糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元的变化

目的：观察糖尿病大鼠下丘脑视上核神经元型一氧化氮合酶(nNOS)免疫阳性神经元数量的变化。方法：用链脲佐菌素诱导建立糖尿病大鼠模型;免疫细胞化学染色显示nNOS免疫阳性神经元,并进行定量分析。结果：糖尿病视上核nNOS免疫阳性神经元着色深浅不一,着色较深的阳性神经元散在分布,神经元的形态多样,突起较少。对照组大鼠视上核nNOS免疫阳性神经元较稀疏,各时期无明显改变。糖尿病2w,nNOS免疫阳性神经元数量与对照组无显著差异;7w,nNOS阳性神经元较密集,明显多于对照组;12w,nNOS免疫阳性神经元数量略低于7w,但仍多于对照组。结论：糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元数量明显增多。     

Nitric oxide modulates the discharge rate of basal forebrain neurones: a study in freely moving rats

In urethane‐anaesthetized rats the infusion of a nitric oxide (NO)‐donor [NOC‐18, 1 mM (DETA/NO); 2,2’‐(hydroxynitrosohydrazino)bis‐ethanamine)] into the basal forebrain (BF) inhibited the discharge rate of most neurones, suggesting that NO may promote sleep via inhibition of wake‐promoting neurones in the BF. However, this hypothesis still needs to be confirmed in freely moving rats. The objective of this study was to examine whether NO modulates the discharge rate of BF neurones in freely moving rats in a similar manner to anaesthetized rats. We measured the discharge rates of BF neurones in freely moving rats during microdialysis infusion of a NO‐donor (1 mm ; NOC‐18) in different vigilance states. Neurones were characterized as wake (W)‐on (51.8%), W‐off (28.6%) and W/non‐rapid eye movement (REM)‐independent (21.4%) based on their discharge profiles during wakefulness (W) and non‐REM sleep. The NO‐donor affected the discharge rate of most BF neurones during quiet wakefulness (QW; 55%) and non‐REM sleep (64%). The most prominent response in all neuronal groups was a decrease in the discharge rate during QW and non‐REM sleep. A small subpopulation of neurones increased the discharge rate. The increase in NO in the BF during prolonged wakefulness may facilitate sleep via inhibition of wake‐promoting neurones.     

Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.     

大鼠下丘脑内的一氧化氮合酶与雌激素受体双标神经元

目的:探讨一氧化氮合酶(NOS)和雌激素受体(ER)在下丘脑诸核团的分布及共存,为揭示雌激素与一氧化氮之间的内在联系提供形态学依据。方法:采用NADPH-d组织化学法并结合免疫组织化学技术,观察雌性大鼠下丘脑内NOS阳性神经元、ER阳性神经元以及NOS/ER双染神经元的形态及分布。结果:NOS阳性神经元主要分布在下丘脑室旁核、视上核、下丘脑外侧区和室周核;ER阳性神经元在下丘脑诸核团的表达不及NOS阳性神经元广泛;NOS与ER双染神经元主要分布在下丘脑的室旁核、视上核、下丘脑外侧区及室周核;其他区域可见散在分布的双染神经元。结论:NOS与ER双染神经元主要集中分布在视上核的背内侧和背外侧部及室旁核小细胞部腹内侧区,在下丘脑外侧区分布较广但比较分散,室周核呈散在分布。     

Possible morphological bases for synchronisation of neuronal firing in the rat supraoptic nucleus during lactation

Oxytocin-containing neurones in the supraoptic and paraventricular nuclei of lactating rats display a periodic activation which results in a pulsatile release of hormone before each reflex milk ejection induced by suckling. This electrical activity occurs in an all-or-none fashion and is synchronised in the whole population of oxytocin neurones in both nuclei. The present report describes changes in the ultrastructure of the supraoptic nucleus of lactating animals which may serve as morphological bases for such a functional synchronisation.In the supraoptic nuclei of normal rats, neurosecretory neurones are usually separated by elements of the neuropil, particularly glial processes. At rare intervals, adjacent neurosecretory somata, and dendrites, are seen to be in direct apposition. The only specialisations apparent between the contiguous membranes are occasional attachment plates. In nuclei of lactating rats, quantitative analysis indicated that 34% of profiles of the sectioned neurosecretory cell bodies were in direct contact with each other and 22% with profiles of dendrites, a 5-fold increase over the corresponding frequencies observed in normal male and virgin female animals. Such contacts involved 10% of the total measured soma surface membrane (compared to 1.5% in the controls). The number of attachment plates supporting the apposing membranes also increased significantly as did the mean size of the individual appositions. There was also a higher incidence of presynaptic terminals contacting more than one post-synaptic element (soma or dendrite) in the same plane of section, a rare phenomenon in the normal nucleus. No further increases were evident in these appositional relations in virgin female and lactating rats deprived of water for one day, a stimulus which enhances vasopressin release.It is postulated that the structural reorganisation observed in the nuclei of lactating animals may lead to electrical interactions between the neurosecretory cells and may thus be one of the factors supporting the synchronisation of neuronal activity during the episodic release of oxytocin.     

Nitric oxide modulates metalloproteinase-2, collagen deposition and adhesion rate after polypropylene mesh implantation in the intra-abdominal wall

Prosthetic meshes are commonly used to correct abdominal wall defects. However, the inflammatory reaction induced by these devices in the peritoneum is not completely understood. We hypothesized that nitric oxide (NO), produced by nitric oxide synthase 2 (NOS2) may modulate the response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall in wild-type and NOS2-deficient (NOS2^−/−) mice. After 15 days tissues around the mesh implant were collected, and inflammatory markers (the cytokine interleukin 1β (IL-1β) and NO) and tissue remodeling (collagen and metalloproteinases (MMP) 2 and 9) were analyzed. The lack of NOS2-derived NO induced a higher incidence of visceral adhesions at the mesh implantation site compared with wild-type mice that underwent the same procedure (P < 0.05). Additionally, higher levels of IL-1β were present in the mesh-implanted NOS2^−/− animals compared with control and wild-type mice. Mesh implantation induced collagen I and III deposition, but in smaller amounts in NOS2^−/− mice. MMP-9 activity after the surgical procedure was similarly increased in both groups. Conversely, MMP-2 activity was unchanged in mesh-implanted wild-type mice, but was significantly increased in NOS2^−/− mice (P < 0.01), due to decreased S-nitrosylation of the enzyme in these animals. We conclude that NOS2-derived NO is crucial for an adequate response to and integration of polypropylene mesh implants in the peritoneum. NO deficiency results in a prolonged inflammatory reaction to the mesh implant, and reduced collagen deposition may contribute to an increased incidence of visceral adhesions.     

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.     

Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study

The gas nitric oxide is a messenger in brain signaling. In the hypothalamo-hypophyseal system nitric oxide is involved in the control of the expression and/or release of peptide hormones (corticotropin-releasing hormone, gonadotropin-releasing hormone, vasopressin and oxytocin). Nitric oxide synthase (NOS), the enzyme generating nitric oxide, is abundantly present in the magnocellular nuclei of the rat hypothalamus. Its localization in the human hypothalamus is less well studied. Hence, we investigated the anatomical distribution of neuronal nitric oxide synthase in the human supraoptic nucleus by use of immunohistochemical and enzyme histochemical techniques. The immunohistochemical localization of NOS was studied in 31 matched human hypothalami (13 control cases, eight depressed patients and ten schizophrenics). NADPH-diaphorase studies were carried out on seven additional hypothalami (three normal brains, four schizophrenics). Apparent inter-individual differences exist with regard to the occurrence of the enzyme in supraoptic neurons. In a majority of cases no immunostaining or histochemical reaction for the enzyme was observed. In seven cases (three controls, two schizophrenics, two depressives) a population of nitrergic nerve cells was seen in the dorsomedial part of the nucleus. This group of cells also stained for NADPH-diaphorase. Also, there were a few NOS-immunopositive neurons scattered throughout the nucleus. Additionally, thin NADPH-diaphorase positive fibers were observed to cross the nucleus. Our data show that, unlike the rat, the human supraoptic nucleus contains only a small number of nitrergic neurons. No correlation was found between the expression of the enzyme in supraoptic neurons and the psychiatric status of the patients.     

自发性高血压大鼠第三脑室触液神经元内一氧化氮合酶与加压素的共表达

目的:观察自发性高血压大鼠(SHR)与SD大鼠第三脑室触液神经元(CSFCN)内一氧化氮合酶(NOS)与加压素(VP)的分布和共存。方法:应用还原型尼克酰胺嘌呤二核苷酸磷酸脱氢酶(NADPH-d)组织化学方法,结合ABC免疫组织化学技术。结果:在SHR视前区至室间核后大细胞亚核平面的第三脑室室壁均有NOS阳性CSFCN的分布;在SHR第三脑室室壁的CSFCN内NOS与VP具有共存性,且SHR组第三脑室的CSFCN内NOS与VP共存率较SD组高。结论:一氧化氮(NO)与VP在下丘脑的血压神经内分泌活动调节中起着重要的介导作用,也可能对高血压的发生发展有影响。     

Nitric oxide synthesis is increased in the endometrial tissue of women with endometriosis

BACKGROUND: Previous studies have shown that peritoneal macrophages from women with endometriosis produce excess nitric oxide (NO). This study was designed to quantify the amount of NO and determine the expression of endothelial (eNOS) and inducible NO synthases (iNOS) in women with and without endometriosis. METHODS: An enzyme-linked immunosorbent assay (ELISA) was performed on endometrial tissues obtained from controls (myoma, n = 30) and on eutopic/ectopic endometrial tissues from endometriosis patients (n = 34) to evaluate eNOS and iNOS protein concentrations in these endometrial tissues. A rapid-response chemiluminescence analyser was used to measure NO directly in fresh endometrial tissues. RESULTS: Mean (+/- SEM) levels of NO were significantly increased in the endometrial tissues of women with endometriosis (13.2 +/- 7.8 versus 19.8 +/- 12.6 nmol/g tissue; P = 0.016). Apparently higher levels of NO were found in ectopic compared with eutopic endometrium (P = 0.057). Endometrial tissues of women with endometriosis appeared to contain more iNOS than those of controls (3.6 +/- 2.2 versus 8.6 +/- 12.2 pg/ microg protein; P = 0.06), but no significant difference was found in eNOS levels. CONCLUSIONS: Greater amounts of NO and NOS are present in the endometrial tissues of women with endometriosis, implying a possible role for NO in the pathogenesis of endometriosis.     

Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli

We investigated the chemical and anatomical features of nitric oxide synthase (NOS)-containing neurons in the paraventricular and supraoptic nuclei in the rat hypothalamus using combinations of enzyme histochemistry, in situ hybridization and immuno-histochemistry. Neurons expressing NOS mRNA completely overlapped with NADPH-diaphorase-positive neurons. Topographical distribution of NOS was segregated from that of CRF-containing parvicellular neurons in the posterior paraventricular nucleus but overlapped with that of magnocellular neurons. In the paraventricular nucleus, 70% of oxytocin neurons contained NOS, which corresponded to one half of NOS neurons. About one third of vasopressin-immunoreactive neurons were NADPH-diaphorase-positive and the same proportion of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive. In the supraoptic nucleus, 50% of oxytocin neurons were NADPH-diaphorase-positive, which corresponded to 40% of NOS neurons. About 25% of vasopressin neurons were NADPH-diaphorase-positive, and 30% of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive. When NADPH-diaphorase histochemistry was performed first, subsequent immunostaining was markedly perturbed. Using fluoro-gold as a retrograde tracer, 4% of NADPH-diaphorase-positive neurons were shown to contribute to the descending projection to the spinal cord. About 40%–50% of NADPH-diaphorase-positive neurons exhibited Fos immunoreactivity after injection of lipopolysaccharide or hypertonic saline, while only 10%–15% of these neurons expressed Fos in response to immobilization or pain. Endogenous NO may be involved in the regulation of magnocellular functions, especially when the internal environment is disturbed.     

Nitric oxide modulates acetylcholine‐induced vasodilatation in the hepatic arterial vasculature of the dual‐perfused rat liver

The role of nitric oxide in the modulation of hepatic arterial vascular reactivity was investigated in an isolated dual‐perfused rat liver preparation. Twelve male Wistar rats (200–250 g) were anaesthetized with sodium pentobarbitone (60 mg kg^–1 i.p.). The livers were then excised and perfused in vitro through hepatic arterial and portal venous cannulae at constant flow rates. Concentration‐dependent dose–response curves to acetylcholine (10^–8–10^–5 M ), sodium nitroprusside (10^–6–5 × 10^–4 M ), and adenosine triphosphate (ATP) (10^–8–10^–5 M ) in the hepatic artery were constructed after the tone was raised by addition of methoxamine (3 μM L^–1). Acetylcholine‐induced vasodilatation in the hepatic artery was significantly attenuated with inhibition of nitric oxide synthase by using N^G‐nitro‐L ‐arginine methyl ester (30 μM ), E_max=51.7 ± 2.8 vs. 32.5 ± 3.1 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. ATP‐induced hepatic arterial vasoconstriction which was significantly enhanced with L ‐NAME, E_max=94.0 ± 9.3 vs. 127.0 ± 8.0 mmHg, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. Sodium nitroprusside‐induced hepatic arterial vasodilatation remained unchanged with N^G‐nitro‐L ‐arginine methyl ester, E_max=57.0 ± 3.4 vs. 57.0 ± 4.1, before vs. after N^G‐nitro‐L ‐arginine methyl ester, respectively. The data from the present study suggest that acetylcholine‐induced vasodilatation in the intrahepatic arterial vasculature of the rat liver is at least, in part, mediated by the release of nitric oxide. In addition, ATP‐induced hepatic arterial vasoconstriction is also modulated by the release of nitric oxide (*P < 0.05, Student’s paired t‐test).     

Partial co-existence of NADPH-diaphorase and acetylcholinesterase in the hypothalamic magnocellular secretory nuclei of the rat

Co-localization of NADPH-diaphorase (ND) and acetylcholinesterase (AChE) activities were explored in the magnocellular secretory nuclei of the rat hypothalamus by means of a double histochemical staining of the same sections. Partial co-existence was found in all the nuclei studied (paraventricular, supraoptic, fornicals and circular nuclei). No particular location of the neurons expressing both markers was found, although in the paraventricular nucleus all of them (ND+, AChE+ and neurons expressing both markers) were preferentially located in the magnocellular subdivisions whereas in the parvicellular ones only some neurons belonging to all three types were detected, mainly located in the periventricular and medial subdivisions. The lowest degree of co-existence was found at the level of the main magnocellular nuclei (supraoptic and paraventricular) when compared with the accessory magnocellular nuclei, especially the posterior fornical and the circular nuclei. These results extend previous data on the chemical nature of the neurons producing nitric oxide in the neurosecretory nuclei and the possible functional role of this atypical messenger in the hypothalamus.     

Met-enkephalin induces fast synaptic plasticity of magnocellular neurons in the rat supraoptic nucleus.

A morphometric-ultrastructural study was made of the supraoptic nucleus of rats of both sexes following central administration of met-enkephalin. Ten minutes after met-enkephalin treatment the number of axo-somatic synapses was significantly increased. This effect was more pronounced in female rats than in males and could be prevented by preceding administration of naloxone. Animals that received naloxone followed by met-enkephalin showed a dilation of the rough endoplasmic reticulum into a vesicular shape. Our results provide preliminary evidence for a fast remodeling of synaptic input to magnocellular hypothalamic neurons. It is likely that the known inhibitory action of opioids on the hypothalamo-neurohypophysial system is partly mediated by this plasticity.     

Antidromic responses in the paraventricular magnocellular neurons of the rat hypothalamus: latency variations correlated with the firing rate

Summary Magnocellular neurosecretory cells were antidromically identified in the hypothalamic paraventricular nucleus (PVN) of urethane-anesthetized, ovariectomized female rats following electrical stimulation of the neurohypophysis. Seventy-one cells with a tonic pattern of spontaneous discharge were distinguished and used to examine the relationships between the measures of antidromic spike latency, activation threshold and discharge rate. The discharge rate was artificially modulated by either microiontophoresis of glutamate or antidromic stimulation of the neurohypophysis. In all the PVN cells with tonic activity, the latency lengthened and the threshold increased as a function of the discharge rate. Activation of individual cells by microiontophoresis of glutamate was effective, as was simultaneous activation of many PVN cells by antidromic stimulus. Similar relationships between the discharge rate and the parameters of antidromic activation were seen in 3 cells, when their rates varied spontaneously over a wide range without manipulation. These data suggest that the excitability of axons of presumed oxytocinergic cells in the PVN-neurohypophyseal system are influenced by their prior activity, probably through metabolic changes in individual axons.     

Interaction between Serotonin and Nitric Oxide (NO) in the Activation of the Serotoninergic System in the Common Snail

The effects of serotonin and NO donors on serotoninergic neurons (more than 60) in the brain of the common snail Helix lucorum were studied. Serotonin and NO donors induced depolarization in all neurons, and increased spike activity and activated the synchronous synaptic input, including train-like input, resulting in the onset of synchronous train activity in all these neurons. The excitatory effect of serotonin was significantly decreased by 5,7-dihydroxytryptamine (5,7-DOT) and monomethylarginine – a blocker of endogenous NO synthesis. Both these substances blocked the serotonin activation of the synchronous train input. 5,7-DOT also blocked the activation of this input by NO donors, but had no effect on their excitatory actions. The effects of 5,7-DOT developed quickly, were reversible, and were comparable to the effects of serotonin receptor antagonists. The data obtained here provide evidence that serotonin and NO have similar regulatory effects on the serotoninergic system in the snail brain. Not only do they excite serotoninergic neurons, but they also coordinate their functioning by activating common synaptic inputs, which are apparently also serotoninergic. It is suggested that NO has the role of a second messenger during serotoninergic excitation and functions as a co-transmitter for the presynaptic input.     

移植视网膜NOS阳性神经元的发育及其与上丘的联系

目的：观察不同年龄组段大鼠正常视网膜及移植视网膜内NOS阳性神经元的发育情况及其定位分布，以及移植视网膜能否与视觉中枢建立功能联系。方法：应用NADPH脱氢酶组织化学方法和c—fos免疫组织化学方法显示。结果：(1)正常大鼠视网膜N0S阳性神经元最早出现于生后第5天，第18d数量达到高峰。(2)移植视网膜具有正常视网膜的各层结构和相似的生长发育规律，移植视网膜NOS阳性神经元在生后第4d出现，第12d数量达到高峰，第22d后降至正常成年鼠水平。(3)移植视网膜接受光刺激后，一侧或双侧上丘浅层出现明显的c-fos免疫反应阳性神经元。结论：根据NOS阳性神经元的定位、分布，推测其为无长突细胞、移位无长突细胞及节细胞；移植视网膜能与宿主视觉中枢上丘建立功能联系。     

Special aspects of firing rates and thermosensitivity of preoptic neurons

Summary Mean firing rates (per 10 sec) of preoptic neurons of the rat were recorded at normal temperature and after central warming and cooling. Thermosensitivity was determined according to the reactions of the mean firing rate. In compatibility with results from other animals we obtained the following percentages (52 neurons): 23% warm-sensitive, 13% cold-sensitive, 64% non-thermosensitive neurons.However, it turned out that a lot of interesting special effects are not taken into account, using exclusively the criterion increasing/decreasing/constant mean firing rate. Particularly we observed long lasting adaption processes and stationary non-linear effects as well as striking oscillations which were dependent on the thermal stimuli, while the mean interval value remained constant.     

Nitric oxide is involved in nicotine-induced burst firing of rat ventral tegmental area dopamine neurons

In the present study, using single cell recordings in vivo and intracellular recordings in vitro from midbrain slices, the role of N-methyl-d-aspartate (NMDA) receptor signaling on firing activity in ventral tegmental area dopamine neurons elicited by nicotine was investigated in the rat. In accordance with previous studies, systemic nicotine (0.5 mg/kg s.c.) increased both firing rate and burst firing of dopamine neurons in vivo, and bath-applied nicotine (10 microM) increased firing rate in vitro. The competitive NMDA receptor antagonist CGP39551 (2.5 mg/kg i.p.) inhibited nicotine's effects on burst firing and also attenuated the nicotine-induced increase in firing rate. Moreover, although the nitric oxide (NO)-synthase inhibitor N-nitro-l-arginine-methyl-ester (l-NAME; 5.0 mg/kg i.p.) had no effect on cell firing by itself, it prevented the response to nicotine in vivo. In contrast, l-NAME (100 microM) did not influence nicotine's effect on dopamine cell firing in vitro, suggesting that the effect of l-NAME seen in vivo is dependent on presynaptic afferent input. The present study confirms previous results suggesting that the effect of systemically administered nicotine is in part presynaptic and mediated via NMDA receptors. The data also indicate that NO plays an important role in the previously demonstrated, indirect, glutamate-mediated excitation of these neurons by nicotine. By inference, our results provide additional support for the involvement of NO in nicotine dependence.