Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms.

Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of vasopressin release.

Hyposmolality differentially and spatiotemporally modulates levels of glutamine synthetase and serine racemase in rat supraoptic nucleus.

Prolonged hyposmotic challenge (HOC) has a dual effect on vasopressin (VP) secretion [Yagil and Sladek (1990) Am J Physiol 258(2 Pt 2):R492-R500]. We describe an electrophysiological correlate of this phenomenon, whereby in vitro HOC transiently reduced the firing activity of VP neurons within the supraoptic nucleus of brain slices, which was followed by a rebound increase of their activity; this was paralleled by changes in the level of proteins relevant to astroglia-neuronal interactions. Hence, in vitro HOC transiently (at 5 min) increased the level of astrocyte-specific glial fibrillary acidic protein (GFAP), which then declined to control or base level (at 20 min); this was blocked by the gliotoxin L-aminoadipic acid, but not by tetanus toxin, which was used to inhibit neurotransmission. Similarly, in vivo HOC led to changes in GFAP level, which after an early increase (10 min) returned to normal (30 min). Immunoassays revealed that neuronal, but not astrocytic, expression of serine racemase (SR) was increased at the late stage of HOC in vivo, whereas at an early stage there was a transient increase in level of the astrocyte-specific glutamine synthetase (GS). Furthermore, there was an increased molecular association between GFAP and GS at 10 min, whereas SR increased its association with the neuronal nuclear antigen NeuN at 30 min. These results suggest that the dual effect of HOC on VP neuronal secretion/activity could be related to metabolic/signaling changes in astrocytes (glutamate-glutamine conversion) and neurons (D-serine synthesis/ammonia production), which may account for the rebound in VP neuronal activity, presumably by promoting the activation of neuronal glutamate receptors.

Apoptosis of supraoptic AVP neurons is involved in the development of central diabetes insipidus after hypophysectomy in rats.

BACKGROUND: It has been reported that various types of axonal injury of hypothalamo-neurohypophyseal tract can result in degeneration of the magnocellular neurons (MCNs) in hypothalamus and development of central diabetes insipidus (CDI). However, the mechanism of the degeneration and death of MCNs after hypophysectomy in vivo is still unclear. This present study was aimed to disclose it and to figure out the dynamic change of central diabetes insipidus after hypophysectomy. RESULTS: The analysis on the dynamic change of daily water consumption (DWC), daily urine volume(DUV), specific gravity of urine(USG) and plasma vasopressin concentration showed that the change pattern of them was triphasic and neuron counting showed that the degeneration of vasopressin neurons began at 10 d, aggravated at 20 d and then stabilized at 30 d after hypophysectomy. There was marked upregulation of cleaved Caspase-3 expression of vasopressin neurons in hypophysectomy rats. A "ladder" pattern of migration of DNA internucleosomal fragments was detected and apoptotic ultrastructure was found in these neurons. There was time correlation among the occurrence of diabetes insipidus, the changes of plasma vasopressin concentration and the degeneration of vasopressin neurons after hypophysectomy. CONCLUSION: This study firstly demonstrated that apoptosis was involved in degeneration of supraoptic vasopressin neurons after hypophysectomy in vivo and development of CDI. Our study on time course and correlations among water metabolism, degeneration and apoptosis of vasopressin neurons suggested that there should be an efficient therapeutic window in which irreversible CDI might be prevented by anti-apoptosis.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Increased nitric oxide synthase activity and expression in the hypothalamus of hindlimb unloaded rats.

Upon return from spaceflight or resumption of normal posture after bed rest, individuals often exhibit cardiovascular deconditioning. Although the mechanisms responsible for cardiovascular deconditioning have yet to be fully elucidated, alterations within the central nervous system have been postulated to be involved. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important brain regions in control of sympathetic outflow and body fluid homeostasis. Nitric oxide (NO) modulates the activity of PVN and SON neurons, and alterations in NO transmission within these brain regions may contribute to symptoms of cardiovascular deconditioning. The purpose of the present study was to examine nitric oxide synthase (NOS) activity and expression in the PVN and SON of control and hindlimb unloaded (HU) rats, an animal model of cardiovascular deconditioning. The number of neurons exhibiting NOS activity as assessed by NADPH-diaphorase staining was significantly greater in the PVN but not SON of HU rats. Western blot analysis revealed that neuronal NOS (nNOS) but not endothelial NOS (eNOS) protein expression was higher in the PVN of HU rats. In the SON, there was a strong trend for an increase in nNOS (p=0.052) and a significant increase in eNOS expression in HU rats. Our results suggest that increased nNOS in the PVN contributes to autonomic and humoral alterations following cardiovascular deconditioning. In contrast, the functional significance of increases in nNOS and eNOS protein in the SON may be related to alterations in vasopressin release observed previously in HU rats.

Age-related changes in oxytocin-, arginine vasopressin- and nitric oxide synthase-expressing neurons in the supraoptic nucleus of the rat.

Many histochemical investigations indicated that the oxytocin (OXY), the arginine vasopressin (AVP) and the nitric oxide synthase (NOS) have been synthesized in the supraoptic nucleus (SON) neurons. The objective of this study was to examine the age-related expression of the OXY, the AVP and the NOS in the SON of the young adult (2-month-old) and the aged (24-month-old) rats. The histochemistry for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d; marker for the NOS) and the double labeling histochemistry for the OXY/NADPH-d or the AVP/NADPH-d were employed, and the quantitative analysis was performed with a computer-assisted image processing system. In comparison of the young adult and the aged group, the cell number, the cell size and the reactive density of the NOS-expressing neurons showed a significant increase along with age, and these evidences suggested the age-related increase of the nitric oxide (NO) production. The age-related significant increase was not detected in the number of the OXY/NOS-expressing neurons in the dorsal part, but was detected in the number of the AVP/NOS-expressing neurons in the ventral part. Based on our histochemical findings and reports demonstrated by other authors, we attempted to discuss the physiological role of NOS for the secretion of posterior pituitary hormones along with age.

Neuronal nitric oxide synthase inhibition differentially affects oxytocin and vasopressin secretion in salt loaded rats.

Nitric oxide, an endogenous gas produced by nitric oxide synthase (NOS), has been described as a neuromodulator of hormone secretion, including the neurohypophysial peptides oxytocin (OT) and vasopressin (AVP), hormones involved in the sodium and water homeostasis. The presence of NOS in the hypothalamic nuclei as well as in the circumventricular organs suggests a nitrergic regulation of OT and AVP secretion. Thus, the aim of this study was to evaluate the effect of 7-nitroindazole (7-NI), a selective inhibitor of neuronal NOS, in the plasma OT and AVP levels in rats submitted to a short and long-term salt loading. We also evaluated the NOS activity in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Our data showed an increase of plasma OT and AVP levels in both short and long-term salt loading. The augment of plasma OT and AVP levels was accompanied by an increase of NOS activity in the SON and PVN. The injection of 7-NI potentiated the increase of plasma OT induced by salt loading, but inhibited the increase of plasma AVP in the same experimental conditions. These results indicate that, under short and prolonged osmotic stimulation, nitric oxide may differentially control the neurohypophysial secretion.

Glucocorticoids regulate peptidyl-glycine alpha-amidating monooxygenase gene expression in the rat hypothalamic paraventricular nucleus.

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254.

Stimulated vasopressin (VP) release from magnocellular neuroendocrine cells in the supraoptic nucleus (SON) of hyperosmotic rats is inhibited by treatment with the industrial polychlorinated biphenyl (PCB) mixture, Aroclor 1254. Because VP responses to hyperosmotic stimulation are regulated by nitric oxide (NO) signaling, we studied NO synthase (NOS) activity in the SON of hyperosmotic rats as potential target of PCB-induced disruption of neuroendocrine processes necessary for osmoregulation. To examine PCB-induced changes in NOS activity under normosmotic and hyperosmotic conditions, male Sprague-Dawley rats were exposed to Aroclor 1254 (30mg/kg/day) in utero and NADPH-diaphorase (NADPH-d) activity was assessed in SON sections at three ages: postnatal day 10, early adult (3-5 months) or late adult (14-16 months). Hyperosmotic treatment increased mean NADPH-d staining density of oil hyperosmotic controls by 19.9% in early adults and 58% in late adulthood vs normosmotic controls. In utero exposure to PCBs reduced hyperosmotic-induced upregulation of NADPH-d activity to control levels in early adults and by 28% in late adults. Basal NADPH-d was reduced in postnatal rats. Rats receiving PCB exposure as early adults orally for 14 days displayed normal responses. Our findings show that developmental but not adult exposure to PCBs significantly reduces NOS responses to hyperosmolality in neuroendocrine cells. Moreover, reduced NADPH-d activity produced by in utero exposure persisted in stimulated late adult rats concomitant with reduced osmoregulatory capacity vs oil controls (375±9 vs 349±5mOsm/L). These findings suggest that developmental PCBs permanently compromise NOS signaling in the activated neuroendocrine hypothalamus with potential osmoregulatory consequences.

Localisation of 11ß-Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular Nuclei.

An accumulating body of evidence suggests that the activity of the mineralocorticoid, aldosterone, in the brain via the mineralocorticoid receptor (MR) plays an important role in the regulation of blood pressure. MR was recently found in vasopressin and oxytocin synthesising magnocellular neurosecretory cells (MNCs) in both the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus. Considering the physiological effects of these hormones, MR in these neurones may be an important site mediating the action of aldosterone in blood pressure regulation within the brain. However, aldosterone activation of MR in the hypothalamus remains controversial as a result of the high binding affinity of glucocorticoids to MR at substantially higher concentrations compared to aldosterone. In aldosterone-sensitive epithelia, the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) prevents glucocorticoids from binding to MR by converting glucocorticoids into inactive metabolites. The present study aimed to determine whether 11ß-HSD2, which increases aldosterone selectivity, is expressed in MNCs. Specific 11ß-HSD2 immunoreactivity was found in the cytoplasm of the MNCs in both the SON and PVN. In addition, double-fluorescence confocal microscopy demonstrated that MR-immunoreactivity and 11ß-HSD2-in situ hybridised products are colocalised in MNCs. Lastly, single-cell reverse transcriptase-polymerase chain reaction detected MR and 11ß-HSD2 mRNAs from cDNA libraries derived from single identified MNCs. These findings strongly suggest that MNCs in the SON and PVN are aldosterone-sensitive neurones.

Amidating processing enzyme complex for bioactive peptides (PAM) shows differences in specific activity and form in secretory granules isolated from the proximal and distal parts of the hypothalamo-neurohypophyseal tract in rats.

In rats the PAM specific activity in hypothalamic and neurohypophyseal extracts was 0.58 +/- 0.8, respectively 1.78 +/- 0.6 nmol.mg prot.-1 x h-1 (n = 5). PHM specific activity in the soluble part of the granules was higher in the neurohypophyseal than in the hypothalamic granules, and the fraction of total PHM and PAL present in the soluble part increased with the distance from the hypothalamus from some 45% to approx. 85%. Western blots of membrane and soluble granule fractions showed prevalence of higher mol. wt. forms in hypothalamic granules. It would appear that higher mol. wt. forms of PAM are processed by proteolytic enzymes during transport in the neuron and that non-neural cells in the neurohypophysis have a considerable PAM activity.

Local synaptic organization of cholinergic neurons in the basolateral hypothalamus.

A monoclonal antibody to choline acetyltransferase (ChAT) was utilized for immunocytochemical identification of cholinergic neurons in the basolateral hypothalamus. Light and electron microscopic examination revealed a network of cell bodies, dendrites, and axonal processes dorsolateral to the supraoptic nucleus. Within this region the cells immunoreactive for ChAT receive numerous unlabeled terminals which contact dendrites, cell soma, axons and occasional somatic spines. In a few cases, small ChAT-immunoreactive terminals were observed contacting a cholinergic cell soma or large dendrite. Many ChAT-immunoreactive fibers were directed toward the supraoptic nucleus forming a dense local network but very few of these fibers penetrated deeper than approximately 20 micron into the supraoptic nucleus. A total of 63 ChAT-immunoreactive terminals were mapped within the basal hypothalamus, of which the vast majority contacted unlabeled dendrites immediately dorsolateral to the supraoptic nucleus. Labeled terminals were rare or nonexistent in the medial portions of the hypothalamus or deep within the supraoptic nucleus. This pattern of ChAT terminal densities correlates with the distribution of binding for the muscarinic cholinergic probe, [3H]quinuclidinylbenzilate, but not the binding of the putative nicotinic cholinergic probe, [125I]alpha-bungarotoxin, which is high within the supraoptic nucleus. Thus, the cholinergic neurons of the basal hypothalamus appear to form a network of intrinsic connections which probably represent input to muscarinic cholinergic receptors. No evidence was found to suggest that cholinergic presynaptic terminals were colocalized with the alpha-bungarotoxin binding protein within the supraoptic nucleus.

Distribution of tyrosine-hydroxylase-immunoreactive neurons in the hypothalamus of rats.

The distribution and morphology of cells containing tyrosine hydroxylase (TH) immunoreactivity in the hypothalamus of rats were studied by using a modified immunoperoxidase technique. The TH cell system is more complexly organized than was previously thought. On the basis of their clustering patterns, hypothalamic TH neurons could be subdivided into two groups: dorsal and ventral. The ventral group consists of a prominent aggregate of cells located in the caudal part of the arcuate nucleus. From here, cells extend around the caudal part of the ventromedial and dorsomedial nuclei and the base of the diencephalon. Tyrosine hydroxylase-positive cells are present throughout the arcuate nucleus, except in its ventromedial part. Anteriorly, immunoreactive cells appear in the suprachiasmatic and supraoptic nuclei, in the retrochiasmatic area, and in the ventral part of the anterior hypothalamic nucleus. The dorsal group has its main concentration of cells in the medial part of the zona incerta, from which two clusters of cells, one medial and one lateral, extend rostralward. The medial group comprises cells in the medial part of the dorsomedial, paraventricular, and anterior hypothalamic nuclei. These cells adjoin the periventricular cells. The lateral group of cells emanating from the zona incerta occupies the lateral part of the dorsomedial and anterior hypothalamic nuclei and the dorsal hypothalamic area. The dorsal and ventral TH cell groups are in continuity medially in the periventricular layer, and laterally through the cells that surround the ventromedial nucleus. Although the cells vary widely in size, shape, and dendritic arborization pattern, there are two main cell types. Small (21 X 11 microns), round to fusiform cells, with two or three dendrites arborizing simply, were frequently seen in the arcuate, suprachiasmatic, periventricular, supramammillary nuclei and at the borders of the ventromedial nucleus. The other cell type is larger (40 X 15 microns) and multipolar, with three to five frequently branching dendrites. The dendritic field is large and the cells are intensely TH-immunoreactive. Although the larger cells occur occasionally in every hypothalamic nucleus, their principal locations are in the dorsal parts of the dorsomedial, posterior hypothalamic nuclei and the dorsal and lateral parts of the zona incerta, and in the areas dorsal and medial to the mammillothalamic tract at caudal hypothalamic levels. In this paper we give a detailed description of TH-immunoreactive fibers and terminals in the hypothalamus and a comparison with previous studies of catecholamine cells in the hypothalamus.

An immunohistochemical study of the organization of catecholaminergic cells and terminal fields in the paraventricular and supraoptic nuclei of the hypothalamus.

The distribution of catecholaminergic fibers and cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus was investigated with immunohistochemical methods in the adult albino rat. Sections through the nuclei were stained with antisera to the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). The results suggest that adrenergic (PNMT-stained) fibers innervate the entire parvocellular division of the paraventricular nucleus, although the highest density of fibers was found in the medial part of the division. Only widely scattered adrenergic fibers are found in the magnocellular division of the nucleus and in the supraoptic nucleus. Noradrenergic fibers appear to innervate the periventricular zone of the paraventricular nucleus and those parts of the paraventricular and supraoptic nuclei that contain predominantly vasopressinergic neurons in both the normal and in the homozygous Brattleboro rat. Significant numbers--somewhat more than 500--of dopaminergic (TH-stained) neurons are found in the paraventricular nucleus; the cells are distributed throughout the nucleus but are concentrated in the medial and periventricular parts of the parvocellular division. Double-labeling experiments with the retrogradely transported tracer true blue indicate that between 4% and 8% of the dopaminergic neurons in the paraventricular nucleus project to the region of the dorsal vagal complex and/or thoracic levels of the spinal cord. It is concluded that adrenergic inputs to the paraventricular nucleus may influence cells that project to the median eminence and to preganglionic autonomic cell groups in the medulla and spinal cord. Noradrenergic inputs to the supraoptic and paraventricular nuclei may influence primarily vasopressinergic cells that project to the posterior lobe of the pituitary, as well as cells in the periventricular part of the paraventricular nucleus that project to the median eminence.

Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: tyrosine hydroxylase and dopamine-beta-hydroxylase.

This study describes the distribution of catecholaminergic neurons in the hypothalamus and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase. Tyrosine hydroxylase-immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10-15 microns) to magnocellular (25-50 microns) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine-beta-hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine-beta-hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine-beta-hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine-synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine-beta-hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters.

Differential distribution of acetylcholinesterase activity among vasopressin- and oxytocin-containing supraoptic magnocellular neurons.

Acetylcholinesterase activity was demonstrated histochemically at light- and electron-microscopic levels, in Vibratome sections of the supraoptic nucleus of fixed hypothalami derived from osmotically stimulated and unstimulated Long Evans rats, from homozygous Brattleboro rats with hypothalamic diabetes insipidus, from lactating rats, from normal adult male house mice (Mus musculus) and from mice with hereditary nephrogenic diabetes insipidus (di/di). Reaction product was located in supraoptic magnocellular neurons; in dorsal and rostral aspects of the supraoptic nuclei lightly stained cells predominate, whereas in ventral and caudal regions densely staining perikarya predominate. Pre- and post-embedding immunocytochemical detection of oxytocin-neurophysin or vasopressin-neurophysin, combined with acetylcholinesterase histochemistry, showed that the lightly staining cells are oxytocinergic, and the densely staining cells vasopressinergic. Osmotic stimulation of the animals, either by substitution of drinking water for 3 days with 2.5% saline or reason of genetic defects which result in diabetes insipidus, enhanced the acetylcholinesterase activity of the vasopressin neurons but had little effect on the weekly acetylcholinesterase-reactive oxytocin cells. Acetylcholinesterase activity was particularly marked in the hypertrophied abnormal magnocellular neurons of homozygous Brattleboro rats which do not release significant amounts of vasopressin. The increased acetylcholinesterase activity in osmotically stimulated animals cannot, therefore, be a function of vasopressin. Acetylcholinesterase activity was also detected in large multipolar neurons lying dorsolateral to the supraoptic nucleus, and in their fine axonal processes which project towards the supraoptic nucleus. A very few synaptic boutons surrounded by acetylcholinesterase reaction product were found in contact with magnocellular neuron basal dendrites. However, much of the punctate acetylcholinesterase reactivity observed at the light microscopic level and previously interpreted as representing the loci of cholinergic synaptic boutons was shown to be intracellular, and probably caused by acetylcholinesterase activity in some large, secondary lysosomes.

Disturbance of fluid homeostasis leads to temporally and anatomically distinct responses in neuropeptide and tyrosine hydroxylase mRNA levels in the paraventricular and supraoptic nuclei of the rat.

The response of six mRNAs (for prepro-corticotropin-releasing hormone, prepro-enkephalin, prepro-vasoactive intestinal polypeptide/peptide histidine isoleucine, prepro-neurotensin/neuromedin N, prepro-cholecystokinin, and prepro-tyrosine hydroxylase) was measured in the hypothalamic paraventricular and supraoptic nuclei after increasing periods of osmotic stimulation caused by the replacement of regular drinking water with hypertonic saline (up to five days) or by forced dehydration (up to three days). In addition, hematocrits and concentrations of corticosterone were determined after the different periods of osmotic stimulation and correlated with the effects on the content of the various mRNAs. The temporal response of the mRNAs within the paraventricular and supraoptic nuclei to osmotic stimulation was different within the three compartments of these nuclei. First, in response to overnight osmotic stimulation, magnocellular neurosecretory neurons increased their mRNA content for two molecules (prepro-corticotropin-releasing hormone and tyrosine hydroxylase). As the stimulus was maintained over the next two to four days, these cells accumulated the mRNAs for at least three other peptides (cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine and enkephalin). Second, the response of peptide-coding mRNAs in parvicellular neurosecretory neurons of the paraventricular nucleus appeared to be slower; no changes could be measured after overnight stimulation. However, after a further two- to four-days of continued osmotic stimulation, the content of the mRNA coding for corticotropin-releasing hormone markedly decreased while that for cholecystokinin increased. No change in the content of the mRNAs coding for prepro-vasoactive intestinal polypeptide/peptide histidine isoleucine, enkephalin, and prepro-neurotensin/neuromedin N could be seen at any time after osmotic stimulation in parvicellular neurosecretory neurons. Third, increases in the content of mRNA coding for corticotropin-releasing hormone in the parvicellular neurons that provide descending projections from the paraventricular nucleus could only be detected after longer periods of osmotic stimulation. The effect of osmotic stimulation on plasma corticosterone concentrations was quickly apparent; plasma corticosterone concentrations were significantly elevated on the first morning after the beginning of salt-loading, and demonstrated the rapid effects of osmotic stimulation on the mechanisms controlling corticosterone release. These results show that the synthetic capability of cells in all three compartments of the paraventricular and supraoptic nuclei are modified by osmotic stimulation over different time scales, thereby allowing differential modulation of the neuroendocrine, autonomic, and behavioral components of the animal's response to disturbances in fluid homeostasis.(ABSTRACT TRUNCATED AT 400 WORDS)

Up-regulation of nitric oxide synthase messenger RNA in an integrated forebrain circuit involved in oxytocin secretion.

The hypothalamo-neurohypophysial system contains high levels of neuronal nitric oxide synthase and this increases further during times of neurohormone demand, such as that following osmotic stimulation. Using double in situ hybridization, we demonstrate here an increase in the expression of nitric oxide synthase messenger RNA by oxytocin neurons, but not vasopressin neurons, of the supraoptic nucleus at the time of lactation, when oxytocin is in demand due to another neuroendocrine stimulus, the milk-ejection reflex. In addition, using immunocytochemical retrograde tracing, we show that neurons of the subfornical organ, median preoptic nucleus and organum vasculosum of the lamina terminalis, which project to the supraoptic nucleus, contain nitric oxide synthase. These three structures of the lamina terminalis, together with the hypothalamo-neurohypophysial system, make up the forebrain osmoresponsive circuit that controls osmotically-stimulated release of oxytocin in the rat. The expression of nitric oxide synthase messenger RNA in the lamina terminalis was also shown to increase during lactation. The increases in nitric oxide synthase messenger RNA were not apparent during pregnancy. These results provide evidence for an integrated nitric oxide synthase-containing neural network involved in the regulation of the hypothalamo-neurohypophysial axis. The expression of nitric oxide synthase messenger RNA increases in this circuit during lactation and correlates with a reduction in the sensitivity of the circuit to osmotic stimuli also present in lactation but not pregnancy. As nitric oxide is believed to attenuate neurohormone release, it seems that the increased nitric oxide synthase messenger RNA expression detected here during lactation at a time of high oxytocin demand may be involved in reducing the sensitivity of the whole forebrain circuit to osmotic stimuli.

Effects of oestrogen upon nitric oxide synthase NADPH-diaphorase activity in the hypothalamo-neurohypophysial system of the rat.

An understanding of the interaction between oestrogen and the nitric oxide synthase/nitric oxide system is important for determining the roles of nitric oxide in central nervous control of osmotic homeostasis and certain aspects of reproduction. The effects of oestrogen on nitric oxide synthase and nitric oxide synthase activity were investigated in the magnocellular neurosecretory system. Ovariectomized female rats were injected subcutaneously with 17beta-estradiol benzoate either 10 microg daily for four days (short-term low-dose) or 200 microg daily for 21 days (long-term high-dose). In the neurohypophysis the density of NADPH-diaphorase staining--a marker for nitric oxide synthase activity--was increased after both short-term low-dose and long-term high-dose estradiol treatment, but no difference in nitric oxide synthase immunoreactivity was observed after either treatment. In the magnocellular supraoptic and paraventricular nuclei, short-term low-dose oestrogen treatment did not induce any detectable changes in nitric oxide synthase gene expression, the proportion of nitric oxide synthase-immunoreactive neurons, or the proportion of NADPH-diaphorase-positive neurons. Long-term high-dose oestrogen treatment also had no effect on nitric oxide synthase gene expression or immunoreactivity, but caused a reduction of the proportion of NADPH-diaphorase-positive neurons in the supraoptic nucleus and a reduction in the intensity of this histochemical staining. Qualitatively similar changes were observed in the magnocellular part of the paraventricular nucleus. The results provide, for the first time, evidence of a complex interaction between oestrogen and nitric oxide synthase in the neuroendocrine system in which nitric oxide synthase activity is regulated differently in the magnocellular cell bodies and axonal terminals and in which the activity of the enzyme rather than its expression is controlled.