In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

AIM： To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS： Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS： The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca^2＋ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION： The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Whole-cell recordings of voltage-gated Calcium,Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective：To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods：Hippocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results：The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca^2＋ currents, delayed rectifier K^＋ current and voltage-gated Na^＋ currents. Conclusion：Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.

在体膜片钳技术的新进展

在体膜片钳是指在整体动物上直接对其中枢神经元进行全细胞膜片钳记录的技术 ,在生理学和药理学研究中具有良好的应用前景 .常规采用的是盲法记录 ,最近出现的可视法记录 ,采用双光子靶向膜片钳 (two photontargetedpatching ,TPTP)技术 ,通过基因操作在动物脑内目标神经元中构建特异表达的荧光标志 ,可以做到对特定神经元亚群的靶向研究 .对这两种方法的原理和操作进行了简单的介绍 .

在体研究前扣带回皮层对伤害性热刺激的反应

目的采用在体穿孔全细胞膜片钳技术,记录麻醉大鼠的前扣带回皮层(ACC)神经元对热刺激的反应,以证实ACC参与热感觉信息的处理。方法成年雄性SD大鼠10只,腹腔注射乌拉坦麻醉后,将大鼠头部固定于立体定位仪上进行在体穿孔膜片钳记录,分别给予大鼠后爪37、60℃的热刺激,记录ACC神经元对两种热刺激的反应。结果麻醉状态下,给予大鼠后爪伤害性热刺激(60℃)后,ACC神经元膜电位活动由低频转变为高频,分布由双峰转变为单峰,平均膜电位上升至(-53.62±6.38)mV,明显高于无刺激时和非伤害性刺激(37℃)时(P<0.01),且平均膜电位绝对值增加(5.43±2.87)mV。非伤害性热刺激(37℃)不能引起ACC电活动变化。结论 ACC参与伤害性热感觉信息的处理。

在体记录大鼠海马神经元对不同单色激光闪光刺激的反应

目的:探讨海马CA1锥体神经元对不同单色激光闪光刺激的反应,为论证激光诱发的神经系统生物学效应提供实验支持。方法:健康成年SD大鼠麻醉后,行气管插管术,开颅钻孔暴露脑面,进行在体膜片钳记录。全细胞记录稳定后,采用4种不同波长的单色激光对其眼球进行闪光刺激,记录大鼠海马神经元的反应。结果:给予大鼠闪光刺激后,其海马神经元对蓝色激光和紫色激光表现出明显的超极化反应,对红色和绿色激光刺激则没有明显的反应。蓝光引起的变化中,超极化的幅值为(8.32±1.10)m V,反应持续时间为(115.32±13.02)ms;紫光引起的超极化的幅值为(9.01±2.25)m V,刺激反应持续时间为(109.27±16.62)ms,蓝光和紫光之间没有统计学差异。50 m W、75 m W、100 m W、125 m W功率的紫色激光刺激引起的超极化幅值,分别为(7.28±0.16)m V、(9.25±0.71)m V、(10.91±0.08)m V、(12.67±0.38)m V,相关性分析显示幅值变化与刺激功率高度正相关。结论:麻醉状态下,蓝光和紫色的闪光刺激可以诱导大鼠海马神经元出现明确的抑制性反应,红光和绿光没有明显的作用。

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal

AIM:To investigate the effects of Lizhong Tang,an herbal product used in traditional Chinese medicine,on mouse small intestine interstitial cells of Cajal(ICCs).METHODS:Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues.The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody.A whole-cell patch-clamp configuration was used to record potentials(current clamp) from cultured ICCs.All of the experiments were performed at 30-32 ℃.RESULTS:ICCs generated pacemaker potentials,and Lizhong Tang produced membrane depolarization in current-clamp mode.The application of flufenamic acid(a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang.Pretreatment with thapsigargin(a Ca 2+-ATPase inhibi-tor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang.However,pacemaker potentials were completely abolished in the presence of an external Ca 2+-free solution,and under this condition,Lizhong Tang induced membrane depolarizations.Furthermore,When GDPβ-S(1 mmol/L) was in the pipette solution,Lizhong Tang still induced membrane depolarizations.In addition,membrane depolarizations were not inhibited by chelerythrine or calphostin C,which are protein kinase C inhibitors,but were inhibited by U-73122,an active phospholipase C inhibitors.CONCLUSION:These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.

Cranial irradiation impairs intrinsic excitability and synaptic plasticity of hippocampal CA1 pyramidal neurons with implications for cognitive function

Radiation therapy is a standard treatment for head and neck tumors.However,patients often exhibit cognitive impairments following radiation therapy.Previous studies have revealed that hippocampal dysfunction,specifically abnormal hippocampal neurogenesis or neuroinflammation,plays a key role in radiation-induced cognitive impairment.However,the long-term effects of radiation with respect to the electrophysiological adaptation of hippocampal neurons remain poorly characterized.We found that mice exhibited cognitive impairment 3 months after undergoing 10 minutes of cranial irradiation at a dose rate of 3 Gy/min.Furthermore,we observed a remarkable reduction in spike firing and excitatory synaptic input,as well as greatly enhanced inhibitory inputs,in hippocampal CA1 pyramidal neurons.Corresponding to the electrophysiological adaptation,we found reduced expression of synaptic plasticity marker VGLUT1 and increased expression of VGAT.Furthermore,in irradiated mice,long-term potentiation in the hippocampus was weakened and GluR1 expression was inhibited.These findings suggest that radiation can impair intrinsic excitability and synaptic plasticity in hippocampal CA1 pyramidal neurons.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).

MicroRNA is a potential target for therapies to improve the physiological function of skeletal muscle after trauma

MicroRNAs can regulate the function of ion channels in many organs.Based on our previous study we propose that miR-142a-39,which is highly expressed in denervated skeletal muscle,might affect cell excitability through similar mechanisms.In this study,we overexpressed or knocked down miR-142a-3p in C2C12 cells using a lentivirus method.After 7 days of differentiation culture,whole-cell currents were recorded.The results showed that overexpression of miR-142a-3p reduced the cell membrane capacitance,increased potassium current density and decreased calcium current density.Knockdown of miR-142a-3p reduced sodium ion channel current density.The results showed that change in miR-142a-3p expression affected the ion channel currents in C2C12 cells,suggesting its possible roles in muscle cell electrophysiology.This study was approved by the Animal Ethics Committee of Peking University in July 2020(approval No.LA2017128).

Chlorogenic acid alters the voltage-gated potassium channel currents of trigeminal ganglion neurons

Chlorogenic acid（5-caffeoylquinic acid, CGA） is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer＇s disease and neuropathic pain disorders. However, its mechanism of action is unclear.Voltage-gated potassium channel（Kv） is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,Aand IK,Vchannels. Trigeminal ganglion（TRG）neurons were acutely disassociated from the rat TRG, and two different doses of CGA（0.2 and 1 mmol·L21） were applied to the cells.Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,Aand IK,Vchannels. The results demonstrated that 0.2 mmol·L21CGA decreased the peak current density of IK,A. Both 0.2 mmol·L21and1 mmol·L21CGA also caused a significant reduction in the activation and inactivation thresholds of IK,Aand IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,Aand IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.

Expression of gamma-aminobutyric acid type A receptor α_2 subunit in the dorsal root ganglion of rats with sciatic nerve injury

The γ-aminobutyric acid neurotransmitter in the spinal cord dorsal horn plays an important role in pain modulation through primary afferent-mediated presynaptic inhibition. The weakening of γ-aminobutyric acid-mediated presynaptic inhibition may be an important cause of neuropathic pain. γ-aminobutyric acid-mediated presynaptic inhibition is related to the current strength of γ-aminobutyric acid A receptor activation. In view of this, the whole-cell patch-clamp technique was used here to record the change in muscimol activated current of dorsal root ganglion neurons in a chronic constriction injury model. Results found that damage in rat dorsal root ganglion neurons following application of muscimol caused concentration-dependent activation of current, and compared with the sham group, its current strength and γ-aminobutyric acid A receptor protein expression decreased. Immunofluorescence revealed that γ-aminobutyric acid type A receptor α2 subunit protein expression decreased and was most obvious at 12 and 15 days after modeling. Our experimental findings confirmed that the y-aminobutyric acid type A receptor α2 subunit in the chronic constriction injury model rat dorsal root ganglion was downregulated, which may be one of the reasons for the reduction of injury in dorsal root ganglion neurons following muscimol-activated currents.

The Effect of Coriaria Lactone on NMDA Receptor Mediated Currents in Rat Hippocampal CA1 Neurons

Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.

Effect of the ginsenoside Rb1 on the spontaneous contraction of intestinal smooth muscle in mice

AIM: To investigate the effect and the possible mechanism of ginsenoside Rb1 on small intestinal smooth muscle motility in mice. METHODS: Intestinal smooth muscle strips were isolated from male ICR mice (5 wk old), and the effect of ginsenoside Rb1 on spontaneous contraction was recorded with an electrophysiolograph. The effect of ginsenoside Rb1 on ion channel currents, including the voltage-gated K + channel current (IK V ), calcium-activated potassium channel currents (IK Ca ), spontaneous transient outward currents and ATP-sensitive potassium channel current (IK ATP ), was recorded on freshly isolated single cells using the whole-cell patch clamp technique. RESULTS: Ginsenoside Rb1 dose-dependently inhibited the spontaneous contraction of intestinal smooth muscle by 21.15% ± 3.31%, 42.03% ± 8.23% and 67.23% ± 5.63% at concentrations of 25 μmol/L, 50 μmol/L and 100 μmol/L, respectively (n=5,P<0.05). The inhibitory effect of ginsenoside Rb1 on spontaneous contraction was significantly but incompletely blocked by 10 mmol/L tetraethylammonium or 0.5 mmol/L 4-aminopyridine, respectively (n=5, P<0.05). However, the inhibitory effect of ginsenoside Rb1 on spontaneous contraction was not affected by 10 μmol/L glibenclamide or 0.4 μmol/L tetrodotoxin. At the cell level, ginsenoside Rb1 increased outward potassium currents, and IK V was enhanced from 1137.71 ± 171.62 pA to 1449.73 ± 162.39 pA by 50 μmol/L Rb1 at +60 mV (n=6, P<0.05). Ginsenoside Rb1 increased IK Ca and enhanced the amplitudes of spontaneous transient outward currents from 582.77 ± 179.09 mV to 788.12 ± 278.34 mV (n=5, P<0.05). However, ginsenoside Rb1 (50 μmol/L) had no significant effect on IK ATP (n=3, P<0.05). CONCLUSION: These results suggest that ginsenoside Rb1 has an inhibitory effect on the spontaneous contraction of mouse intestinal smooth muscle mediated by the activation of IK V and IK Ca , but the K ATP channel was not involved in this effect.

乙醇对小脑皮层浦肯野细胞复杂性峰电位活动的影响

[目的]探讨乙醇对小鼠小脑皮层浦肯野细胞（PC）复杂性峰电位（CS）活动的影响.[方法]取6～8周龄昆明种小鼠,腹腔注射给予乌拉坦（1.3g/kg）施行麻醉,在小脑Vermis区施行直径为1.0～1.5mm的开颅术,揭除硬脑膜,在暴露的脑表面区域持续灌流给予含氧的人工脑脊液（ACSF）;电生理记录选用在体膜片钳记录,并结合生物素染色技术,记录电极内充装20～30μL电极內液,阻抗为4～6 MΩ;通过膜片钳放大器及数据采集软件记录小脑皮层PC自发性活动.[结果]给予乌拉坦施行麻醉后,小鼠小脑皮层Vermis区PC表现为规律的单纯性峰电位（SS）放电,平均频率为（19.00±4.20）Hz;同时伴有不规律的CS活动,平均频率为（0.31±0.12）Hz;在电流钳记录模式下,乙醇（300 mmol/L）可缩短放电暂停时间,减少后超极化振幅;在电压钳下,乙醇可显著抑制CS的小穗个数及波形下面积,但对CS发生频率即小穗间隔频率未见有影响;乙醇对CS的抑制作用具有一定的剂量依存性;阻断NMDA和mGluR1受体对乙醇所起的CS抑制作用未见有影响,但乙醇对CS的抑制作用可被CB1受体阻断剂所阻断.[结论]乙醇通过活化CB1受体抑制小鼠小脑皮层PC的CS活动,提示过量饮酒可能通过激活攀缘纤维-PC突触前CB1受体抑制外周信息向小脑皮层PC的传入,进而影响运动调节功能.

Inhibitory effects of synthetic cannabinoid WIN55,212-2 on nicotine-activated currents in rat trigeminal ganglion neurons

Cannabinoid and nicotinic acetylcholine receptors are strongly associated with algesia. Previous studies in our laboratory have reported inhibitory effects of synthetic cannabinoid WIN55, 212-2 on nicotine-activated currents （Inic）, but the underlying mechanisms remain poorly understood. The present study used whole-cell patch clamp techniques to investigate the modulatory effects of synthetic cannabinoid WIN55, 212-2 on Inic in cultured rat trigeminal ganglion neurons. The results revealed several major findings： WIN55, 212-2 inhibited Inic in rat trigeminal ganglion neurons. In addition, when WIN55, 212-2 （3 μmol/L） was applied simultaneously with nicotine （100 μmol/L）, the inhibition of WIN55, 212-2 on Inic was reversible, concentration-dependent and voltage-independent This effect was not mediated by CB1, CB2 or VR1 receptors; neither the selective CB1 receptor antagonist AM281, CB2 receptor antagonist AM630 nor VR1 receptor antagonist capsazepine reduced the inhibitory effect of WIN55, 212-2. Further, the inhibition of nicotinic responses by WIN55, 212-2 was not sensitive to the membrane permeable cyclic adenosine monophosphate （cAMP） analog 8-Br-cAMP. The G-protein inhibitor GDP-I3-S （1 mmol/L） did not block the inhibitory effects of WIN55, 212-2 on/n^c, excluding the involvement of G-protein mediation. The results suggested that WIN55, 212-2 inhibits/n^o directly via the neuronal nicotinic acetylcholine receptor, and that this inhibition is non-competitive. WIN55, 212-2 did not act as an open channel blocker of the neuronal nicotinic acetylcholine receptor, and did not affect the desensitization of Into. The results suggest that nicotine receptors may be physically plugged from outside the membrane by drugs containing WIN55, 212-2.

Inhibition of 5-HT_3 Receptors-activated Currents by Cannabinoids in Rat Trigeminal Ganglion Neurons

This study investigated the modulatory effect of synthetic cannabinoids WIN55,212-2 on 5-HT3 receptor-activated currents (I5-HT3) in cultured rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The results showed that: (1) The majority of examined neurons (78.70%) were sensitive to 5-HT (3–300 μmol/L). 5-HT induced inward currents in a concentration-dependent manner and the currents were blocked by ICS 205-930 (1 μmol/L), a selective antagonist of the 5-HT3 receptor; (2) Pre-application of WIN55,212-2 (0.01–1 μmol/L) significantly inhibited I5-HT3 reversibly in concentration-dependent and voltage-independent manners. The concentra-tion-response curve of 5-HT3 receptor was shifted downward by WIN55,212-2 without any change of the threshold value. The EC50 values of two curves were very close (17.5±4.5) mmol/L vs. (15.2±4.5) mmol/L and WIN55,212-2 decreased the maximal amplitude of I5-HT3 by (48.65±4.15)%; (3) Neither AM281, a selective CB1 receptor antagonist, nor AM630, a selective CB2 receptor antagonist reversed the inhibition of I5-HT3 by WIN55,212-2; (4) When WIN55,212-2 was given from 15 to 120 s before 5-HT application, inhibitory effect was gradually increased and the maximal inhibition took place at 90 s, and the inhibition remained at the same level after 90 s. We are led to concluded that-WIN55,212-2 inhibited I5-HT3 significantly and neither CB1 receptor antagonist nor CB2 receptor antagonist could reverse the inhibition of I5-HT3 by WIN55,212-2. Moreover, WIN55,212-2 is not an open channel blocker (OCB) of 5-HT3 receptor. WIN55,212-2 significantly inhibited 5-HT-activated currents in a non-competitive manner. The inhibition of I5-HT3 by WIN55,212-2 is probably new one of peripheral analgesic mechanisms of WIN55,212-2, but the mechanism by which WIN55,212-2 inhibits I5-HT3 warrants further investigation.

Mechanism of action of two insect toxins huwentoxin-Ⅲ and hainantoxin-Ⅵ on voltage-gated sodium channels

Selenocosmia huwena and Selenocosmia hainana are two tarantula species found in southern China.Their venoms contain abundant peptide toxins.Two new neurotoxic peptides,huwentoxin-Ⅲ(HWTX-Ⅲ) and hainantoxin-VI(HNTX-VI),were obtained from the venom using ion-exchange chromatography and reverse-phase high performance liquid chromatography(RP-HPLC).The mechanism of action of HWTX-Ⅲ and HNTX-VI on insect neuronal voltage-gated sodium channels(VGSCs) was studied via whole-cell patch clamp techniques.In a fashion similar to δ-atracotoxins,HNTX-VI can induce a slowdown of current inactivation of the VGSC and reduction in the peak of Na+ current in cockroach dorsal unpaired median(DUM) neurons.Meanwhile,10 μmol/L HNTX-IV caused a positive shift of steady-state inactivation of sodium channel.HWTX-ⅡI inhibited VGSCs on DUM neurons(concentration of toxin at half-maximal inhibition(IC50)≈1.106 μmol/L) in a way much similar to tetrodotoxin(TTX).HWTX-Ⅲ had no effect on the kinetics of activation and inactivation.The shift in the steady-state inactivation curve was distinct from other depressant spider toxins.The diverse effect and the mechanism of action of the two insect toxins illustrate the diverse biological activities of spider toxins and provide a fresh theoretical foundation to design and develop novel insecticides.

Effects of SO_(2)derivatives on sodium currents in acutely isolated rat hippocampal lead-exposed neurons

In this study, the effects of acute SO_2 derivatives and chronic lead exposure together on sodium cur-rents (INa) were investigated in acutely isolated rat hippocampal neurons by using the whole-cell patch clamp techniques. We found that chronic lead exposure hardly reduced the amplitudes of INa. In the normal condition, sodium current started to appear at around ?70 mV, and reached the peak current at around ?40 mV. After chronic lead exposure, the data changed to ?70 and ?30 mV. After adding SO2 derivatives, the data changed to ?80 and ?40 mV, respectively. SO_2 derivatives caused a significant in-crease of INa in hippocampal chronic-lead exposed neurons. Chronic lead exposure induced a right shift of the activation curve and a left shift of the inactivation curve of sodium channels. SO_2 derivatives caused negative shifts of the activation and inactivation curves of INa in hippocampal chronic-lead ex-posed neurons. Lead exposure put off the time reaching the peak of INa activation. SO_2 derivatives in-creased the time constants of inactivation after lead exposure. The interaction of lead and SO_2 deriva-tives with voltage-dependent sodium channels may lead to changes in electrical activity and contribute to worsening the neurotoxicological damage.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells:Implications for Tinnitus Generation

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.