大鼠延髓内脏带与下丘脑室旁核、视上核往返投射通路参与高渗调节反应的研究

目的探讨延髓内脏带(MVZ)与下丘脑室旁核(PVN)和视上核(SON)之间是否存在往返渗透压投射通路。方法通过给予大鼠饮用3%氯化钠的方法制作高渗刺激模型,并用WGA-HRP逆行追踪、抗Fos、抗酪氨酸羟化酶(TH)或加压素(VP)及胶质纤维酸性蛋白(GFAP)免疫组织化学相结合的四重标记方法,观察MVZ、PVN和SON中WGA-HRP、Fos、TH、VP和GFAP阳性分布及表达状况。结果高渗刺激后MVZ、PVN和SON内Fos阳性细胞明显增多;GFAP阳性结构也明显增多,其分布与Fos阳性细胞分布基本一致,表现为胞体肥大、突起粗长。星形胶质细胞(AST)紧密包绕在神经元周围形成神经元-AST复合体(N-ASC)。结论神经元和AST以N-ASC的形式共同参与渗透压调节反应,体内存在MVZ和SON或PVN之间往返的渗透压调节通路。     

Fos expression variances in mouse hypothalamus upon physical and osmotic stimuli: co-staining with vasopressin, oxytocin, and tyrosine hydroxylase

Fos expression in the hypothalamus and its quantification in vasopressinergic (AVP), oxytocinergic (OXY) and tyrosine hydroxylase (TH) immunoreactive cells in the hypothalamic paraventricular (PVN), supraoptic (SON), suprachiasmatic (SCh), and arcuate (Arc) nuclei was performed in response to physiologically two different, i.e. osmotic (i.p. hypertonic saline, HS) and immobilization (IMO), stimuli in mouse using a dual Fos-neuropeptide immunohistochemistry. Both 60 min of HS and 120 min of IMO evoked Fos induction in many hypothalamic structures, whereas, HS evoked more extensive Fos labeling than IMO in the SON, ventromedial (VMN) and dorsomedial (NDM) hypothalamic nuclei and the retrochiasmatic area (RCh). Other hypothalamic structures including the anterior hypothalamic area (AHA), the latero-anterior hypothalamic nucleus (LA), the Arc, the perifornical nucleus (PeF), and the lateral hypothalamic area (LH) showed similar Fos incidence after both HS and IMO. However, after both stimuli explicitly most extensive Fos expression was observed in the PVN. In addition, in the PVN substantially more Fos-AVP (62-67% versus 10-15%) and Fos-OXY (38-45% versus 4-8%) perikarya were observed after HS than IMO, respectively. Incidence of TH-immunoreactive Fos labeled cells in the PVN was also more frequent after HS. In the SON, HS activated more than 50% of AVP and OXY neurons while IMO less than 4%. The number of TH activated neurons in Arc was also higher after HS (11%) than IMO (4%). Lowest number of colocalizations was revealed in the SCh where both HS and IMO activated around 2% of AVP neurons. The present data demonstrate that both HS and IMO are powerful stimuli for the majority of hypothalamic structures displaying considerable topographic similarity in Fos expression suggesting their multifunctional involvement. The quantity and phenotypic differences of activated hypothalamic neurons may speak out for functional dissimilarities in response to HS and IMO.     

Functional and anatomic relationship between cholinergic neurons in the median preoptic nucleus and the supraoptic cells

The median preoptic nucleus (MePO) has been suggested to be an important area in the brain for the regulation of vasopressin (VP) release under the condition of osmotic stimulation. Fos immunoreactivity (Fos-ir), choline acetyltransferase (ChAT) immunoreactivity and retrograde labeling with fluoro-gold were used in this study to determine whether cholinergic neurons in the MePO can be activated by hypertonic NaCl, and to characterize the specific MePO cells that have anatomic projections to the supraoptic nuclei (SON). The results showed that c-fos expression specifically induced by hypertonic NaCl was found in the ChAT cells of the MePO. A retrograde tracing experiment demonstrated that the MePO neurons projecting to the SON were cholinergic. In addition, hypertonic saline-induced Fos-ir was colocalized with the MePO neurons back labeled with fluoro-gold from the SON. Together, these data provide evidence that the MePO cholinergic neurons are activated by osmotic stimulation, and suggest that cholinergic cells in the MePO are functionally important in the control of the SON neurons under the condition of hypertonic stimulation.     

Astrocytic Fos expression in the rat posterior pituitary following LPS administration

Systemic lipopolysaccharide (LPS) administration has been shown to cause profound Fos expression in multiple regions of the brain. In the present experiment, Fos expression in the hypothalamic supraoptic nucleus (SON), posterior pituitary, and anterior pituitary was investigated using quantitative immunohistochemistry. In the SON and anterior pituitary, a large number of Fos-positive cells were observed by restraint stress, hyperosmotic administration (1.5, 3, and 9% NaCl), and LPS administration (5, 25, and 125 microg/kg). In the posterior pituitary, LPS administration caused a significant increase in the number of Fos-positive nuclei in a dose-dependent manner, whereas restraint stress and hyperosmotic stimulation (1.5 and 3% NaCl) did not increase the number of Fos-positive cells and 9% NaCl administration induced weak Fos immunoreactivity. Moreover, a dual-labeling study using a confocal microscope revealed that Fos-positive cells in the posterior pituitary were astrocytes using MAP2, an astrocytic marker in the posterior pituitary. Here, we demonstrated that the astrocytes of the posterior pituitary expressed Fos in response to LPS administration, which suggests that Fos expression participates in the activation of astrocytes during acute-phase responses with LPS administration.     

Time course of Fos and Fras expression in the hypothalamic supraoptic neurons during chronic osmotic stimulation

The Fos family comprises Fos and several subtypes of Fos-related proteins (Fras) such as FosB, Fra-1, Fra-2, DeltaFosB, and chronic Fras. Changes in the expression of Fos family proteins with time are not well elucidated, particularly during chronic stimulation. In the present experiments, we investigated quantitatively the time course changes in Fos, FosB and Fras immunoreactivity in the magnocellular neurons of the supraoptic nucleus (SON) during acute and chronic osmotic stimulation. A small number of Fos- and FosB-positive neurons were observed in the SON of control rats, while many Fras-positive neurons were seen in control animals. Significant increases in the numbers of Fos-, FosB-, and Fras-positive neurons were observed 2 h after acute osmotic stimulation by intraperitoneal (i.p.) injection of 3% NaCl solution. Although the number of Fos-positive neurons returned to the control level 4 h after i.p. injection, a significant number of FosB- and Fras-positive neurons were still observed 8 h after i.p. injection. During chronic osmotic stimulation by giving 2% NaCl solution for 2 and 5 days, a large number of Fos-positive neurons were observed, but the cessation of chronic osmotic stimulation by normal water drinking immediately decreased the number of Fos-positive neurons to the control level within 2 h. The number of FosB-positive neurons was increased with period of chronic osmotic stimulation, and a significant number were observed 2-8 h after the cessation of the stimulation. The number of Fras-positive neurons was also significantly higher during chronic osmotic stimulation, and this number was significantly high 2-8 h after the cessation of the stimulation. RT-PCR analysis demonstrated the persistent expression of c-fos mRNA in the SON during chronic osmotic stimulation. These results suggest that c-fos mRNA and Fos protein are constitutively elevated during chronic osmotic stimulation and the time course changes in Fos are different from those seen in FosB and Fras.     

c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress

The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.     

Neuronal expression of Fos protein in the hypothalamus of rats with heart failure

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.     

Neuroanatomical and biochemical evidence for the involvement of the area postrema in the regulation of vasopressin release in rats

Studies were carried out in the rat to determine if the area postrema (AP), a medullary circumventricular organ, might be involved in the control of vasopressin (VP) release. The data from this study demonstrate the existence of direct neural connections between the AP and the hypothalamic VPergic neurons of the supraoptic nucleus (SON) as showed by the retrograde tracer horseradish peroxidase (HRP). Labeled neurons were observed in the AP following HRP injections into the SON. In addition, rats with AP lesions showed an impaired ability to conserve water and concentrate their urine in response to an hypertonic NaCl load. They, also, failed to maintain sodium retention and showed an attenuation of VP release during intracellular dehydration. These findings indicate that AP plays an important role in the regulation of VP release during changes in osmotic environment and suggest that this medullary circumventricular organ is a part of central circuitry subserving salt-water balance.     

Glial limitans elasticity subjacent to the supraoptic nucleus

Two previous studies from our laboratory have indicated that the ventral glial limitans subjacent to the hypothalamic supraoptic nucleus (SON-VGL) undergoes a reversible thinning upon chronic activation of the magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus (SON). Numerous other studies have shown that MNC somata hypertrophy with activation. One aim of the current study was to understand better how SON-VGL thinning occurs. A second aim was to quantify overall changes of the MNC somata region due to cellular hypertrophy to compare relative changes in dimensions. Here, we undertook a light microscopic stereological investigation of the SON and the subjacent SON-VGL of Nissl stained material under basal and activated conditions. Astrocyte numbers in the underlying SON-VGL remained stable across hydration state as did the overall volume of the SON-VGL and dendritic zone reference area. How these data are consistent with our earlier observations of SON-VGL thinning was resolved by the finding of a highly significant, 30% increase in the mediolateral dimension of the SON-VGL in dehydrated rats. These observations fit well with previous work from our laboratory that demonstrates a reorientation of SON-VGL astrocytes, from vertical to horizontal, which occurs in the activated SON-VGL. We found a significant, approximately 54%, increase in the overall volume of the MNC region of the SON. No significant rostrocaudal lengthening of the SON was detected, although a trend was evident. All the observed changes reversed with rehydration. These data indicate that elasticity of the SON-VGL acts to accommodate the volume expansion of the MNCs and enables the SON-VGL to continue as an interface between the underlying cerebrospinal fluid in the subarachnoid space and the expanded SON above.     

高渗刺激后大鼠视上核星形胶质细胞和神经元的反应及其相互关系的光、电镜研究

目的探讨大鼠尾静脉注射高渗盐水(9％NaCl，5．5mL／kg)后，视上核(SON)内星形胶质细胞和神经元的可塑性反应及相互间的关系。方法用免疫组织化学和免疫电镜技术，观察刺激后15，45，90，180和360minSON内缝隙连接蛋白43(Cx43)和蛋白32(Cx32)的变化及超微结构。结果光镜下观察到Cx43阳性星形胶质细胞在15min出现，45min达到高峰；Cx32阳性神经元90min达到高峰。电镜下在SON内，观察到下列四种超微结构：(1)突触样结构(synapse like structure)，位于神经元的轴突末梢与Cx43阳性的星形胶质细胞突起之间；(2)三成分的突触复合体(tripartite synaptic structure)，由突触前膜、突触后膜和靠近此突触的星形胶质细胞突起共同组成；(3)同源性缝隙连接(gap junction，GJ)，位于星形胶质细胞突起之间，两侧均为Cx43；(4)“异源性缝隙连接样结构”(heterotypic gap junctions，HGJ)，是由Cx32阳性神经元和Cx43阳性星形胶质细胞突起组成的一种超微结构。结论高渗刺激后，SON内Cx43阳性星形胶质细胞和Cx32阳性神经元明显增加，前者出现和高峰的时间早于神经元；两者之间的HGJ数量明显增加，其他结构的数量变化不明显，因此两者可能是通过HGJ进行快速的信息交流。     

Acute hyperosmotic stimulus‐induced Fos expression in neurons depends on activation of astrocytes in the supraoptic nucleus of rats

Acute hyperosmolarity induced a time‐dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expresion occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons. © 2009 Wiley‐Liss, Inc.     

Differential expression of tenascin by astrocytes associated with the supraoptic nucleus (SON) of hydrated and dehydrated adult rats

The present study evaluated the expression of tenascin by astrocytes in the supraoptic nucleus and associated ventral glial limitans (SON-VGL) under conditions that induce reversible changes in neuronal organization (dehydration and rehydration). Immunostaining of astroglia cultured from rat neonatal SON-VGL confirmed that these cells are capable of both expressing and secreting tenascin. Observations of immunostained tissue sections from adult rats revealed tenascin immunoreactivity primarily in the VGL and dendritic zone, subjacent to SON neuronal somata. Comparison of immunostained tissues from hydrated and dehydrated animals showed an apparent decrease in the intensity of immunostaining with dehydration. Subsequent Western blots of similar tissues confirmed the presence of the 210–220-kDa tenascin protein in the SON-VGL. SON-VGL tissues from control, dehydrated, and rehydrated rats were then studied by using SDS-PAGE and quantitative gel densitometry. A consistent decrease in tenascin concentration was observed by 6 days of dehydration that, with rehydration, reversed back toward or beyond control levels. Together, these observations indicate that SON-VGL astrocytes variably express tenascin and that this protein may play a role in adult SON plasticity. © 1996 Wiley-Liss, Inc.     

Circadian modulation of osmoregulated firing in rat supraoptic nucleus neurones

The antidiuretic hormone vasopressin (VP) promotes water reabsorption from the kidney and levels of circulating VP are normally related linearly to plasma osmolality, aiming to maintain the latter close to a predetermined set point. Interestingly, VP levels rise also in the absence of an increase in osmolality during late sleep in various mammals, including rats and humans. This circadian rhythm is functionally important because the absence of a late night VP surge results in polyuria and disrupts sleep in humans. Previous work has indicated that the VP surge may be caused by facilitation of the central processes mediating the osmotic control of VP release, and the mechanism by which this occurs was recently studied in angled slices of rat hypothalamus that preserve intact network interactions between the suprachiasmatic nucleus (SCN; the biological clock), the organum vasculosum lamina terminalis (OVLT; the central osmosensory nucleus) and the supraoptic nucleus (SON; which contains VP-releasing neurohypophysial neurones). These studies confirmed that the electrical activity of SCN clock neurones is higher during the middle sleep period (MSP) than during the late sleep period (LSP). Moreover, they revealed that the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT was greater during the LSP than during the MSP. Activation of clock neurones by repetitive electrical stimulation, or by injection of glutamate into the SCN, caused a presynaptic inhibition of glutamatergic synapses made between the axon terminals of OVLT neurones and SON neurones. Consistent with this effect, activation of clock neurones with glutamate also reduced the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT. These results suggest that clock neurones in the SCN can mediate an increase in VP release through a disinhibition of excitatory synapses between the OVLT and the SON during the LSP.     

Enhanced expression of heme oxygenase-1 and carbon monoxide excitatory effects in oxytocin and vasopressin neurones during water deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 μm) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.     

Oestradiol potentiates hormone secretion and neuronal activation in response to hypertonic extracellular volume expansion in ovariectomised rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 μg/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c‐Fos‐VP and c‐Fos‐OT double‐labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c‐Fos‐OT and c‐Fos‐VP double‐labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.     

Systemic Osmotic Stimulation Increases Vasopressin and Oxytocin Release Within the Supraoptic Nucleus

Vasopressin (VP) and oxytocin (OT) are released within the hypothalamic nuclear region in response to direct microdialysis with hypertonic solutions. Experiments were performed to determine whether systemic osmotic stimulation causes changes in intranuclear peptide release within the supraoptic nucleus (SON). A hypertonic sodium chloride solution was injected intraperitoneally (ip) or intravenously (iv) and microdialysis techniques were used to simultaneously monitor central and peripheral peptide release in urethane anesthetized rats. Systemic osmotic stimuli elicited increases in intranuclear peptide release which were delayed and long-lasting, occurring over a 2.5 h period. In contrast, plasma peptide levels peaked at 30-min after the stimulus. The results demonstrate that increased plasma sodium elicits an increase in VP and OT release into the extracellular space of the hypothalamic SON. The different patterns of peptide release in plasma and brain point toward the possibility of independently regulated release into the different compartments.