在爪蟾卵母细胞中表达大鼠耳蜗核钾通道的方法学研究

目的研究在爪蟾卵母细胞中表达的大鼠耳蜗核电压依赖性离子通道的特性，为揭示感音神经性聋听觉中枢离子通道的变化奠定方法学基础。方法采用异硫氰酸胍－苯酚氯仿法和美国新磁珠技术，从５０只大鼠耳蜗核提取多聚腺嘌呤ｍＲＮＡ，注入非洲爪蟾卵母细胞表达有功能的离子通道，并利用电压钳方法记录电压依赖性的离子通道电流。结果在注射大鼠耳蜗核ｍＲＮＡ的１６个卵母细胞膜上记录到电压依赖性的瞬间外向型钾离子通道电流，其电流最大幅度的平均值为３６４±４２ｎＡ。结论本实验建立的模型在听觉中枢的生理、病理及药理学研究中起到一定的作用。     

豚鼠耳蜗外毛细胞的钾通道

目的研究外毛细胞钾离子通道的生物物理学和药理学特性.方法用细胞贴附式和内面向外式膜片钳技术,研究豚鼠耳蜗单离外毛细胞底侧膜的钾离子单通道电流.结果记录到两种钾离子单通道电流:(1)大电导型钙激活钾通道,在等渗140mmol/L KC1溶液时电导为161 26pS(内面向外式)或155±27pS(细胞贴附式),反转电位为0mV;浴液为Hanks液而电极内液为140mmo/L KC1液时,电导为133±9pS(细胞贴附式).当膜内面浴于无钙液时通道活动消失.通道呈簇状发放或快速簇状发放方式,其开启和关闭时程直方图均可用三阶指数方程拟合.双氢链霉素能够可逆性地抑制外毛细胞的大电导型钙激活钾通道,表现为电流幅度减少,在链霉素浓度增大以及细胞膜电位偏正时更明显,新霉素的抑制作用更强.(2)～44pS钾通道.结论豚鼠耳蜗外毛细胞底侧膜具有两种钾离子通道,研究了其大电导型钙激活钾通道的生物物理学和药理学特性.     

不同年龄大鼠耳蜗核细胞的定量观察

分别对幼年，成年及老年大鼠耳蜗核的体积，神经元的核仁体积和耳蜗核神经元的数量进行检测。结果示：老年大鼠耳蜗核体积与成年组相关无显著性意义；神经地核仁体积，老年组较其它两组有极显著性缩小；老年组球形细胞和多极细胞无明显减少；梭形细胞与其它两组相差无显著意义，而胶质细胞极明显增多，平均增多４８．５％。表明老年性聋的病理变化不仅发生在耳蜗，而且发生在蜗神经核。     

豚鼠耳蜗损伤对耳蜗核神经元细胞面积的影响

为了解耳蜗损伤对中枢听觉系统形态和功能的改变及在改变过程中的意义，采用计算机图像处理技术观察５只新生和１３只成年豚鼠损伤耳蜗后不同时间腹侧耳蜗核神经元细胞面积的改变。     

豚鼠耳蜗损伤对耳蜗核神经元细胞面积的影响

为了解耳蜗损伤对中枢听觉系统形态和功能的改变及在改变过程中的意义，采用计算机图像处理技术观察了５只新生和１３只成年豚鼠损伤耳蜗后不同时间腹侧耳蜗核神经元细胞面积的改变。发现新生豚鼠耳蜗损伤后２４小时前腹侧耳蜗核和后腹侧耳蜗核神经元细胞面积无明显改变，成年豚鼠损伤耳蜗后４、７、６０天前腹侧耳蜗核神经元细胞面积较对侧分别缩小２０．９３％、２５．７０％、２８．７２％，６０天组较正常对照组减小５１％；后腹侧耳蜗核损伤侧较对照侧分别缩小１７．５８％、２０．３０％、３８．５５％，６０天组较正常对照组减小３２．７５％。证实耳蜗损伤可迅速引起腹侧耳蜗核神经元细胞面积的改变，提示临床听力损失早期开始听神经刺激相当重要。     

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     

机械敏感性钾通道TREK-1在耳蜗血管纹内皮细胞的表达

目的研究机械敏感性钾通道TREK-1在大鼠耳蜗血管纹血管内皮细胞的表达情况,探讨其在内耳血流局部调节的作用与意义。方法原代培养Wistar大鼠血管纹血管内皮细胞并进行传代纯化,逆转录-聚合酶链反应（RT-PCR）与免疫荧光分别检验TREK-1在此传代细胞mRNA转录及蛋白水平上的表达。结果RT-PCR特异性扩增出TREK-1cDNA片段,细胞免疫荧光呈阳性反应。结论大鼠耳蜗血管纹血管内皮细胞有机械敏感性钾通道TREK-1表达,推断与耳蜗局部血流调节有关。     

甘氨酸受体α1亚单位在单侧聋大鼠耳蜗核不同发育时间的表达CSCD

目的 观察单侧耳蜗毁损后1、2、4周甘氨酸受体α1亚单位（glycine receptor alpha1 subunit,GlyRα1）在大鼠耳蜗核的表达变化,为探讨单侧聋听觉中枢重组的分子机制提供参考。方法 SD大鼠在出生后12 d,随机分为实验组（左侧耳蜗毁损）及对照组,术后1、2、4周取耳蜗核,切片后采用免疫组化方法观察GlyRα1的表达变化。结果 GlyRα1在两组双侧耳蜗核中的表达在造模后2周出现升高,4周降低;实验组术后2周双侧表达差异有统计学意义（P<0.05）,术后1、4周及对照组各时间段双侧无统计学差异（P>0.05）。对照组左侧与实验组术侧相比术后1、4周差异有统计学意义（P<0.05）,术后2周差异无统计学意义（P>0.05）;实验组及对照组右侧术后1、2、4周均有统计学差异（P<0.05）。结论 单侧听觉剥夺导致大鼠双侧耳蜗核接收不对称的信息,观察到的耳蜗核GlyRα1在发育中的不对称改变可能为初级听觉中枢重组所必需。     

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     

Developmental time course of potassium channel expression in the rat cochlear nucleus

Voltage gated potassium channels play critical roles in determining the responses of auditory brainstem neurons to acoustic stimuli. In the present study, we examined the developmental expression of potassium channels in rat cochlear nucleus. Quantitative RT-PCR revealed that Kv1.1 , Kv1.2 and Kv3.1 showed a monotonic increase in mRNA levels from postnatal days 3-28 (P3-P28), after which mRNA level was relatively constant until P56. In contrast, Kv4.2 mRNA levels were lower on average by a factor of 2 after P28 than before P28. Relative to Kv1.1, Kv3.1 and Kv1.2 mRNA were more abundant before P10 and less abundant thereafter. To address the relationship between message and protein levels, we performed semi-quantitative Western blotting for Kv1.2. The message for Kv1.2 increased earlier in development than the protein levels. Immunocytochemistry revealed a broad expression of Kv1.1 and Kv1.2 in the VCN. Staining intensity increased from 7-28 days postnatal. Kv1.2 immunostaining was less variable across cells than Kv1.1 staining. We conclude that maturation of potassium channel expression in the rat cochlear nucleus continues until at least 4 weeks postnatal.     

Deafferentation-induced changes in protein kinase C expression in the rat cochlear nucleus

Isoforms of the signal transducing molecule, protein kinase C (PKC), may play a role in neural plasticity following sensory deafferentation. To explore the role of PKC in central auditory plasticity, we studied the effect of auditory deafferentation on the expression of PKC betaI, betaII, gamma, and delta in the rat dorsal (DCN) and ventral cochlear nucleus (VCN), using immunocytochemistry. Male rats were treated with kanamycin and furosemide to induce hair cell loss. At various intervals post-treatment, brains were perfusion-fixed and processed for immunocytochemistry. Following deafferentation, we observed a gradual increase in PKC betaI immunoreactivity (ir) in the deepest layers of the DCN, possibly representing synapses of primary afferents or parallel fibers on unlabeled neurons. Correlated with this, we observed an increase in the number of neurons in the deep DCN that showed PKC delta ir. In controls, we observed PKC gamma ir in small ovoid cells concentrated in the middle layer of the DCN. From days 4 through 14 after deafferentation, we found an increase in the intensity of staining of these cells, with a return toward control levels by day 28. Finally, Purkinje-like cells (PLC) in the VCN, which express only PKC delta in control rats, began to express PKC gamma after deafferentation, correlated with increased expression of calbindin D28k in PLC. Thus PKC isoforms are differentially regulated in the CN following deafferentation, supporting a role for PKC in auditory plasticity.     

Voltage-gated and background K+ channel subunits expressed by the bushy cells of the rat cochlear nucleus

Bushy cells of the ventral cochlear nucleus produce a single, short latency action potential at the beginning of long depolarisations. In the present work an immunochemical survey was performed to detect the presence of K+ channel subunits which may contribute to the specific membrane properties of the bushy cells. The immunocytochemical experiments conducted on enzymatically isolated bushy cells indicated positive immunolabelling for several subunits known to be responsible for the genesis of rapidly inactivating K+ currents. Bushy cells showed strong expression of Kv3.4, 4.2 and 4.3 subunits, with the lack of Kv1.4 specific immunoreaction. The Kv3.4-specific immunoreaction had a specific, patchy appearance. Bushy cells also expressed various members of the Kv1 subunit family, most notably Kv1.1, 1.2, 1.3 and 1.6. Weak positivity could be observed for Kv3.2 subunits. The positive immunolabelling for Kv3.4, Kv4.2 and Kv4.3 was confirmed in free-floating tissue slices. Voltage-clamp experiments performed on positively identified bushy cells in brain slices corroborated the presence and activity of Kv3.4 and Kv4.2/4.3 containing K+ channels. Bushy cell showed strong immunopositivity for TASK-1 channels too. The results presented in this work indicate that bushy cells possess several types of voltage-gated K+ channel subunits whose activity may contribute to the membrane properties and firing characteristics of these neurones.     

Metabolism of the dorsal cochlear nucleus in rat brain slices

In vitro brain slices of the cochlear nucleus have been used for electrophysiological and pharmacological studies. More information is needed about the extent to which the slice resembles in vivo tissue, since this affects the interpretation of results obtained from slices. In this study, some chemical parameters of the dorsal cochlear nucleus (DCN) in rat brain slices were measured and compared to the in vivo state. The activities of malate dehydrogenase and lactate dehydrogenase were reduced in some DCN layers of incubated slices compared to in vivo brain tissue. The activities of choline acetyltransferase and acetylcholinesterase were increased or unchanged in DCN layers of slices. Adenosine triphosphate (ATP) concentrations for in vivo rat DCN were similar to those of cerebellar cortex. Compared with in vivo values, ATP concentrations were decreased in the DCN of brain slices, especially in the deep layer. Vibratome-cut slices had lower ATP levels than chopper-cut slices. Compared with the in vivo data, there were large losses of aspartate, glutamate, glutamine, gamma-aminobutyrate and taurine from incubated slices. These amino acid changes within the slices correlated with the patterns of release from the slices.     

光学成像蜗核谷氨酸受体的兴奋性传导

目的在神经细胞群的水平上二维动态观测蜗核(CN)神经元兴奋性谷氨酸递质受体的传导机制.方法自新生小鼠制备脑干切片,用吸光性电压敏感染料RH 155染色,并成像于16×16 elements Photodiode Arrays光学记录系统,电刺激位听神经(第8颅神经,nⅧth)断端.为了观察谷氨酸是否为耳蜗核神经元的兴奋性递质,用受体拮抗剂(NMDA受体拮抗剂APV,non-NMDA受体拮抗剂CNQX)灌流脑片.结果①电刺激nⅧth断端后光学记录显示兴奋传导至CN区域;②CN神经兴奋有激发延迟和高峰延迟,DCN和VCN之间激发和高峰延迟之间比较,差异有显著性意义(P＜0.01);③光学记录可以同步观察氨基酸拮抗剂对多个神经元核团兴奋性传递的作用,NMDA受体拮抗剂APV和non-NMDA受体拮抗剂CNQX对EPSP的抑制作用及其在VCN和DCN的作用方式不同.APV灌流后神经元的EPSP被抑制,计算其减低比率,在VCN为99.10±0.02%(n=18 elements),DCN为76.00±19.20%(n=46 elements).同时,CNQX灌流后神经元的EPSP被抑制,用相同方法计算其减低比率,在VCN为0.90±0.02%(n=18elements),DCN为24.00±19.20%(n=46 elements).结论光学记录膜电位方法可以在神经细胞群的水平上直观观察CN神经电活动的时空二维方式及其兴奋性突触传递过程;药理实验证实,谷氨酸是CN的神经元突触后电位传导的兴奋性递质;谷氨酸两种受体NMDA和non-NMDA均参与介导CN神经元EPSP;NMDA受体和non-NMDA受体的作用在VCN和DCN的不同神经元核团之间有区别.     

Expression of glycine receptor subunit mRNAs in the rat cochlear nucleus

The distribution of glycine receptor subunit mRNAs in the cochlear nucleus of the adult rat was examined using radioactive in situ hybridization. Expression was compared among six cell types by counting silver grains over somata. Expression of the immature alpha2 subunit was not above the threshold for detection in any neurons. Levels of expression of mature subunits varied among different cell types. Spherical bushy, small cell cap/shell neurons and fusiform cells had high expression of glycine receptor mRNA for alpha1, alpha 3 and beta subunits. Octopus cells and corn cells had high expression for alpha 3 and beta subunits, and only moderate expression for alpha1 subunit. Granule cells located between the dorsal and ventral cochlear nucleus had moderate expression of alpha 3 and beta subunits and no detectable alpha1 expression. These patterns of expression predict differences in glycinergic pharmacological properties between the cochlear nucleus neurons.     

Modulation of spontaneous activity by acetylcholine receptors in the rat dorsal cochlear nucleus in vivo

In vitro studies have implicated muscarinic acetylcholine receptors (mAChRs) in the modulation of spontaneous activity (SA) of neurons in the rat dorsal cochlear nucleus (DCN) (Chen et al., 1994,1998). Early studies suggest that cholinergic pathways also modulate SA in vivo, but these effects have not been investigated pharmacologically. The purpose of the present study was to determine whether multiunit SA can be modulated in vivo by application of cholinergic agents to the surface of the DCN. Sprague Dawley rats were used in the current experiment. The influence of cholinergic activation on SA was tested by applying carbachol (5–500 μM) to the DCN surface while recording multiunit SA at a depth of 250 μm. Out of a total of 32 sites tested, all but 2 (94%) showed well-defined responses to carbachol, characterized by suppression, activation or a combination of both (two-component responses). The most common responses were pure suppression and suppression accompanied by transient activation. Both the proportion of sites showing suppressive responses and the magnitude of suppression averaged across sites increased with dose. Although the proportion of sites showing pure activation in response to carbachol decreased with dose, there was no clear trend in the magnitude of activation with dose. The suppressive responses to high doses of carbachol were blocked by pre-application of atropine. These results extend previous work by suggesting that muscarinic receptors play an important role in the modulation of SA in vivo.