Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH?+?sham group, ischemia group and IH?+?ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (P_B?=?404 mmHg, PO₂?=?84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, l-NAME?+?ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of l-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.     

Leptin counteracts the hypoxia-induced inhibition of spontaneously firing hippocampal neurons: a microelectrode array study

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O(2)). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca(2+)-activated K(+) channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 μM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons.     

Cerebrovascular inflammation after brief episodic hypoxia: modulation by neuronal and endothelial nitric oxide synthase.

Obstructive sleep apnea, apnea of prematurity, and sudden infant death syndrome are associated with a high risk of morbidity and mortality secondary to the neuronal and cerebrovascular consequences of the associated intermittent hypoxia. We hypothesized that episodic hypoxia (EH) promotes inflammation in the cerebral microcirculation and that nitric oxide (NO) produced by the endothelial and neuronal isoforms of NO synthase (eNOS and nNOS, respectively) modulates this response. Anesthetized and ventilated Swiss-Webster ND4 mice, wild-type mice, and NO synthase knockout mice were subjected to a 1-h period of EH (twelve 30-s periods of hypoxia every 5 min). Four, 24, or 48 h later, mice were reanesthetized for imaging of leukocyte dynamics in the cortical venular microcirculation by epifluorescence videomicroscopy through closed cranial windows. In Swiss-Webster ND4 mice, leukocyte adherence increased 2.1-fold at 4 h, 3.4-fold at 24 h, and 1.8-fold at 48 h relative to time-matched, normoxic controls; there was no evidence of delayed hippocampal CA1 pyramidal cell death. A similar response was noted in wild-type mice. However, in eNOS knockouts, leukocyte-endothelial cell adherence was elevated to 4.4-fold over baseline 24 h after EH, and a significant fraction of these animals showed evidence of delayed CA1 cell death. Conversely, in nNOS knockouts, no increase in adherence was noted at 24 h and CA1 viability remained unaffected. We conclude that NO derived from nNOS promotes an inflammatory response in the cerebrovascular microcirculation after short-term EH and that NO produced by eNOS blunts the extent of this response and exerts neuroprotective effects.     

Carotid body function and ventilatory responses in intermittent hypoxia. Evidence for anomalous brainstem integration of arterial chemoreceptor input

Obstructive sleep apnea is a frequent medical condition consisting in repetitive sleep-related episodes of upper airways obstruction and concurrent events of arterial blood hypoxia. There is a frequent association of cardiovascular diseases and other pathologies to this condition conforming the obstructive sleep apnea syndrome (OSAS). Laboratory models of OSAS consist in animals exposed to repetitive episodes of intermittent hypoxia (IH) which also develop cardiovascular pathologies, mostly hypertension. The overall OSAS pathophysiology appears to be linked to the repetitive hypoxia, which would cause a sensitization of carotid body (CB) chemoreflex and chemoreflex-driven hyperreactivity of the sympathetic nervous system. However, this proposal is uncertain because hyperventilation, reflecting the CB sensitization, and increased plasma CA levels, reflecting sympathetic hyperreactivity, are not constant findings in patients with OSAS and IH animals. Aiming to solve these uncertainties we have studied the entire CB chemoreflex arch in a rat model of IH, including activity of chemoreceptor cells and CB generated afferent activity to brainstem. The efferent activity was measured as ventilation in normoxia, hypoxia, and hypercapnia. Norepinephrine turnover in renal artery sympathetic endings was also assessed. Findings indicate a sensitization of the CB function to hypoxia evidenced by exaggerated chemoreceptor cell and CB afferent activity. Yet, IH rats exhibited marked hypoventilation in all studied conditions and increased turnover of norepinephrine in sympathetic endings. We conclude that IH produces a bias in the integration of the input arising from the CB with a diminished drive of ventilation and an exaggerated activation of brainstem sympathetic neurons.     

Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.     

Proteomic analysis of CA1 and CA3 regions of rat hippocampus and differential susceptibility to intermittent hypoxia

The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia that characterizes sleep apnea. Proteomic approaches were used to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia (IH). Ninety-nine proteins were identified, and 15 were differentially expressed in the CA1 and the CA3 regions. Following IH, 32 proteins in the CA1 region and only 7 proteins in the more resistant CA3 area were up-regulated. Hypoxia-regulated proteins in the CA1 region included structural proteins, proteins related to apoptosis, primarily chaperone proteins, and proteins involved in cellular metabolic pathways. We conclude that IH-mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress-induced proteins and apoptosis, and, ultimately, disruption of structural proteins and cell integrity. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue, and may allow for future delineation of interventional strategies aiming to enhance neuronal adaptation to IH.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英文)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠 15min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高     

Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia

Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the β1 and β4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular β subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory β subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.     

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.     

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the Ub^G76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.     

Calcium store depletion induces persistent perisomatic increases in the functional density of h channels in hippocampal pyramidal neurons

The regulation of intracellular calcium by the endoplasmic reticulum (ER) plays a critical role in neuronal function. While the consequences associated with depleting calcium from the ER have been studied in multiple systems, it is not known whether the intrinsic properties of a neuron change in response to such perturbations. In this study, we demonstrate that the depletion of calcium from the ER of hippocampal CA1 pyramidal neurons induces a persistent, perisomatic increase in the density of functional h channels resulting in a reduction in intrinsic excitability and an increase in the optimal response frequency. This form of intrinsic plasticity is dependent on the elevation of cytoplasmic calcium, inositol triphosphate receptors, store-operated calcium channels, and the protein kinase A pathway. We postulate that this form of depletion-induced intrinsic plasticity is a neuroprotective mechanism that reduces excitability after depletion of calcium stores triggered through altered network activity during pathological conditions.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠15 min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高.     

BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.     

BK Channels Reveal Novel Phosphate Sensitivity in SNr Neurons

Whether large conductance Ca²⁺-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca²⁺]_i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the K_ATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with K_ATP channels under ischemic stresses.     

Mechanisms of neuronal death in brain aging and Alzheimer's disease: role of endocrine-mediated calcium dyshomeostasis.

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electrophysiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease.     

钾通道在培养大鼠海马神经元凋亡性容积减少中的作用

为探讨钾通道参与神经元凋亡的可能机制,在星形孢菌素（STS)诱导的培养海马神经元凋亡模型上,研究了凋亡时神经细胞容积的动态变化及钾通道在其中的作用.实验结果显示,钾通道阻断剂四乙铵或升高细胞外K⁺均能够明显抑制STS诱导的神经元凋亡,并且大电导钙激活钾通道（BK）选择性阻断剂iberiotoxin和paxilline具有同样程度的抗细胞凋亡作用,表明钾通道（可能主要是BK通道）参与了STS诱导的培养海马神经元凋亡.在STS诱导神经元凋亡的早期就出现了细胞容积的显著减少,而钾通道阻断剂或升高细胞外K⁺均可阻断该细胞容积减少.研究结果提示细胞内钾离子的外流可能参与了凋亡性细胞容积减少,这也可能是钾通道介导细胞凋亡的重要机制之一.     

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.     

Mechanisms of neuronal death in brain aging and alzheimer's disease: Role of endocrine-mediated calcium dyshomeostasis

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electro-physiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease. © 1992 John Wiley & Sons, Inc.     

Raphe serotonergic neurons modulate genioglossus corticomotor activity in intermittent hypoxic rats

Background

Genioglossus activity is greater during wakefulness but decreases to a weaker state during sleep in obstructive sleep apnea syndrome (OSAS) patients, compared to healthy subjects. Previous studies suggested that the corticomotor control of the genioglossus was modified in OSAS patients. Intermittent hypoxia (IH), the typical pathophysiological change in OSAS, can induce genioglossus facilitation. The serotonergic neurons of the raphe dorsal (DRN) and magnus nuclei (RMg) are responsive to hypoxia and play important roles in the control of the genioglossus. However, it remains unknown whether DRN and RMg serotonergic neurons are responsible for the facilitated corticomotor activity of the genioglossus during IH. This study explored the influence of IH on the corticomotor activity of the genioglossus by transcranial magnetic stimulation (TMS), and the role of DRN and RMg serotonergic neurons in this effect.

Methods

Rats were exposed to IH and divided into two groups. In one group, anti-SERT-SAP was microinjected into the DRN and RMg respectively to kill serotonergic neurons. In the other group, artificial cerebrospinal fluid (ACSF) was injected. Comparisons were conducted between the two groups during four weeks of IH and four weeks after IH.

Results

Compared to the corresponding ACSF-injected group, the DRN lesion group and RMg lesion group showed longer TMS latencies and lower amplitudes during IH from the 1^st to the 28^th day. After 28 days of IH, longer latencies and lower amplitudes were seen only in the DRN lesion group.

Conclusion

These results indicate that DRN and RMg serotonergic neurons play different roles in the facilitation of genioglossus corticomotor activity induced by IH.