fold increase over control in the number of coupled cells per injection. Effects of TM stimulation on coupling also were blocked by bathing slices in a guanylate cyclase inhibitor (10 microM LY83583). In contrast to cGMP, 1 mM 8-bromo-cAMP significantly reduced coupling. We conclude that synaptically released histamine increases coupling via cGMP-dependent mechanisms.

The study was carried out on lines of rats selected for high (KHA) and low (KLA) rates of development of an active avoidance conditioned reflex in a shuttle box. The volume of nucleoli of oxytocinergic cells of the paraventricular and supraoptic nuclei of the hypothalamus, as well as the content of oxytocin-immunopositive material in the posterior lobe of the hypophysis, were measured. The volume of the nucleoli in the intact KHA rats was smaller, and the amount of oxytocin-immunopositive material in the posterior lobe of the hypophysis greater, than in the KLA rats. A stressor (unavoidable electrodermal stimulation) exerts an opposite effect on the oxytocinergic system of the hypothalamus in rats of the two lines: the volume of the nucleoli increases in the KHA rats, while the amount of oxytocin-immunopositive material in the posterior lobe of the hypophysis decreases; the changes are opposite in the KLA rats. These results suggests that the reactivity of the oxytocinergic system is closely associated with the specific features of the animal's adaptive behavior, in particular, with the capacity for learning.

Recent studies have supported the existence of projections to the paraventricular and supraoptic nuclei of the hypothalamus that arise from non-catecholaminergic neurons in the nucleus of the solitary tract, whose terminal distribution is suggestive of interactions with both parvocellular and magnocellular neurosecretory neurons. Pre-embedding immunolabeling methods were used to compare and characterize the termination patterns of axons immunoreactive for two putative markers for this projection system, inhibin beta and somatostatin-28, at the ultrastructural level. Axon terminal profiles stained for either peptide were found to form symmetric or asymmetric junctions predominantly with the shafts of unlabeled dendrites of varying caliber. A small percentage of peptidergic terminals was found in both hypothalamic nuclei to engage in so-called 'shared synapses', where a single terminal profile contacted two postsynaptic elements. Axo-somatic terminations were relatively rarely seen in the supraoptic nucleus, but were somewhat more abundant in the paraventricular nucleus. These comprised principally symmetric junctions onto the somatic membranes of an ostensibly mixed population of cells, some of which bore apparent neurosecretory specializations. Combined immunoperoxidase and immuno-autoradiographic staining methods were used to estimate the extent to which either terminal type interacts with oxytocin neurons. Oxytocin stained elements comprised a minority of the postsynaptic targets of both peptidergic terminal types in the paraventricular nucleus, and a scant majority of those in the supraoptic nucleus. These results support the view that peptidergic neurons in the caudal nucleus of the solitary tract interact synaptically with multiple cell types in the parvocellular division of the paraventricular nucleus, and preferentially with oxytocinergic elements in the

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

The postsynaptic AMPA/kainate and N-methyl-D-aspartate-selective glutamate receptors are formed by several different subunits and the overall subunit composition of the receptor appears to determine its physiological and pharmacological properties. Although glutamatergic mechanisms have been implicated in various forms of hippocampal stress responses, the impact of stress on glutamate receptor subunit composition has not yet been elucidated. We have used cell-by-cell quantitative in situ hybridization to assess stress-induced changes in transcript levels of N-methyl-D-aspartate and AMPA receptor subunit genes in subdivisions of the rat hippocampus and hypothalamus that are implicated in the stress response. We found that 24 h after a single immobilization stress there was a significant increase in the cellular level of NR1 subunit messenger RNA (about 35-45% above control values) in hippocampal CA3 and CA1 pyramidal cells as well as in neurons of the hypothalamic supraoptic and paraventricular nuclei. Moreover, in the CA3 area we have detected a concomitant increase (50% above controls) in the level of NR2B subunit messenger RNA, while the expression of NR2A subunit gene did not change after stress. Stress induced a selective decrease in the level of AMPA receptor subunit glutamate receptor A messenger RNA in neurons of both the CA3 and CA1 areas (18 and 24%, respectively, below control values). These results suggest that the regulation of specific subunit messenger RNAs of the N-methyl-D-aspartate and AMPA receptors may be involved in altered hippocampal and hypothalamic responsiveness to glutamate and thus could play a critical role in stress-induced changes in their function.

The cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptor, GluR3, was identified using antibodies that recognize the N-terminus of the predicted polypeptide sequence of GluR3. Regional immunoblot analysis of monkey brain homogenates identified a protein of approximately 102,000 mol. wt that was enriched in hypothalamus. Immunocytochemistry demonstrated that GluR3 was enriched within the hypothalamic magnocellular neurosecretory nuclei and axons of the hypothalamo-neurohypophysial tract in rat and monkey. GluR3 immunoreactivity co-localized to oxytocin-containing, but not vasopressin-containing, neurons of the hypothalamic paraventricular nucleus, supraoptic nucleus and accessory magnocellular nuclei. Ultrastructurally, GluR3 immunoreactivity was enriched throughout cytoplasm of the somatodendritic compartment and was associated with postsynaptic and presynaptic structures. GluR3 immunoreactivity was frequently observed to be clustered at the plasma membrane of the somatodendritic compartment, consistent with the predicted localization of a membrane-bound ion channel. Additionally, GluR3-immunoreactive axon terminals in synaptic contact with unlabeled dendrites within the retrochiasmatic area and bed nucleus of the stria terminalis were observed, providing morphological evidence for a presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor. By immunoblot analysis and immunocytochemistry using antibodies directed against a specific alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor in rat and monkey brain, our findings suggest a highly selective hypothalamic distribution of the GluR3 subunit that may have functional significance in the glutamatergic regulation of oxytocinergic neurons.

The adult oxytocinergic system undergoes extensive synaptic and neuronal-glial remodelling in response to differing conditions of secretion and has become a remarkable example of activity-dependent structural plasticity in the adult mammalian brain. Under stimulation (parturition, lactation, chronic dehydration), glial coverage of oxytocin neurons is significantly reduced and their surfaces become extensively juxtaposed; concurrently, they are contacted by an increased number of synapses. These changes are reversible with cessation of stimulation. We here present observations showing that putative inhibitory and excitatory afferents contribute to this synaptic plasticity. The data are derived from several different comparative analyses of ultrathin sections of the rat supraoptic nucleus (SON) in which presynaptic (gamma-amino butyric acid (GABA) or glutamate) and postsynaptic (oxytocin or vasopressin) partners were identified with postembedding immunogold staining. We thus found that in virgin rats, under basal conditions of oxytocin release, 30-40% of synapses on oxytocinergic or vasopressinergic somata in the SON are GABAergic and about 20% glutamatergic. On the other hand, in lactating rats, in which oxytocin secretion is greatly enhanced, there was an increase in the incidence of both types of synapses, and in particular, on those impinging on oxytocinergic somata.

Central to this investigation are several basic hypotheses that are designed to test the role of nitric oxide (NO) in the complex process of central regeneration and plasticity in a well established model system of the mammalian brain. We have employed histochemical techniques at the light and ultrastructural level coupled with correlative scanning electron microscopy, immunoelectron microscopy, and in situ hybridization in order to determine the functional significance of the increased expression of nitric oxide synthase (NOS) in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei which accompanies regeneration of their axotomized neurites following hypophysectomy. The aim of this investigation was to determine the potential role and temporal up-regulation of NOS in this basic regenerative process and to establish the ultrastructural and neuroanatomical correlates during critical periods of regeneration and regrowth of SON and PVN axons following hypophysectomy in the endocrine hypothalamus of the rat. Our data support the hypothesis that NO may serve as a second messenger molecule that may act in some fashion to govern not only the process of central regeneration and regrowth of magnocellular (SON/PVN) axons into the median eminence, neural stem, and neural lobe (the neurohypophyseal system) but may also influence the regeneration of neurites into new neuroanatomical domains such as the adjacent lumen of the third cerebral ventricle. We have demonstrated a distinct temporal relationship between injury (axotomy) of SON/PVN axons and the establishment of new neurovascular zones following hypophysectomy with the up-regulation of NOS. This up-regulation appears to correlate well with successful regeneration in the mammalian neurohypophyseal system. We have also successfully inhibited axonal regeneration with the use of nitroarginine, a competitive antagonist of NO. NOS up-regulation attendant to regeneration of SON and PVN axons may have inestimable clinical implications, particularly with respect to closed head injury and cerebral contusion that involves the mechanical shearing of the infundibular stalk. In addition, this investigation has reaffirmed that large numbers of bona fide neurons migrate and emerge upon the floor of the adjacent third cerebral ventricle shortly following hypophysectomy (within 2 weeks). The origin and mechanisms of neuronal migration and plasticity following hypophysectomy are the subject of interpretation and discussion in this investigation.

In the present experiments, we examined the effect of ovariectomy on the increases in litter weight and structural plasticity of MNCs in the supraoptic nucleus (SON) during lactation. Female rats were ovariectomized 2 days after parturition, and the increases in litter weight were measured as the index of milk let-down from dams during lactation. The lactation period was elongated up to 6 weeks by providing new litter to obtain more apparent effects of the ovariectomy. There was no significant difference in the increases in litter weight between non-operated and ovariectomized females. After lactation for 6 weeks, the ultrastructures such as juxtaposition (surface membrane apposition) and multiple synapses (terminals contacting with two or more postsynaptic elements) of MNCs in the SON in nonoperated and ovariectomized females were examined to compare with those of virgins. The percentage of juxtaposition and the number of multiple synapses significantly increased in nonoperated lactating females as compared with those of virgins. Ovariectomized rats showed similar structural changes to those of nonoperated females during lactation. Therefore, we conclude that ultrastructural plasticity of MNCs in the SON is maintained even in the absence of an ovary, and direct or indirect actions of suckling stimulation may be important in maintaining the plasticity during lactation.

The distribution of vasopressin (VP)-like and oxytocin (OX)-like immunoreactivities was investigated in the chameleon brain with antisera specific for either peptide. VP-like and OX-like neurons are found only in two nuclei: in the supraoptic and in the paraventricular nuclei, whereas both peptidergic innervations are restricted to some ventral areas, especially to the hypothalamus. However, in these areas, OX-like innervation is less dense than the VP-like innervation. Even though, no sex differences were noted for the two systems, differences are present between the normal female and the preoviposit one concerning VP-like system. In the latter female, numerous VPergic-like cell bodies occurred in the ependymal layer, making likely direct contact with the cerebrospinal fluid (CSF) of the third ventricle. In addition, some VP-like nerve fibers seem to be in supraependymal position at the septal wall, probably also in contact with CSF.

Background: The present paper describes the cytoarchitectonic, morphometric, and three-dimensional characteristics of the golden hamster supraoptic nucleus (SON) in order to provide an anatomical basis for subsequent morphofunctional studies that use this species as an experimental animal. The dimensions (volume and length) and the number of cells of each part of the supraoptic nucleus were obtained, as well as morphometric parameters of their neurons (cross-sectional area and maximum and minimum diameters). A three-dimensional reconstruction of hamster SON has been made in order to know the spatial morphology of this nucleus and to reveal the structural differences between both parts., Methods: Ten male adult golden hamsters (Mesocricetus auratus) were used. Animals were anaesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2. The hypothalamic area from seven animals was dissected out, dehydrated, and embedded in paraffin. Serial sections of 10 microns were cut in a coronal plane. Sections were stained with thionin, dehydrated, cleared in eucalyptol, and mounted with Eukitt. To prove the neurosecretory nature to the SON, every fourth section was immunostained against neurophysin by using the peroxidase-antiperoxidase method. To study the neuronal morphometric parameters, all magnocellular neurons of the SON were drawn in sections separated 80 microns with the aid of a camera lucida under 500x magnification. Serial 50 microns thick frozen sections of the hypothalamus from three animals were drawn with camera lucida to determine the volume of the two parts of the SON and to make the three-dimensional reconstruction., Results: The SON extends rostrocaudally 1.98 +/- 0.03 mm from the preoptic area to the tuberal hypothalamic area. Two classical parts can be clearly delimited: principal (SONp) and retrochiasmatic (SONr). The neuronal population of the two parts of the SON appears constituted only by magnocellular neurons. The volume of the SONp is 0.039 +/- 0.03 mm3 and contains about 762 +/- 93 magnocellular cells, with a density of 19,151.8 cells/mm3. The volume of the SONr is 0.126 +/- 0.03 mm3 and contains about 1,296 +/- 132 neurons with a density of 10,536.6 cells/mm3. The three-dimensional reconstruction reveals that the SONp appears located in a more cephalic, lateral, and dorsal position than the SONr, and a clear discontinuity between the two parts is observed., Conclusions: The present study shows that the classically termed SON, in the hamster, clearly consists of two spatially separated neural populations. The SONr is longer than SONp and has the larger volume and higher number of neurons; however, the neurons of the SONr are smaller in cell area than those of the SONp.

The supraoptic (SON) and paraventricular nuclei (PVN) of the human hypothalamus are production sites of vasopressin (AVP) and oxytocin (OXT). Although the hypothalamus is affected in Alzheimer's disease (AD), previous work has not only shown that in these two nuclei no neurons are lost, neither during aging nor in AD, but that the number of AVP-expressing neurons and their nucleolar size had even increased with age. These observations indicated that the peptide synthesis of the AVP neurons was activated in the oldest age-groups. Recently published, qualitative observations, using the area of the Golgi Apparatus (GA) as a sensitive parameter for neurosecretory activity, confirmed the activation of SON and PVN neurons with age in human; however, in this report the neurons were not identified according to their neuropeptide content. In the present quantitative study we determined whether the AVP neurons were indeed activated as a result of the aging process in controls and AD patients. We applied a polyclonal antiserum directed against the medial cisternae of the GA on formalin-fixed, paraffin-embedded tissue sections taken from the dorsolateral SON (dl-SON) of 10 controls and 10 AD patients, and performed our measurements in this area that is known to be predominantly occupied (90-95%) by AVP neurons. In addition, the sparse OXT cells present in the area of study, were excluded from the measurements on the basis of alternative sections stained for OXT. In the dl-SON, the area occupied by the GA and the cellular profile area per patient were quaNtified by means of image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Efferent projections of the lateral septal nucleus (LS) to the preoptic area and the hypothalamus were identified in 20 female guinea pigs after iontophoretic injection of the anterograde axonal tracer Fluoro-Ruby. Tubero-infundibular (TI) neurons of the preoptic area and the hypothalamus were retrogradely labeled after intracardiac injection of Granular Blue or Fluoro-Gold. Magnocellular neurons of the supraoptic and paraventricular nuclei were also labeled. The double labeling procedure allowed an estimation of the extent of the direct relationship between LS efferents and TI neurons. Contacts between lateral septal fibers and TI cell bodies were mainly observed at the light-microscopical level in the preoptic area. A group of labeled fibers coursing along the third ventricle established sparse connections with hypothalamic periventricular TI neurons. A few appositions was observed in the infundibular (arcuate) nucleus, suggestive of a monosynaptic regulation of TI neurons by a septo-arcuate tract. Close association with labeled magnocellular neurons was also noted at the edge of the supraoptic and paraventricular nuclei. The sparse but direct connections between LS and TI neurons may be involved in the neuroendocrine functions of the LS.