Rat Supraoptic Nucleus Research Articles

Amplification of Transducer Gain by Angiotensin II-Mediated Enhancement of Cortical Actin Density in Osmosensory Neurons

Osmosensory neurons transduce osmotic signals into a neural spike code that commands behavioral and endocrine responses that mediate body fluid homeostasis. Although changes in osmoregulatory reflex gain are known to occur under physiological and pathological conditions, the basis for this modulation is unknown. Here, we show that angiotensin II amplifies osmosensory transduction by enhancing the proportional relationship between osmolality, receptor potential, and action potential firing in rat supraoptic nucleus neurons. This effect is mediated by a phospholipase C- and protein kinase C-dependent increase in cellular mechanosensitivity that is associated with a rapid increase in cortical actin filament density. Preventing this increase with cytochalasin D eliminated the enhancement of mechanosensitivity, whereas enhancing actin filament density with jasplakinolide potentiated mechanosensitivity and occluded the effects of angiotensin II. These results indicate that a receptor-mediated increase in cortical actin density can enhance osmosensitivity in acutely isolated supraoptic neurons.

Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus.

In this study, we test the hypothesis that there are differential splicing patterns between the expressed oxytocin (OT) and vasopressin (VP) genes in the rat supraoptic nucleus (SON). We quantify the low abundance, intron-containing heteronuclear RNAs (hnRNAs) and the higher abundance mRNAs in the SON using two-step, quantitative SYBR Green real-time reverse transcription (RT)-PCR and external standard curves constructed using synthetic 90 nt sense-strand oligonucleotides. The levels of OT and VP mRNA in the SON were found to be similar, approximately 10(8) copies/SON pair, whereas the copy numbers of VP hnRNAs containing intron 1 or 2 and the OT hnRNA containing intron 1 are much lower, i.e., approximately 10(2)-10(3) copies/rat SON pair. However, the estimated copy number of the intron 2-containing OT hnRNA is much larger, approximately 10(6) copies/SON pair. The relative distributions of all the OT and VP RNA species were invariant and independent of the physiological status of the rats (e.g., osmotically stimulated or lactating rats). Using intron-specific riboprobes against hnRNAs, we demonstrate by fluorescence in situ hybridization strong signals of OT hnRNA containing intron 2 predominantly in the cytoplasm, in contrast to the localization of the VP hnRNA found only in the nuclei. Taken together, these data support the view that the splicing patterns between OT and VP gene transcripts are different and show that there is a selective cytoplasmic retention of OT intron 2.

The Effects of Apelin on the Electrical Activity of Hypothalamic Magnocellular Vasopressin and Oxytocin Neurons and Somatodendritic Peptide Release

Apelin, a novel peptide originally isolated from bovine stomach tissue extracts, is widely but selectively distributed throughout the nervous system. Vasopressin and oxytocin are synthesized in the magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, which are apelin-rich regions in the central nervous system. We made extracellular electrophysiological recordings from the transpharyngeally exposed SON of urethane-anaesthetized rats to assess the role of apelin in the control of the firing activity of identified magnocellular vasopressin and oxytocin neurons in vivo. Apelin-13 administration onto SON neurons via microdialysis revealed cell-specific responses; apelin-13 increased the firing rates of vasopressin cells but had no effect on the firing rate of oxytocin neurons. A direct excitatory effect of apelin-13 on vasopressin cell activity is also supported by our in vitro studies showing depolarization of membrane potential and increase in action potential firing. To assess the effects of apelin-13 on somatodendritic peptide release, we used in vitro release studies from SON explants in combination with highly sensitive and specific RIA. Apelin-13 decreases basal (by 78%; P < 0.05; n = 6) and potassium-stimulated (by 57%; P < 0.05; n = 6) vasopressin release but had no effect on somatodendritic oxytocin release. Taken together, our data suggest a local autocrine feedback action of apelin on magnocellular vasopressin neurons. Furthermore, these data show a marked dissociation between axonal and dendritic vasopressin release with a decrease in somatodendritic release but an increase in electrical activity at the cell bodies, indicating that release from these two compartments can be regulated wholly independently.

MiXED Messages in Ion Channel Modulation

Ion channels are modulated by multiple molecular mechanisms. In this issue of Neuron, Pietrzykowski et al. expand the mechanistic repertoire by demonstrating that ethanol-induced microRNA can modulate the pattern of mRNA splice variants from which BK potassium channels are constructed. Because BK channels are important targets of ethanol, this finding has implications for mechanisms of ethanol sensitivity and tolerance.

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

TRPV1 Gene Required for Thermosensory Transduction and Anticipatory Secretion from Vasopressin Neurons during Hyperthermia

Increases in core body temperature promote thermoregulatory cooling by stimulating sweat production and preemptive renal water reabsorption through the release of vasopressin (VP, antidiuretic hormone). The mechanism by which the hypothalamus orchestrates this anticipatory VP release during hyperthermia is unknown but has been linked to a central thermosensory mechanism. Here, we report that thermal stimuli spanning core body temperatures activate a calcium-permeable, ruthenium red- and SB366791-sensitive nonselective cation conductance in hypothalamic VP neurons. This response is associated with a depolarizing receptor potential and an increase in action potential firing rate, indicating that these neurons are intrinsically thermosensitive. The thermosensitivity of VP neurons isolated from trpv1 knockout (Trpv1(-/-)) mice was significantly lower than that of wild-type counterparts. Moreover, Trpv1(-/-) mice showed an impaired VP response to hyperthermia in vivo. Channels encoded by the trpv1 gene thus confer thermosensitivity in central VP neurons and contribute to the thermal control of VP release in vivo.

Role of FosB in the supraoptic nucleus of rats with hepatic cirrhosis

Bile duct ligation (BDL), a model of hepatic cirrhosis, is associated with inappropriate vasopressin release and increased FosB expression in vasopressin neurons in the supraoptic nucleus (SON). BDL rats were injected bilaterally in the SON (n = 5) with 400 nl of an adeno‐associated virus (AAV) vector expressing a dominant negative construct for FosB (AAV‐ΔJunD) plus GFP. AAV injected BDL rats, control BDL rats, and sham ligated rats were instrumented with radio telemetry transmitters for measuring blood pressure and heart rate. Three weeks after BDL surgery, rats were individually housed in metabolism cages for 1 week. Daily average mean arterial pressures and heart rates were not different among the groups. Average daily water intake was significantly greater in AAV‐BDL rats (55 ± 7 ml) and BDL rats (47 ±4) compared to sham ligated controls (35 ± 1). Urine output was significantly greater in AAV‐treated BDL rats (38 ± 5 ml) than control BDL rats (21 ± 2) or sham controls (23 ± 1). Urine sodium was significantly lower in AAV‐treated BDL rats (47 ± 13 mmol/L) than the other groups (BDL 89 ± 12; Sham 136 ± 22) although average daily Na excretion was not different among the groups. SON injections of the FosB dominant null produced a diuresis in BDL rats that may be related to decreased circulating levels of vasopressin. FosB expression in vasopressin SON neurons may contribute to inappropriate vasopressin release in BDL rats. (HL62579)

Age-impaired fluid homeostasis depends on the balance of IL-6/IGF-I in the rat supraoptic nuclei

Adaptive metabolic changes associated with bacterial infections are likely to cause dehydration. Activation of hypothalamic neurons in the supraoptic nucleus that release anti-diuretic arginine-vasopressin in plasma provides water retention. Aging is characterized by arginine-vasopressin neuron hyper-activity and over-expression of pro-inflammatory cytokines like interleukin (IL)-6. Conversely, insulin-like growth factor (IGF)-I, known to exhibit anti-inflammatory properties, decreases with age. We compared activation of arginine-vasopressin neurons in adult (3 months) and aged (22 months) Wistar rats by measuring not only c-fos expression, plasma arginine-vasopressin and diuresis but also the expression of IL-6 and IGF-I in the supraoptic nuclei after intraperitoneal lipopolysaccharide injection. Aged rats displayed a heightened, shorter lasting activation of arginine-vasopressin neurons following lipopolysaccharide as compared to adults. IL-6 mRNA was 3-fold higher while IGF-I mRNA was 10-fold lower in aged than in adult rats. Brain pre-treatment with neutralizing anti-IL-6 antibodies or recombinant IGF-I in aged rats reversed lipopolysaccharide-induced anti-diuresis. These data extend the concept of neuroendocrine-immune interactions to the arginine-vasopressin neuronal system by establishing a relationship between brain IL-6/IGF-I balance and age-associated arginine-vasopressin neuronal dysfunction.

Effect of ghrelin on the exitatory synaptic inputs to the magnocelluer neurosecretory neurons in the supraoptic nucleus of rat

Effect of ghrelin on the exitatory synaptic inputs to the magnocelluer neurosecretory neurons in the supraoptic nucleus of rat

Combined microdialysis with single cell recording to study mechanisms and consequences of dendritic peptide release within the rat supraoptic nucleus in vivo

Combined microdialysis with single cell recording to study mechanisms and consequences of dendritic peptide release within the rat supraoptic nucleus in vivo

Effect of brain-derived neurotrophic factor on inhibitory synaptic input into the magnocellular neurosecretory cells in the rat supraoptic nucleus

Effect of brain-derived neurotrophic factor on inhibitory synaptic input into the magnocellular neurosecretory cells in the rat supraoptic nucleus

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.

Differential Kinetics of Oxytocin and Vasopressin Heteronuclear RNA Expression in the Rat Supraoptic Nucleus in Response to Chronic Salt Loading In vivo

Previous studies have shown that the secretion of oxytocin and vasopressin from the posterior pituitary always accompanies systemic hyperosmotic stimuli in rats, and that oxytocin and vasopressin mRNAs consistently increase in response to prolonged hyperosmotic stimuli. Hence, it has been widely interpreted that oxytocin and vasopressin secretion and gene expression are closely coupled. In the present study, we used both vasopressin and oxytocin intron- specific probes to measure vasopressin and oxytocin heteronuclear RNA (hnRNA) levels, respectively, by in situ hybridisation in the rat supraoptic nucleus (SON) in conjunction with radioimmunoassays of vasopressin and oxytocin peptide levels in plasma and in the posterior pituitary in normally hydrated rats and after 1-5 days of salt loading. Increased oxytocin secretion in response to hyperosmotic stimuli exceeded vasopressin secretion at every time point studied. Vasopressin hnRNA in the SON increased to near maximal levels within minutes after the hyperosmotic stimulus, and was maintained throughout all 5 days of salt loading. By contrast, oxytocin hnRNA did not significantly change from control levels until approximately 2 days after hyperosmotic stimulation, and was not maximal until 3 days. In summary, increases in oxytocin gene transcription in response to osmotic stimuli are dramatically delayed compared to increases in vasopressin gene transcription under the same conditions. These data indicate that oxytocin gene transcription is not as closely correlated with pituitary peptide secretion as is vasopressin gene transcription, and suggests that there is a fundamental difference in excitation-secretion-transcription coupling mechanisms that regulate these two closely related genes in the rat magnocellular neurones in the SON.

Chronic Osmotic Stimuli Increase Salusin‐β‐Like Immunoreactivity in the Rat Hypothalamo‐Neurohypophyseal System: Possible Involvement of Salusin‐β on [Ca2+]i Increase and Neurohypophyseal Hormone Release from the Axon Terminals

Salusin-alpha and -beta were recently discovered as bioactive endogenous peptides. In the present study, we investigated the effects of chronic osmotic stimuli on salusin-beta-like immunoreactivity (LI) in the rat hypothalamo-neurohypophyseal system. We examined the effects of salusin-beta on synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and neurohypophyseal hormone release from both freshly dissociated SONs and neurohypophyses in rats. Immunohistochemical studies revealed that salusin-beta-LI neurones and fibres were markedly increased in the SON and the magnocellular division of the paraventricular nucleus after chronic osmotic stimuli resulting from salt loading for 5 days and dehydration for 3 days. Salusin-beta-LI fibres and varicosities in the internal zone of the median eminence and the neurohypophysis were also increased after osmotic stimuli. Whole-cell patch-clamp recordings from rat SON slice preparations showed that salusin-beta did not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. In vitro hormone release studies showed that salusin-beta evoked both arginine vasopressin (AVP) and oxytocin release from the neurohypophysis, but not the SON. In our hands, in the neurohypophysis, a significant release of AVP and oxytocin was observed only at concentrations from 100 nm and above of salusin-beta. Low concentrations below 100 nm were ineffective both on AVP and oxytocin release. We also measured intracellular calcium ([Ca(2+)](i)) increase induced by salusin-beta on freshly-isolated single nerve terminals from the neurohypophysis devoid of pars intermedia. Furthermore, this salusin-beta-induced [Ca(2+)](i) increase was blocked in the presence of high voltage activated Ca(2+)channel blockers. Our results suggest that salusin-beta may be involved in the regulation of body fluid balance by stimulating neurohypophyseal hormone release from nerve endings by an autocrine/paracrine mechanism.

The Transcriptome of the Rat Hypothalamic‐Neurohypophyseal System is Highly Strain‐Dependent

We have used microarrays to comprehensively describe the transcriptomes of the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and the neurointermediate lobe of adult male Sprague-Dawley (SD) and Wistar-Kyoto (WKY) rats, as well as the PVN of Wistar rats. Comparison of these gene lists has enabled us to identify surprisingly large differences in hypothalamic-neurohypophyseal system gene expression patterns in these three strains. We have also shown that different transcript populations are enriched in the PVN and the SON of SD and WKY rats. The transcriptome differences catalogued here may be molecular substrates for the neuro-humoral phenotypic differences exhibited by different strains of rats.

14‐3‐3 Proteins Within the Hypothalamic‐Neurohypophyseal System of the Osmotically Stressed Rat: Transcriptomic and Proteomic Studies

The hypothalamic-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the actions of the antidiuretic hormone vasopressin (VP) and the natriuetic peptide oxytocin (OT). VP and OT are synthesised as separate prepropeptide precursors in the cell bodies of magnocellular neurones in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, the axons of which innervate the posterior pituitary gland (PP). Dehydration evokes a massive release of both peptides into the circulation, and this is accompanied by a function-related remodelling of the HNS. Microarray studies on mRNAs differentially expressed in the SON revealed that transcripts encoding the Ywhag and Ywhaz isoforms of the 14-3-3 family of regulatory proteins, are increased in the rat SON by 3 days of water deprivation; findings that we have confirmed by the real-time polymerase chain reaction. Because there is no necessary proportionality between transcript and protein abundance, we next examined Ywhag and Ywhaz translation products throughout the HNS in parallel with 14-3-3 post-translational modification, which is known to be an important determinant of functional activity. Both proteins are robustly expressed in the SON in VP- and OT-containing neurones, but the abundance of neither changes with dehydration. However, the total level of Ywhaz protein is increased in the neurointermediate lobe of the pituitary (NIL, which includes the PP), in parallel with a basic post-translationally modified isoform, suggesting transport from the cell bodies of the SON of newly-synthesised protein and changes in its activity. The level of an acidic, probably phosphorylated, Ywhag isoform is down-regulated in the SON by dehydration, although total levels are unchanged. Finally, based on the presence of a phosphorylated 14-3-3 binding motif, we have identified a 14-3-3 binding partner, proteasome subunit, beta type 7, in the NIL. Thus, we suggest that, through complex transcriptional, and post-translational processes, 14-3-3 proteins are involved in the regulation or mediation of HNS plasticity following dehydration.

Synergistic activation of astrocytes by ATP and norepinephrine in the rat supraoptic nucleus

Supraoptic nucleus (SON) neurons receive a dense innervation from noradrenergic fibers, the activity of which stimulates vasopressin (VP) and oxytocin (OT) release, notably during homeostatic regulation of blood pressure and volume. This regulation is known to involve the co-release of norepinephrine (NE) and ATP, which act in synergy to stimulate Ca 2+ increase in SON neurons and to enhance release of VP and OT from hypothalamo-neurohypophysial explants. We here demonstrate that both ATP and NE also trigger transient intracellular Ca 2+ rise in rat SON astrocytes, the two agonists showing a synergistic action similarly to what has been reported in SON neurons. The responses to both agonists are not or are only moderately affected after blockade of neuronal activity by tetrodotoxin, or of neurotransmitter release by removal of extracellular Ca 2+, suggesting that the receptors involved are located on the astrocytes themselves. ATP acts via P2Y 1 receptors, as indicated by the pharmacological profile of Ca 2+ responses and the strong immunolabeling for this receptor in SON astrocytes. Responses to NE involve both α and β adrenergic receptors, the latter showing a permissive role on the former. These results point to further implication of SON astrocytes in the regulation of VP and OT secretion, and suggest that they are potentially important elements participating in all regulatory processes of hypothalamo-neurohypophysial function that involve activation of noradrenergic pathways.

The role of the actin cytoskeleton in oxytocin and vasopressin release from rat supraoptic nucleus neurons

Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K(+), 50 mm) or TG (200 nm) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 microm) or depolymerizing agents (latrunculin B, 5 microm) to treat SON and neural lobe (NL) explants in vitro and measure high K(+)-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K(+)-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K(+)-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.

Actin Filaments Mediate Mechanical Gating during Osmosensory Transduction in Rat Supraoptic Nucleus Neurons

Osmosensory transduction is a bidirectional process displayed by neurons involved in the control of thirst and antidiuretic hormone release, and is therefore crucial for body fluid homeostasis. Although this mechanism is known to involve the activation of nonselective cation channels during hypertonicity-evoked shrinking, and the inhibition of these channels during hypotonicity-evoked swelling, the basis for this regulation is unknown. Here, we investigated this process using whole-cell patch-clamp recordings from neurons acutely isolated from the supraoptic nucleus of adult rats. The mechanosensitivity index, defined as the ratio of conductance change to normalized volume change, was quantitatively equivalent whether cell volume was increased or decreased by changes in extracellular fluid osmolality, or by changes in pipette pressure. Moreover, responses induced by hyperosmotic or hypo-osmotic media could be reversed by increasing or decreasing pipette pressure, respectively. The mechanosensitivity index was significantly reduced in neurons treated with cytochalasin-D, a compound that promotes the depolymerization of actin filaments. Conversely, cells treated with jasplakinolide, a compound that promotes actin polymerization, showed a significant increase in mechanosensitivity index. Finally, the depolarizing and excitatory effects of hypertonic stimuli were significantly enhanced by jasplakinolide and reduced by cytochalasin-D. We conclude that osmosensory transduction in these neurons is a reversible mechanical process that depends on an intact actin cytoskeleton, and the sensitivity of the transducer appears to vary in proportion with the density of actin filaments.

In vivo brain-derived neurotrophic factor release and tyrosine kinase B receptor expression in the supraoptic nucleus after osmotic stress stimulus in rats

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family involved in plasticity and neuroprotective processes. In recent years, we have reported the presence of BDNF mRNA in the supraoptic nucleus (SON) as well its sensitivity to osmotic stress. The rat SON is a relatively homogenous nucleus mainly consisting of magnocellular soma with their dendritic processes. BDNF may be released from dendrites to the extracellular space to stimulate tyrosine kinase (Trk) B receptors which are hypothetically present on these subcellular SON compartments. The main goal of this work was thus to study the presence and the in vivo BDNF-IR release from SON using the push–pull perfusion technique following systemic (i.p.) or local (within the SON) osmotic stimulation. BDNF was detected by immunocytochemistry and its release was measured by immunological assay (ELISA). Likewise, TrkB receptor localization in the SON—mRNA and their respective proteins—were studied by in situ hybridization and immunohistofluorescence techniques, respectively. Phosphorylation of CREB was detected by immunohistofluorescence.We present here direct evidence of in vivo dendritic BDNF release from SON which is highly sensitive to osmotic stress. The osmotic response latency period clearly depends on the mode of stimulus application (210 min for i.p. route vs. 15 min for intra-SON administration). The fact that BDNF is released as a very rapid peak when osmotic stimulation is locally applied is strong evidence in favor of an intra-SON origin of this secretion. Osmotic stress also increased phosphorylated cAMP response element binding protein immunoreactivity in the SON. In addition, we show in control rats that truncated forms of tyrosine kinase B receptor 2 mRNA represent the most abundant messenger in the SON as compared with brain-derived neurotrophic factor full-length catalytic receptor or truncated forms of tyrosine kinase B receptor 1 mRNA. In conclusion, it is likely that BDNF and their receptors are involved in neuronal plasticity changes induced by osmotic stress in the SON.