水杨酸钠对大鼠下丘神经元电压门控性钠通道的抑制作用

目的:了解电压门控性钠通道在水杨酸钠导致耳鸣的机制中所起的作用。方法:利用全细胞膜片钳技术研究水杨酸钠对急性分离的大鼠下丘神经元钠通道的影响。结果:水杨酸钠抑制钠通道电流(INa),而且此抑制作用具有浓度依赖性(0.1～10.0mmol/L)。水杨酸钠抑制INa的50%抑制浓度(IC50)值为1.43mmol/L。水杨酸钠不影响INa的电导-电压曲线和稳态激活曲线,将INa的稳态失活曲线向超极化方向移动9mV。此外,水杨酸钠还延长INa的失活后恢复的时间。结论:水杨酸钠以浓度依赖的方式抑制INa,并且影响INa的稳态失活和失活后恢复的动力学特征,这可能与水杨酸钠导致耳鸣的机制有关。     

水杨酸钠对大鼠下丘神经元L-型钙通道的影响

目的 了解L-型钙通道在水杨酸钠导致耳鸣的机制中所起的作用.方法 利用全细胞膜片钳技术研究水杨酸钠对急性分离的大鼠下丘神经元L-型钙通道的影响.结果 水杨酸钠抑制L-型钙通道电流(ICa,L),且具有浓度依赖性.水杨酸钠抑制ICa,L的半抑制浓度值为1.99 mmol/L.水杨酸钠不影响ICa,L的电导-电压曲线和稳态激活曲线,水杨酸钠将ICa,L的稳态失活曲线向超极化方向移动8 mV,并且延长ICa,L失活后恢复的时间.结论 水杨酸钠对ICa,L的抑制作用,可能通过减少下丘部位γ-氨基丁酸的释放,从而导致耳鸣.     

豚鼠耳蜗单离外毛细胞的外向整流钾电流

目的 :观察豚鼠耳蜗单离外毛细胞 (OHC)的电生理特性 ,记录不同长度OHC的外向整流钾电流 ,分析区分外向整流钾电流所包含的通道电流成分 ,研究外向整流钾电流的动力学特征。方法 :采用酶消化法及机械分离OHC。运用全细胞膜片钳技术 ,在电压钳下记录K+ 通道电流。结果 :OHC的全细胞膜电容为 (30 .96±2 .79) pF(n =2 9) ,零电流电位 (30± 2 .1)mV(n =16 ) ,反转电位为 (- 5 1.6 7± 1.84 )mV(n =9)。不同长度OHC的外向整流钾电流存在系统差异 ,短OHC表现出大的钾电导 ,长OHC则相反。 10 0 μmol/L的氯化镉 (Cd Cl2 )抑制了OHC外向整流钾电流的最大电流幅度的 6 0 % ,且改变了电流的动力学特征 ,对峰电流的影响明显大于稳态电流 (P<0 .0 1,n =5 ) ;1mmol/L的四氨基吡啶 (4 AP)抑制了最大电流幅度的 4 3% ,没有改变电流的动力学特征。外向整流钾电流的激活符合Boltzmann方程 ,V1/ 2 =(- 11.0 7± 0 .2 6 )mV ,S =(6 .6 2± 1.74 )mV(n=13)。结论 :外向整流钾电流包含有钙离子激活的钾离子电流、外向延迟整流钾电流和A型电流     

顺铂对大鼠耳蜗螺旋神经节细胞外向延迟整流钾电流的影响

目的:观察记录顺铂作用下急性分离新生大鼠耳蜗螺旋神经节细胞(SGNs)延迟整流钾通道的电流曲线,分析顺铂对SGNs钾电流激活动力学的影响,并初步探讨其耳毒性机制。方法:采用全细胞膜片钳技术记录SGNs外向延迟整流钾电流及顺铂对此电流的影响。结果:钳制电压为-60mV,刺激电压从-60mV到 80mV逐渐去极化,阶跃电压为10mV,持续时间为500ms,可在SGNs上记录到外向钾电流,该电流对TEA-Cl(4-乙基胺)敏感,具有延迟整流特性;在细胞外液中加入10μmol/L顺铂,能明显抑制SGNs延迟整流钾电流;顺铂对此电流的抑制作用与细胞外液中顺铂的浓度呈剂量依赖性;外液洗脱后SGNs电流可基本恢复正常。结论:钾通道与SGNs动作电位的产生密切相关,顺铂可抑制SGNs钾通道电流,导致听觉功能障碍。     

乙酰胆碱对豚鼠外毛细胞的电压依赖性外向整流钾电流的影响

目的 观察乙酰胆碱（ＡＣｈ）对不同长度豚鼠耳蜗外毛细胞（ＯＨＣ）电压依赖性外向整流钾电流的影响，分析 ＡＣｈ对钾电流激活动力学的影响。方法 全细胞膜片钳技术。结果１００μｍｏｌ／Ｌ的ＡＣｈ对短ＯＨＣ电压依赖性外向整流钾电流的影响较大，刺激电压为５０ｍＶ时，最大外向电流的幅度增加了３４．８％。ＡＣｈ对峰电流的影响大于稳态电流，改变了外向整流钾电流的动力学特征。ＡＣｈ将ＯＨＣ的零电流电位向超极化方向移位约５ｍＶ。1００μｍｏｌ／Ｌ的ＡＣｈ使ＯＨＣ电压依赖性外向整流钾电流的激活动力学发生改变，Ｖ（１／２）＝（－５２．３８±３．９８）ｍＶ，较作用前明显超极化，激活的电压敏感性也提高，Ｓ＝（４０±４．１４）ｍＶ（ｎ＝５）。结论ＡＣｈ增加了ＯＨＣ电压依赖性外向整流钾通道的电导，使通道的激活电压向超极化方向移位。ＡＣｈ的作用是使ＯＨＣ超极化。     

豚鼠耳蜗单离Hensen细胞钾电流特性及三磷酸腺苷对其影响

目的 研究豚鼠耳蜗单离Hensen细胞的钾离子电流及其特性以及三磷酸腺苷(adenosine triphosphate，ATP)对Hensen细胞电生理特性的影响。方法 采用传统全细胞膜片钳技术，对单离Hensen细胞进行记录。ATP通过压力注射仪对单离Hensen细胞给药。结果 Hensen细胞的钾电流呈明显的外向整流性，只有延迟整流性钾电流(delayed rectification potassiu mcurrent，IK)，没有瞬间外向性钾电流(transient outward potassium current，IA)。低浓度(0．1μmol/L，1μmol／L，10μmol／L)ATP可以使Hensen细胞的钾电流的幅度明显降低，且呈浓度依赖性，浓度越高，降幅越大。高浓度ATP(100μmol／L，1mmoL／L，10mmol／L)可以引起Hensen细胞的内向离子流，呈浓度依赖性，浓度越高，升幅越大。ATP的这两种作用均可被ATP受体拮抗剂(100μmol／L舒拉明)所逆转。结论 对Hensen细胞不同电压的刺激可以诱发出延迟整流性钾电流，低浓度ATP对Hensen细胞的钾电流有明显抑制作用，且呈浓度依赖性。高浓度ATP可以引起Hensen细胞的非选择性内向离子流，为钾离子依赖性，而且是通过ATP受体起作用。     

硝苯地平对三磷酸腺苷引起的Hensen细胞内向性离子流的抑制作用

目的探讨硝苯地平对高浓度三磷酸腺苷（ATP）引起的豚鼠耳蜗单离Hensen细胞非选择性内向性离子流的影响。方法采用酶消化和机械分离的方法单离豚鼠耳蜗Hensen细胞，选择胞膜清晰、胞质透明的单离细胞通过压力注射仪分别给予0．1mmol／LATP、1mmol／LATP、10mmol／LATP、0．1mmol／LATP＋0．1mmol／L舒拉明、单独细胞外液、140mmol／LCsCl＋1mmol／LATP、40mmol／L四乙基铵（tetraethylammonium，TEA）＋1mmol／LATP、1mmol／LATP＋10μmol／L硝苯地平，并行全细胞膜片钳记录。结果当分别给予Hensen细胞0．1mmol／L（n=10），1mmol／L（n=10），10mmol／L（n=6）的ATP刺激时，均可记录到内向电流，并随ATP浓度的增加而增强，呈现浓度依赖性。该内向电流可被0．1mmol／L舒拉明（n=5），140mmol／LCsCl（n=5）和40mmol／LTEA（n=5）所抑制。单独给予细胞外液刺激后未见内向及外向离子流。当同时给予1mmol／LATP和10μmol／L硝苯地平刺激时，内向性电流消失，转而出现外向性电流。结论高浓度ATP可引起Hensen细胞的内向性电流，此电流与钾通道密切相关，并无机械转化通道参与，硝苯地平可抑制这种内向性电流并出现与正常情况相似的外向性电流。提示硝苯地平可通过Hensen细胞改善钾离子循环，起到部分保护耳蜗功能的作用。     

豚鼠耳蜗单离Deiters细胞的钾电流

目的　研究豚鼠耳蜗单离Deiters细胞的钾电流及其特性。方法　运用膜片钳技术 ,在全细胞模式下记录正常细胞外液中钾电流 ,不同K 浓度的细胞外液对细胞反转电位和外向钾电流的影响 ,四氨基吡啶 (4 aminopyridine ,4 AP)和四乙基胺 (tetraethylammonium ,TEA)对钾电流成分的阻滞作用 ,探讨外向钾电流通道的激活和失活动力学。结果　单离Deiters细胞具有电压依赖的外向整流离子选择性通道 ;钾通道阻滞剂 4 AP和TEA可使峰电流和迟电流幅度下降 ,表明存在两种类型的钾通道 ,或者这种通道有两种不同的状态 ;通道的激活和失活符合Boltzmann方程。未记录到Deiters细胞的内向钙电流。结论　Deiters细胞外向整流钾电流的作用可能是缓冲细胞周围间隙钾离子浓度     

利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的 观察KCNQ家族钾通道特异性阻滞剂利诺吡啶(linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞(支持细胞)总钾电流的影响，初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。方法 运用膜片钳技术，在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流，并观察100μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果 在正常细胞外液中，单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential，IKn)两种钾电流，而单离Deiters细胞中只记录到外向整流性钾电流。加入100μmol／L利诺吡啶后，外毛细胞中的四乙基二乙胺敏感的钾电流减小，IKn被完全抑制；而Deiters细胞中的外向整流性钾电流大小无变化。结论 KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞，其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分，并构成全部的IKn；但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

豚鼠耳蜗单离Deiters细胞的钾电流

目的 研究豚鼠耳蜗单离Ｄｅｉｔｅｒｓ细胞的钾电流及其特性。方法 运用膜片钳技术,在全细胞模式下记录正常细胞外液中钾电流,不同Ｋ＾＋浓度的细胞外液对细胞反转电位和外向钾电流的影响,四氨基吡啶（４－ａｍｉｎｏｐｙｒｉｄｉｎｅ,４－ＡＰ）和四乙工胺（ｔｅｔｒａｅｔｈｙｌａｍｍｏｎｉｕｍ,ＴＥＡ）对钾电流成分的阻滞作用,探讨外向钾流通道的激活和失活动力学。结果 单离Ｄｅｉｔｅｒｓ细胞具有电压依赖的外向整流     

Effects of salicylate on voltage-gated sodium channels in rat inferior colliculus neurons

To investigate the effects of the tinnitus inducer, sodium salicylate, on voltage-gated sodium channels, we studied freshly dissociated inferior colliculus neurons of rats by the whole-cell voltage clamp method. Salicylate blocked sodium channels in concentration-dependent manner (0.1-10 mM), and the IC50 value of salicylate was estimated to be 1.43 mM after application. The sodium conductance-voltage curve did not shift along the voltage axis with salicylate application. In contrast, the steady-state sodium channel inactivation curve was shifted by about 9 mV in the hyperpolarizing direction. In addition, salicylate delayed the sodium channel recovery from inactivation by increasing the slow time constant. It was concluded that salicylate bound to the resting and inactivated sodium channels to cause blocking, with a higher affinity for the latter state. Our results suggest that salicylate causes a concentration-dependent blockade of voltage-gated sodium channels and shifts the inactivation curve to more hyperpolarized potentials, which could be related to the mechanism of salicylate-induced tinnitus.     

Salicylate blocks L-type calcium channels in rat inferior colliculus neurons

To investigate the effects of the tinnitus inducer sodium salicylate on L-type voltage-gated calcium channels, we studied freshly dissociated inferior colliculus neurons of rats by the whole-cell voltage clamp method. Salicylate's blocking of L-type calcium channels was concentration dependent, and the IC(50) value of salicylate was estimated to be 1.99 mM. An amount of 1 mM salicylate significantly shifted the steady-state inactivation curve of L-type calcium channels about 9 mV in the hyperpolarizing direction and significantly delayed calcium channel recovery. Our results suggest that salicylate's blocking of L-type calcium channels may contribute to salicylate-induced tinnitus by decreasing GABA release in the inferior colliculus.     

水杨酸钠对大鼠下丘内谷氨酸和γ-氨基丁酸的影响

目的探讨水杨酸钠诱导产生耳鸣动物模型时神经递质在其中枢发病机制中的作用。方法利用微透析技术,在活体清醒的状态下检测水杨酸钠诱导的耳鸣动物模型,研究水杨酸钠对听觉中枢核团下丘的神经递质谷氨酸和γ-氨基丁酸的影响。结果腹腔注射10%水杨酸钠（350mg/kg）引起下丘谷氨酸水平的显著性升高,最高达到基础值的236%&#177;19%;γ-氨基丁酸水平显著性降低,最低达到基线水平的50%&#177;12%。对照组（注射生理盐水）并未引起任何显著改变。结论微透析技术活体检测数据表明,下丘内谷氨酸水平的升高和γ-氨基丁酸水平的降低可能和耳鸣的产生有关。     

川芎嗪拮抗链霉素耳毒性作用及其离子通道机制

目的 研究川芎嗪（tetraethylplyrazine，TMP）拮抗链霉素耳毒性作用及其对耳蜗外毛细胞外向K^＋通道的影响，寻求两者的相关性，旨在探讨川芎嗪拮抗耳中毒作用的离子通道机制。方法 选取豚鼠60只，随机分为6组，即对照组、链霉素组、川芎嗪低浓度组、川芎嗪高浓度组、川芎嗪低浓度＋链霉素组和川芎嗪高浓度＋链霉素组，分别注射生理盐水（2．5ml/kg）、链霉素（450mg／kg）、川芎嗪（12mg／kg）、川芎嗪（60ms／ks）、川芎嗪（12mg／kg）＋链霉素（450mg／kg）、川芎嗪（60mg／kg）＋链霉素（450mg／kg），用药10天后检测各组豚鼠ABR反应阈，并采用全细胞膜片钳技术观察川芎嗪对耳蜗外毛细胞Ca^2＋敏感K^＋电流和延迟外向K^＋电流的影响。结果 结果表明川芎嗪明显降低链霉素所致的豚鼠ABR反应阈升高，提示川芎嗪具有明显的拮抗链霉素耳毒性作用；川芎嗪能明显增大豚鼠耳蜗外毛细胞Ca^2＋敏感K^＋电流和延迟外向K^＋电流，并呈浓度依赖关系。结论 川芎嗪可能通过增大K^＋通道电流而发挥其降低链霉素耳毒性作用，推测这是其抗耳毒性作用机制之一。     

Long-term administration of salicylate enhances prestin expression in rat cochlea

Salicylate, a common drug frequently used long term in the clinic, is well known for causing reversible hearing loss and tinnitus. Our previous study, however, demonstrated that chronic administration of salicylate progressively raised the amplitude of distortion product of otoacoustic emissions (DPOAEs), which are mainly caused by (outer hair cell) OHC electromotility. How salicylate affects OHC electromotility to cause this paradoxical increase remains unclear. One possibility is that it could affect prestin, which is a motor protein that contributes to the mechano-electrical properties of OHCs. In this experiment, we assessed the effect of acute and chronic salicylate treatment on prestin expression. Interestingly, after long-term salicylate injection (200 mg/kg, twice daily for 14 days), prestin gene and protein levels were up-regulated about twofold. These levels returned to baseline 14 days after treatment stopped. Acute injection of salicylate (single injection, 400 mg/kg) did not affect prestin levels. These data reveal that chronic salicylate administration markedly, but reversibly, increased prestin levels which may contribute to the enhanced DPOAE amplitudes we observed previously with similar salicylate treatment, which may be responsible for salicylate-induced tinnitus generation.     

Effects of salicylate on serotoninergic activities in rat inferior colliculus and auditory cortex

In vivo microdialysis offers a unique approach to monitor biochemical events related to brain function and metabolism, and has been used extensively in many systems to measure the release of endogenous transmitters and other neuroactive substances during normal and pathological conditions. The characterization of neurotransmitters' changes induced by salicylate in the inferior colliculus (IC) and the auditory cortex (AC) may provide insight into the action of salicylate on the auditory system and, through this, provide a better understanding of neurological mechanism of salicylate-induced tinnitus. In the present study, the effect of salicylate on 5-HT system in IC and AC has been monitored by microdialysis in salicylate-induced tinnitus animal models. Glucose and lactate levels in IC and AC were significantly increased after application of salicylate (350 mg/kg, i.p.), indicating a salicylate-related increase in regional neuronal activity. The 5-HT level increased to a maximum of 268+/-27% basal level in IC 2 h after application and of 277+/-24% basal level in AC around 3 h after application. These data suggest that the increases of 5-HT levels in IC and AC may be involved in the tinnitus generation.     

Salicylate- and quinine-induced tinnitus and effects of memantine

CONCLUSION: Memantine, an antiglutamatergic drug, has been proposed as a treatment for tinnitus. OBJECTIVES: The purpose of this study was to determine if memantine would prevent salicylate-induced tinnitus. Local field potentials were also recorded from auditory cortex to determine what effect salicylate, memantine, and the combination of both drugs would have on evoked potential amplitudes. MATERIALS AND METHODS: Schedule induced polydipsia-avoidance conditioning was used to identify the doses of salicylate or quinine that reliably induced tinnitus in rats. Rats were trained to lick for water during quiet intervals and avoid licking during sound intervals. RESULTS: Rats injected with saline or a low dose of sodium salicylate or quinine failed to develop tinnitus-like behaviors. However, high doses of salicylate (150-300 mg/kg/day) or quinine (100-150 mg/kg/day) greatly reduced licks-in-quiet, behavior consistent with the presence of tinnitus. Licks-in-quiet increased slightly when memantine (1.5 or 3 mg/kg/day) was co-administered with salicylate; however, the effect was not statistically significant or dose-dependent. These results indicate that memantine does not completely suppress salicylate-induced tinnitus. Cortical auditory evoked potential amplitude increased after salicylate treatment; co-administration of memantine failed to block this salicylate-induced increase.     

Differential gene expression profiles in salicylate ototoxicity of the mouse

CONCLUSION: This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. OBJECTIVES: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. MATERIALS AND METHODS: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. RESULTS: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.