Effects of ginkgolide B on neuronal discharges in paraventricular nucleus of rat hypothalamic slices

Objective To study the central role of ginkgolide B （BN52021） in regulating cardiovascular function of nerve center by examining the effects of ginkgolide B on the electrical activity of rat paraventricular nucleus （PVN） neurons in hypothalamic slice preparation and to elucidate the mechanism involved. Methods Extracellular single-unit discharge recording technique. Results （1） In response to the application of ginkgolide t3 （0.1, 1, 10 μmol/L; n = 27） into the perfusate for 2 rain, the spontaneous discharge rates （SDR） of 26 （26/27, 96.30%） neurons were significantly decreased in a dose-dependent manner. （2） Pretreatment with L-glutamate （L-Glu, 0.2 mmol/L） led to a marked increase in the SDR of all 8 （100%） neurons in an epileptiform pattern. The increased discharges were suppressed significantly after ginkgolide B （1 μmol/L） was applied into the perfusate for 2 min. （3） In 8 neurons, perfusion of the selective L-type calcium channel agonist, Bay K 8644 （0.1 μmol/L）, induced a significant increase in the discharge rates of 8 （8/8, 100%） neurons, while ginkgolide B （1μmol/L） applied into the perfusate, could inhibit the discharges of 8 （100%） neurons. （4） In 8 neurons, the broad potassium channels blocker, tetraethylammonium （TEA, 1 mmol/L） completely blocked the inhibitory effect of ginkgolide B （1 μmol/L）. Conclusion These results suggest that ginkgolide B can inhibit the electrical activity of paraventricular neurons. The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and potentially concerned with delayed rectifier potassium channel （KDR）.

Mechanism of hypothalamic paraventricular nucleus in regulating asthmatic attack

BACKGROUND:It has been confirmed that c-fos expression increased markedly in hypothalamic paraventricular nucleus(PVN)during asthmatic attack in rats,and PVN has extensive physiological functions,involving in the regulation of respiratory system,etc.OBJECTIVE:To observe the alteration of electroencephalogram(EEG)and power spectra in PVN during the asthmatic attack,and the alteration of lung function and diaphragmatic muscle discharge after bilateral PVN lesion in asthmatic rats.DESIGN:A randomized control study.SETTING:Laboratory of Physiology and Pharmacology,School of Basic Medical Sciences,Southeast University.MATERIALS:Forty-eight male adult SD rats of 260-300 g were used.The rats were randomly divided into 6 groups(n=8):control group,asthma group,electrolytic lesion of PVN group,KA-induced lesion of PVN group,sham electrolytic lesion of PVN group and sham kainic acid(KA)-induced lesion of PVN group.KA,chicken ovalbumin and aluminum hydroxide were purchased from American Sigma Company.Bordetella pertussis vaccine(Institute of Biological Products of Shanghai);stereotaxic apparatus(JiangwanⅡ,China);lesion-producing device(YC-2 programmable stimulato,Chengdu Instrument Company);MD2000 signal processing system(Nanjing Medical School);data acquisition system(RM6240B,Chengdu Instrument Company).METHODs:The experiments were carried out in the Laboratory of Physiology and Pharmacology,School of Basic Medical Sciences,Southeast University from January to August in 2006.①Rats except for control group were sensitized with an intraperitoneal injection of 100 mg chicken ovalbumin and 100 mg aluminum hydroxide and Bordetella pertussis vaccine containing 5×10^(9) heat-killed in 1 mL of sterile saline.From the fifteenth to seventeenth days rats received three times aerosolized ovalbumin challenge.In rats of the control group and asthma group three steel electrodes were placed into the left PVN(AP-1.8 mm,LR 0.4 mm,OH-7.9 mm),parietal cortex and subcutaneous tissue in lower limb.Lung function tests were carried out simultaneously.Small holes were drilled in the skull to introduce a concentric bipolar electrode in the direction of the PVN in order to perform electrolytic lesion.The electrodes were connected to a lesion-producing device and a current of 1.0-1.5 mA was passed over a period of 10-15 s on each side of the PVN.The rats received 0.5μg/0.5μL of KA in phosphate buffer(0.1 mol/L,pH 7.4),and the speed of infusion was 0.1μL per minute in order to perform KA-induced lesion of PVN.②Three days after operation of lesion,lung function tests were carried out.All the electrode and transducer were connected with data acquisition system.This technique yielded airway resistance(Raw),dynamic compliance(Cdyn),the expiratory time(Te)/the inspiratory time(Ti),minute ventilation volume(MVV),EMGdi frequency and EMGdi integral.③The differences of the measurement data were compared using the t test.MAIN OUTCOME MEASURES:①The alteration of EEG and power spectrum of PVN during asthmatic attack in sensitized rats;②The effects of electrolytic lesion or KA-induced lesion of PVN on lung function in asthmatic rats.RESULTS:All the 48 rats were involved in the analysis of results.①Alteration of EEG and power spectrum:Five minutes after injection of ovalbumin into caudal vena,the breathing rate of the rat was obviously speeded up and the total power spectrum was increased[(18476.71±2140.94),(13838.75±2983.26)mV^(2),P<0.01],the percentage of theδpower andθpower decreased significantly(P<0.01),while the percentage ofαpower andβ1 power were enhanced(P<0.05,0.01).Ten minutes after injection,the EEG power spectrum of PVN further shifted rightward,the total power gradually increased(P<0.01)which suggesting that the intensive hypersynchrony activities of PVN neurons.The percentage ofδpower was decreased significantly(P<0.01),but theα,β1 andβ2 were increased(P<0.01).Twenty-five minutes later,the breathing movements became steady,and the EEG power spectrum of PVN returned to the control level step by step.②The alteration of lung function was detected during asthmatic attack after electrolytic lesion or KA-induced lesions of PVN respectively.It was found that EMGdi frequency,Te/Ti and RL were all decreased(P<0.01),EMGdi integral,MVV and Cdyn were all enhanced(P<0.01),while there were no significant changes in the sham surgery group(P>0.05).CONCLUSION:The excitability of PVN is increased during the asthmatic attack.PVN plays a key role in the regulation of asthma.Both electrolytic and KA lesions of PVN can significantly relieve the asthmatic symptoms of rats,and improve their lung function.

Inhibitory effects of ketamine and L--NAME on expression of c-fos and NOS in hypothalamic paraventricular nucleus after acute hypothermia and hypoxia in rats

Objective: To determine whether NMDA receptor activation mediates the expression of c--fos and NOS and study the relationship between the expression of c--fos and NOS in the hypothalamic paraventricularnucleus (PVN) following acute hypothermia and hypoxia. Methods: Fos immunohistochemistry, NADPH--d histochemistry and Fos/NADH--d double labeling were used. Results: Acute hypothermia and hypoxia induced the overexpression of on fos and NOS in PVN in rats. Pretreatment with ketamine, a NMDA receptor antagonist, resulted in partial inhibition of the expression of c--fos and NOS and that with blocker of NOS resulted in significant inhibition of the expression of c--fos. Conclusion: The activation of NMDA receptor is involved in the expression of c- fos and NOS in PVN in the rats subjected to acute hypothermia and hypoxia.Meanwhile, hypothalamic endogenous NO participates in adaptive reaction to hypothermia and hypoxia,which might be related to the modulation of c- fos expression.

Fos expression incatecholaminergic medullary neu-rons induced by chemical stimulation of stomach projecting to the paraventricular nucleus of hypothalamus in rats

AIM To determine whether medullary catecholaminergic neurons expressing Fos induced by chemical stimulation of the stomach project to the paraventricular nucleus of hypothalamus (PVH) in rats. METHODS A triple labeling method of horseradish peroxidase (HRP) retrograde tracing combined with Fos (ABC method) and tyrosin hydroxylase (TH) (PAP method) immunohistochemical stainings was used in the present study. RESULTS Seven kinds of labeled neurons were found in the nucleus tractus solitarii (NTS), the ventrolateral medulla (VLM) and the reticular formation of the medulla (RF): Fos like immunoreactive (LI) neurons, TH LI neurons and HRP retrogradely single labeled neurons, Fos/HRP, Fos/TH and HRP/TH double labeled neurons, and Fos/HRP/TH triple labeled neurons. CONCLUSION Ascending projections from the NTS, VLM and RF to the PVH might be involved in the transmitting process of the visceral noxious stimulation.

Electroacupuncture preconditioning alleviates myocardial ischemia-reperfusion injury through the hypothalamic paraventricular nucleus-interposed nucleus nerve pathway

OBJECTIVE:To explore whether the paraventricular nucleus(PVN)participates in regulation of the antimyocardial ischemia-reperfusion injury(MIRI)effect of electroacupuncture(EA)and whether this is achieved through the PVN-interposed nucleus(IN)neural pathway.METHODS:The modeling method of myocardial ischemia reperfusion injury was achieved by ligating the left anterior descending coronary artery in SpragueDawley rats.We used the Powerlab multi-channel physiological recorder system to record electrocardiograms and analyze the changes in ST segment displacement;2,3,5-Triphenyltetrazolium chloride staining was used to observe the percentage of myocardial infarction areas.Detecting cardiac troponin I(cTnI),lactate dehydrogenase(LDH)in serum was done with an enzyme-linked immunosorbent assay kit.Morphological changes in the myocardium were detected in each group with hematoxylin-eosin staining of paraffin sections.Detection of c-fos protein expression in the PVN of the hypothalamus was done with the immuneofluorescence method.The Plexon multi-channel acquisition system recorded PVN neuron discharges and local field potentials in each group of rats.Offline Sorter software was used for cluster analysis.Neuro Explorer software was used to perform autocorrelation,raster and frequency characteristics and spectral energy analysis of neuron signals in each group.RESULTS:Compared with the MIRI model group,the areas of myocardial infarction in the EA group were significantly reduced;the expression of cTnI,LDH in serum was decreased significantly.The firing frequency of pyramidal cells in the PVN was significantly increased and the spectrum energy map showed energy was reduced,c-fos expression in PVN was reduced,this indicated that neuronal activity in the PVN participates in the effect of EA improving myocardial injury.In addition,we used the kainic acid method to lesion the IN and observed that the effect of EA was weakened.For example,the area of myocardial infarction of lesion IN+EA group in rats was significantly increased compared with that resulting from EA group,the expression of cTnI,LDH in serum was significantly increased,the firing frequency of pyramidal cells in the PVN was significantly reduced.A spectral energy diagram shows that the energy after damage was higher than that of EA group.At the same time,the expression of c-fos in the PVN increased again.CONCLUSION:Our results indicated that the PVN-IN nerve pathway may participate as an effective pathway of EA to improve the effect of myocardial injury.

Effects of needling acupoints at different nerve segments on oxytocin neurons in rat’s hypothalamic paraventricular nucleus and intragastric pressure

Objective:To compare and explore the effects of needling acupoints at different nerve segments on the oxytocin(OT)neurons in the paraventricular nucleus of hypothalamus(PVN)and the intragastric pressure,and discuss the possible mechanisms.Methods:Thirty-two healthy adult Sprague-Dawley(SD)rats were numbered and divided into 4 groups according to the random number table,a Zusanli(ST 36)group,a Neiguan(PC 6)group,a Weishu(BL 21)group and a control group,with 8 rats in each group.Except the control group,rats in the other three groups received acupuncture at the corresponding acupoints.To observe the differences in double-labeled OT neurons and c-fos neurons of the hypothalamic PVN and the intragastric pressure after acupuncture among the three groups of needling acupoints at different nerve segments.Results:Compared with the control group,the numbers of double-labeled cells in the PVN of the Zusanli(ST 36)group and the Neiguan(PC 6)group decreased significantly,while the intragastric pressure increased significantly(all P<0.05),and the inter-group differences were statistically significant(P<0.05).The intragastric pressure in the Weishu(BL 21)group decreased significantly,and the inter-group difference was statistically significant(P<0.05).Compared with the Weishu(BL 21)group,the numbers of OT/c-fos double-labeled cells in PVN of the Zusanli(ST 36)group and the Neiguan(PC 6)group decreased significantly,and the intragastric pressure increased significantly,the inter-group differences were statistically significant(all P<0.01).Conclusion:Acupoints at different nerve segments have different regulation effects on intragastric pressure.The difference may be related to the different nerve conduction pathways by acupoints at different nerve segments in regulating the intragastric pressure.The PVN may be one common integration center for the regulation of gastric function in the three acupoints[Zusanli(ST 36),Neiguan(PC 6)and Weishu(BL 21)]at different nerve segments.

大鼠脑缺血再灌流后下丘脑室旁核形态学改变

目的 :观察大鼠脑缺血再灌流后下丘脑室旁核的形态学改变。方法 :将大鼠脑缺血动物模型分 3、4、5、6和 7d 5组。经心脏灌流固定后取材 ,石蜡包埋、切片 ,常规 HE染色 ,光镜观察。结果 :脑缺血再灌流 (再灌注 )后 ,下丘脑室旁核的神经元数量减少 ,细胞固缩 ,细胞间隙增大。神经胶质细胞增多 ,无炎性细胞。这些变化随时间的延长而改变明显。结论

Blockade of c-Src Within the Paraventricular Nucleus Attenuates Inflammatory Cytokines and Oxidative Stress in the Mechanism of the TLR4 Signal Pathway in Salt-Induced Hypertension

Toll-like receptor 4 (TLR4) and cellular Src (cSrc) are closely associated with inflammatory cytokines and oxidative stress in hypertension, so we designed this study to explore the exact role of c-Src in the mechanism of action of the TLR4 signaling pathway in salt-induced hypertension. Salt-sensitive rats were given a high salt diet for 10 weeks to induce hypertension. This resulted in higher levels of TLR4, activated c-Src, pro-inflammatory cytokines, oxidative stress, and arterial pressure. Infusion of a TLR4 blocker into the hypothalamic paraventricular nucleus (PVN) decreased the activated c-Src, while microinjection of a c-Src inhibitor attenuated the PVN levels of nuclear factor-kappa B, pro-inflammatory cytokines, and oxidative stress. Our findings suggest that a longterm high-salt diet increases TLR4 expression in the PVN and this promotes the activation of c-Src, which upregulates the expression of pro-inflammatory cytokines and results in the overproduction of reactive oxygen species.Therefore, inhibiting central c-Src activity may be a new target for treating hypertension.

Effects of the aqueous extract of Schizandra chinensis fruit on ethanol withdrawal-induced anxiety in rats

Background We previously demonstrated that the aqueous extract of the Schizandra chinensis fruit （AESC） ameliorated Cd-induced depletion of monoamine neurotransmitters in the brain through antioxidant activity.In the present study,we investigated the effect of AESC on anxiety-like behavior and the levels of norepinephrine and 3-methoxy-4-hydroxyphenylglycol （a metabolite of norepinephrine） in different brain regions during ethanol withdrawal in rats.Methods Male Sprague-Dawley rats were treated with 3 g/kg of ethanol （20％,w/v） or saline by daily intraperitoneal injection for 28 days followed by three days of withdrawal.During withdrawal,rats were given AESC （100 mg.kg 1.d-1 or 300 mg.kg 1·d1,P.O.） once a day for three days.Thirty minutes after the final dose of AESC,the anxiogenic response was evaluated using an elevated plus maze,and the plasma corticosterone levels were examined by radioimmunoassay.Meanwhile,the concentrations of norepinephrine and 3-methoxy-4-hydroxy-phenylglycol in the hypothalamic paraventricular nucleus and hippocampus were also measured by high performance liquid chromatography.Results Rats undergoing ethanol withdrawal exhibited substantial anxiety-like behavior,which was characterized by both the decrease in time spent in the open arms of the elevated plus maze and the increased level of corticosterone secretion,which were greatly attenuated by doses of AESC in a dose-dependent manner.The high performance liquid chromatography analysis revealed that ethanol withdrawal significantly increased norepinephrine and 3-methoxy-4-hydroxy-phenylglycol levels in the hypothalamic paraventricular nucleus,while not significantly altering them in the hippocampus.Similar to the results from the elevated plus maze test,the AESC significantly inhibited the elevation of norepinephrine and its metabolite in the hypothalamic paraventricular nucleus in a dose-dependent manner.Conclusions These results suggest that AESC attenuates anxiety-like behavior induced by ethanol withdrawal through modulation of the hypothalamic norepinephrine system in the brain.

Electrical Stimulation of Deep Peroneal Nerve Mimicking Acupuncture Inhibits the Pressor Response via Capsaicin-lnsensitive Afferents in Anesthetized Rats

Objective： To assess the inhibitory modulation of blood pressure by stimulation of the deep peroneal nerve （DPN） and to determine the involvement of nociceptive fibers in the modulation. Methods： All the animals were divided into six groups （A-F）. The rats in groups A and B received no pretreatment. The rats in groups C and D received subcutaneous injection of capsaicin or control vehicle, respectively, near the DPN for 2 days. Those in groups E and F had the DPN exposed to capsaicin or control vehicle, respectively, for 20 min. Subsequently, pressor responses were induced by stimulation of paraventricular nucleus （PVN） either electrically （groups A and C-F） or chemically via injection of glutamate （group B）. After two stable pressor responses （baseline）, all groups were subject to 5-min DPN stimulation followed by PVN stimulation for 10 s. Arterial blood pressure, heart rate, and electrocardiogram were recorded. The pressor response was calculated as the difference in the mean arterial pressure （MAP） before and after PVN stimulation, and changes from baseline in pressor response after DPN stimulation were compared between the groups. Results： Increases of MAP of 22.88 ＋ 2.18 mm Hg and 20.32 ＋ 5.25 mm Hg were induced by electrical （group A） or chemical （group B） stimulation of the PVN, respectively. These pressor responses were inhibited by stimulation of the DPN, and the MAP was reduced to 12.00 _＋ 2.10 mm Hg in group A （n=6, P〈0.01） and 7.00 ＋ 2.85 mm Hg in group B （n=6, P〈0.01）. Subcutaneous injection of capsaicin （125 mg/kg） near the DPN in group C （n=7） had no effect on the inhibitory effect of DPN stimulation compared with the group D （n=9）, and neither did blockade of nociceptive fibers with capsaicin in group E （n=6） compared with group F （n=8）. Conclusion： Stimulation of the DPN mimicking acupuncture has an inhibitory effect on the pressor response, and the effect is mediated by capsaicin-insensitive afferent fibers in the DPN.