谷氨酸的释放与其兴奋性毒性对耳蜗的作用

谷氨酸是内耳毛细胞与传入神经树突之间主要的神经递质，在介导突触兴奋性活动和突触可塑性方面起着重要作用。但是谷氨酸同时也是一种潜在的神经毒性物质，可对耳蜗产生毒害作用。本文就谷氨酸在耳蜗的来源、影响耳蜗内谷氨酸浓度的因素，作用机制及对抗谷氨酸毒性的药物作一综述。     

谷氨酸—耳蜗传入神经活性物质的研究进展

耳蜗传入神经活性物质目前尚不清楚。众多实验表明,谷氨酸可能为耳蜗传入神经活性物质。本文就谷氨酸在内耳的合成、释放、高亲和的摄取系统及酶活性测定、电生理及免疫细胞化学研究作一综述。     

阿米卡星对幼年大鼠内耳毒性实验观察

目的探讨阿米卡星对幼年大鼠内耳的毒性作用。方法实验组9天龄sD大鼠皮下注射500mg·k^-1·d^-1阿米卡星连续1周,对照组皮下注射0．9％生理盐水连续1周。分别于用药后l天、1周和3周行听觉脑干反应测听（ABR）仪测试实验组和对照组大鼠耳蜗听功能,并对耳蜗进行扫描电镜观察和常规切片观察。结果实验组大鼠ABR阈值,用药后1天与对照组比较、用药后1周与用药后1天和对照组比较,差异均有统计学意义（P〈0．01）．而用药后3周和用药后1周比较,差异尢统计学意义（P〉0．05）。形态学观察,随用药后时间延长,何替器细胞损伤从感觉毛细胞到非感觉支持细胞,用药后3周出现立方上皮样结构。实验组大鼠无前庭功能障碍。结论阿米卡星连续用药可导致幼年大鼠耳蜗毛细胞的严重损伤,而对前庭功能影响较小。     

谷氨酸相关的耳蜗病理

兴奋性氨基酸具有神经毒性作用 ,能选择性地损伤可被其兴奋的神经元。有学者认为急慢性神经病变的最终共同通路都是谷氨酸类受体受到过度刺激 ,进而认为 ,研究和开发临床实用的有效的谷氨酸类受体拮抗剂将引起神经科学治疗的革命[1] 。研究表明 ,耳蜗内毛细胞可能释放谷氨酸作为传入神经递质[2 ] 。谷氨酸及其受体在各种耳蜗病变的发生与发展中所起的作用正成为耳神经科学关注的一个问题。1　谷氨酸相关的耳蜗病理研究发现 ,耳蜗Ⅰ型螺旋神经节细胞存在三种类型亲离子谷氨酸受体 ,即 :Ｎ -甲基 -Ｄ -天门冬氨酸 (Ｎ -ｍｅｔｈｙ1-Ｄ-ａｓｐ…     

谷氨酸的神经毒性对内耳的影响

谷氨酸是听觉系统最重要的传入神经递质，也有神经毒性作用，可特异性地损伤内毛细胞。本文重点综述谷氨酸的神经毒性作用及机制，以及听神经病的诸多病因，试图探讨谷氨酸的神经毒性对内耳的影响及听神经病诸多病因之间可能存在的相关性。     

外源性谷氨酸对豚鼠耳蜗内、外毛细胞易损性的比较

目的观察外源性谷氨酸对豚鼠耳蜗电位及耳蜗内、外毛细胞的影响。方法将健康豚鼠30只随机分为3组,应用豚鼠全耳蜗灌流技术,分别经耳蜗灌流人工外淋巴液和不同浓度的谷氨酸2小时,记录灌流前和灌流后的耳蜗电位(CM、CAP);同时应用透射电镜技术观察灌流前后耳蜗形态学的变化。结果灌流人工外淋巴液后豚鼠耳蜗电位及形态学无改变;灌流10 mmol/L谷氨酸后CM幅度虽有下降,其非线性特点无改变,CAP阈值平均升高了35 dB;灌流20 mmol/L谷氨酸后CM幅度明显下降但仍保持其非线性特点,CAP阈值平均升高了48 dB,灌流谷氨酸后耳蜗内毛细胞及传入神经纤维出现空化。结论谷氨酸是耳蜗主要的兴奋性传入神经递质,应用外源性谷氨酸可以引起耳蜗内毛细胞及传入神经的损伤,但对耳蜗外毛细胞无影响。     

神经营养素-3对豚鼠耳蜗传入神经元兴奋毒性损伤的保护作用

目的 观察谷氨酸（glutamate，Glu）致豚鼠耳蜗Ⅰ型螺旋神经节细胞兴奋毒性损伤后，耳蜗局部应用神经营养素-3（neurotrophin-3，NT-3）对Ⅰ型螺旋神经节细胞的保护作用及对复合动作电位（CAP）反应阈的影响。方法 28只豚鼠左耳安置圆窗电极检测CAP的变化。然后分为NT-3组（实验组5只）、Glu组（实验对照组8只）、Hank’s液组（HBSS组）（正常对照组5只）、Blank组（空白对照组10只），前三组分别在药物灌注术后1天及4周测CAP反应阈，术后4周处死，Blank组术后1天测CAP反应阈后处死，观察Ⅰ型螺旋神经节细胞和神经纤维的显微和超微结构变化。结果 NT-3组、Glu组术后1天CAP反应阈明显增高，NT-3组术后4周CAP反应阈明显降低，与HBSS组及Blank组比较均有显著性差异（P〈0．01）；术后4周NT-3组Ⅰ型螺旋神经节细胞数目的减少幅度明显小于Glu组（P〈0．01），NT-3组与HBSS组和Blank组相比细胞数有显著差异（P〈0．01）。HBSS组与Blank组相比，细胞数、CAP反应阈和组织学改变均无显著差异（P〉0．05）。结论 经外淋巴腔灌注谷氨酸能导致豚鼠耳蜗CAP反应阈提高和Ⅰ型螺旋神经节细胞死亡。耳蜗兴奋性损伤发生后60分钟，局部应用NT-3（15μg／ml）可减轻谷氨酸对耳蜗螺旋神经节细胞兴奋性毒性损伤，提示NT-3可用于神经性耳聋的治疗。     

谷氨酸调节耳蜗内毛细胞游离钙的实验观察

目的 探讨谷氨酸（glutamate,Glu）对离体耳蜗内毛细胞（inner hair cells,IHC)内钙信号的调控作用及其生理病理意义。方法 在激光共聚焦显微镜下用钙敏荧光探针Fluo－3作为指示剂,观察外源性谷氨酸对分离的10个豚鼠耳蜗IHC胞内游离钙离子浓度（[Ca^2 ]i)的影响。结果 分离好的正常IHC呈烧瓶形态,有明显的颈部,皮板上可观察到静纤毛,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活2h,Fluo-3 钙敏荧光探 针染色后IHC胞体、胞核及表皮板有明显的染色梯度,表明游离Ca^3 浓度从细胞 核向细胞逐渐减少。终浓度为3.85umol/L的Glu对游离IHC内[Ca^2 ]i有增高趋势,而对游离外毛细胞（outer hair cells,OHC)内[Ca^2 i]浓度无影响。观察10个IHC,发现9个[Ca^2 ]i浓度增加,1个无变化;观察10个OHC,发现7个[Ca^2 ]i无变化,3无略有下降。当Glu浓度增高后,IHC内[Ca^2 ]i先是迅速升高,继而逐渐下降,IHC外形由烧瓶状逐渐变成球形,提示IHC水种变性。结论 Glu可选择性调控IHC内[Ca^2 ]i ,而对OHC内[Ca^2 ]i无影响,而过量的Glu刺激,可造成IHC[Ca62 ]i的堆积,从而IHC水肿变性。     

大鼠单侧耳蜗损伤后蜗核中GluR2/3受体表达的变化

目的观察单侧耳蜗损伤后不同时段α-氨基-3-羧基-5-甲基异唑-4-丙酸(alpha-amino-3-hydoxy-5-methyl-4-isoxazolepropionate,AMPA)受体亚型谷氨酸受体2/3(glutamate receptor2and3,GluR2/3)在大鼠耳蜗核的表达的变化,为外周损伤后听觉中枢重组的分子机制提供可能的实验依据。方法手术破坏耳蜗各回,制造单侧耳蜗损伤模型;FITC标记免疫组织化学方法检测损伤后2、4、6、8、12、24h和3、7d的GluR2/3亚型在SD大鼠听觉中枢中的表达。结果①损伤后各组健侧GluR2/3受体表达的强弱各组间以及与对照组无显著性差异;②术后2h即可观察到术侧蜗核胞浆中受体表达增强,6h达到高峰,以后逐渐减弱,至第3d时低于正常水平;③正常对照组和健侧的蜗核中,GluR2/3受体主要分布在胞膜,膜周胞浆和突触上;而术侧蜗核中GluR2/3大都分布在胞浆中,胞膜上表达减弱。结论单侧耳蜗损伤后可导致术侧蜗核中GluR2/3在胞膜上的表达降低。     

耳蜗中的谷氨酸-谷氨酰胺循环

谷氨酸是耳蜗内主要的传入神经递质,其对听觉的产生具有重要的作用,同时过量释放的谷氨酸引起的兴奋性毒性作用与许多内耳疾病的发生有关.耳蜗中可能存在谷氨酸摄取系统,即谷氨酸-谷氨酰胺循环.本文对耳蜗中可能存在的谷氨酸-谷氨酰胺循环学说的来源、作用机理、及相关分子的分布特点、临床意义等进行简要综述.     

GluR2/3在不同周龄大鼠耳蜗核中的表达

目的检测α-氨基羟甲基恶唑丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid,AMPA)受体亚型GluR2/3在不同周龄大鼠耳蜗核的表达及其与听性脑干反应(auditorybrainstemresponse,ABR)的关系,为听觉中枢发育的分子机制提供可能的实验依据。方法分别测定1、4、9、15周龄SD大鼠ABR反应阈;FITC标记免疫组织化学方法检测GluR2/3亚型在不同周龄SD大鼠耳蜗核中的表达。结果1周龄SD大鼠未引出明显的ABR波形,4周起能引出稳定的ABR波形,且4、9、15周龄SD大鼠ABR反应阈无显著性差异。GluR2/3在不同周龄大鼠蜗核神经元中均有表达。1周龄SD大鼠背侧蜗核(dorsalcochlearnucleus,DCN)中神经元表达较少,位于胞膜,较弱。4周龄时表达强,主要位于胞膜;9周龄时较弱,位于胞膜及胞浆;15周龄时可见于胞膜及核周胞质,但胞质较强,胞膜含量低。4周龄与1、9、15周龄胞膜相比,有显著性差异;1周龄与9、15周龄胞膜比较无显著性差异。结论出生后GluR2/3在耳蜗核的含量及分布部位均随年龄变化而变化,这种改变可能与耳蜗核的发育相关。     

Onset Neurones in the Anteroventral Cochlear Nucleus Project to the Dorsal Cochlear Nucleus

Considerable circumstantial evidence suggests that cells in the ventral cochlear nucleus, that respond predominantly to the onset of pure tone bursts, have a stellate morphology and project, among other places, to the dorsal cochlear nucleus. The characteristics of such cells make them leading candidates for providing the so-called wideband inhibitory input which is an essential part of the processing machinery of the dorsal cochlear nucleus. Here we use juxtacellular labeling with biocytin to demonstrate directly that large stellate cells, with onset responses, terminate profusely in the dorsal cochlear nucleus. They also provide widespread local innervation of the anteroventral cochlear nucleus and a small innervation of the posteroventral cochlear nucleus. In addition, some onset cells project to the contralateral dorsal cochlear nucleus.     

光学记录蜗核和前庭核核团神经元电活动

目的 在神经细胞群的水平上研究脑干蜗核(cochlear nucleus，CN)和前庭核(vestibular nucleus，VN)神经元电活动。方法 自新生小鼠(1—5天)制备离体脑干切片，用吸光性电压敏感染料RHl55染色20分钟。采用光学记录膜电位(optical recording membrane potential)技术，观察电刺激位听神经(第8颅神经，nVⅢ)后脑干CN和VN的神经电活动。结果 ①电刺激nVⅢ断端后光学记录显示兴奋传导至CN和VN核团(n=40)；②CN和VN神经兴奋有激发延迟(onset latency)和高峰延迟(peak latency)；③所记录的光学信号具有光吸收波长特性，表明光学记录的可靠性；④光学信号包括峰样快反应信号(spike—like fast signal)和持续较长时间的慢反应信号(slow signal)；⑤连续刺激nVⅢ后发现慢反应信号大小递减，为突触疲劳(synaptic fatigue)现象。结论 本研究表明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脑干蜗核和前庭核神经电活动的时空二维方式及其兴奋性突触传递过程，为听觉和平衡觉中枢生理研究提供了新的手段。     

噪声对豚鼠耳蜗外淋巴谷氨酸含量及耳蜗电位的影响

目的观察噪声暴露对豚鼠耳蜗外淋巴液中谷氨酸含量及耳蜗电位的影响.方法应用高效液相色谱仪检测豚鼠噪声暴露耳与对照耳耳蜗外淋巴液中谷氨酸的含量,同时记录噪声暴露前及噪声暴露2小时的耳蜗电位(CM、CAP).结果对照耳外淋巴液中谷氨酸含量为4.27±0.40 μmol/L,噪声暴露耳为8.15±0.78 μmol/L,提示噪声暴露2小时后外淋巴液中谷氨酸含量明显增加(P<0.05);CM相对幅度下降,并且其非线性特点消失,CAP阈值平均升高了约50 dB.结论噪声暴露可以使豚鼠耳蜗外淋巴液中的谷氨酸含量明显增加,并引起耳蜗功能的改变.提示噪声不仅损伤外毛细胞,还可以使内毛细胞谷氨酸过度释放产生兴奋性毒性,直接引起内毛细胞及耳蜗传入神经的损伤.     

Transient Expression of Acetylcholinesterase in the Posterior Ventral Cochlear Nucleus of Rat Brain

In this report we partially characterize a pathway projecting to the posterior ventral cochlear nucleus (PVCN) of the rat brain that transiently expresses a high level of acetylcholinesterase (AChE). The AChE-positive axons form a network that envelops a discrete region of the PVCN that includes the octopus cell region and some cells rostral to it. AChE is first detectable by postnatal day 3 (P3), peaks in expression at about P7–10, and is barely detectable in our preparations by P15. We previously reported that neurons in the octopus cell region express high levels of 7 nAChR mRNA and -bungarotoxin binding during the same time period. In light microscopic immunocytochemical studies using antibodies to the vesicular acetylcholine transporter (VAChT), we could not identify immunopositive boutons in the developing regions of the PVCN that express high levels of AChE-positive fibers despite distinct punctate labeling in other brain regions. Systematic electron microscopic examination of AChE histochemical staining throughout the PVCN revealed intense labeling of axons, but synaptic sites were devoid of reaction product. The source of the AChE-positive fibers is not known, but the fibers are not auditory nerve axons and probably not collaterals of the olivocochlear bundle.     

豚鼠上橄榄外侧核γ－氨基丁酸阳性神经元向耳蜗和耳蜗核的分支投射

为探讨豚鼠上橄榄外侧核内γ－氨基丁酸（ＧＡＢＡ）阳性神经元是否同时支配同侧Ｃｏｒｔｉ器和耳蜗核，采用逆行追踪的方法，将Ｆａｓｔｂｌｕｅ（ＦＢ）和Ｄｉａｍｉｄｉｎｅ黄（ＤＹ）两种不同性质的荧光素分别注入耳蜗鼓阶及同侧耳蜗核，观察上橄榄外侧核内ＧＡＢＡ阳性神经元的分支投射。结果发现，同侧上橄榄外侧核内ＦＢ单标细胞占标记细胞的８０．８％；ＤＹ单标细胞占１２．４％；ＦＢ－ＤＹ双标细胞占６％；ＧＡＢＡ、ＦＢ、ＤＹ三标阳性细胞占０．７％。对侧上橄榄外侧核内ＦＢ、ＤＹ单标细胞均较少，未见双标细胞。结果表明，豚鼠上橄榄外侧核内有ＧＡＢＡ免疫反应阳性神经元向同侧的Ｃｏｒｔｉ器和耳蜗核并行投射，但数量较少，支配Ｃｏｒｔｉ器和耳蜗核的传出神经纤维，分别来自不同的听觉传出神经核团。     

Commissural Neurons in the Rat Ventral Cochlear Nucleus

Commissural neurons connect the cochlear nucleus complexes of both ears. Previous studies have suggested that the neurons may be separated into two anatomical subtypes on the basis of percent apposition (PA); that is, the percentage of the soma apposed by synaptic terminals. The present study combined tract tracing with synaptic immunolabeling to compare the soma area, relative number, and location of Type I (low PA) and Type II (high PA) commissural neurons in the ventral cochlear nucleus (VCN) of rats. Confocal microscopic analysis revealed that 261 of 377 (69%) commissural neurons have medium-sized somata with Type I axosomatic innervation. The commissural neurons also showed distinct topographical distributions. The majority of Type I neurons were located in the small cell cap of the VCN, which serves as a nexus for regulatory pathways within the auditory brainstem. Most Type II neurons were found in the magnocellular core. This anatomical dichotomy should broaden current views on the function of the commissural pathway that stress the fast inhibitory interactions generated by Type II neurons. The more prevalent Type I neurons may underlie slow regulatory influences that enhance binaural processing or the recovery of function after injury.     

Gene Expression Profiles of the Rat Cochlea,Cochlear Nucleus,and Inferior Colliculus

High-throughput DNA microarray technology allows for the assessment of large numbers of genes and can reveal gene expression in a specific region, differential gene expression between regions, as well as changes in gene expression under changing experimental conditions or with a particular disease. The present study used a gene array to profile normal gene expression in the rat whole cochlea, two subregions of the cochlea (modiolar and sensorineural epithelium), and the cochlear nucleus and inferior colliculus of the auditory brainstem. The hippocampus was also assessed as a well-characterized reference tissue. Approximately 40% of the 588 genes on the array showed expression over background. When the criterion for a signal threshold was set conservatively at twice background, the number of genes above the signal threshold ranged from approximately 20% in the cochlea to 30% in the inferior colliculus. While much of the gene expression pattern was expected based on the literature, gene profiles also revealed expression of genes that had not been reported previously. Many genes were expressed in all regions while others were differentially expressed (defined as greater than a twofold difference in expression between regions). A greater number of differentially expressed genes were found when comparing peripheral (cochlear) and central nervous system regions than when comparing the central auditory regions and the hippocampus. Several families of insulin-like growth factor binding proteins, matrix metalloproteinases, and tissue inhibitor of metalloproteinases were among the genes expressed at much higher levels in the cochlea compared with the central nervous system regions.     

The Structural Development of the Mouse Dorsal Cochlear Nucleus

The dorsal cochlear nucleus (DCN) is a major subdivision of the mammalian cochlear nucleus (CN) that is thought to be involved in sound localization in the vertical plane and in feature extraction of sound stimuli. The main principal cell type (pyramidal cells) integrates auditory and non-auditory inputs, which are considered to be important in performing sound localization tasks. This study aimed to investigate the histological development of the CD-1 mouse DCN, focussing on the postnatal period spanning the onset of hearing (P12). Fluorescent Nissl staining revealed that the three layers of the DCN were identifiable as early as P6 with subsequent expansion of all layers with age. Significant increases in the size of pyramidal and cartwheel cells were observed between birth and P12. Immunohistochemistry showed substantial changes in synaptic distribution during the first two postnatal weeks with subsequent maturation of the presumed mossy fibre terminals. In addition, GFAP immunolabelling identified several glial cell types in the DCN including the observation of putative tanycytes for the first time. Each glial cell type had specific spatial and temporal patterns of maturation with apparent rapid development during the first two postnatal weeks but little change thereafter. The rapid maturation of the structural organization and DCN components prior to the onset of hearing possibly reflects an influence from spontaneous activity originating in the cochlea/auditory nerve. Further refinement of these connections and development of the non-auditory connections may result from the arrival of acoustic input and experience dependent mechanisms.     

Contralateral Effects and Binaural Interactions in Dorsal Cochlear Nucleus

The dorsal cochlear nucleus (DCN) receives afferent input from the auditory nerve and is thus usually thought of as a monaural nucleus, but it also receives inputs from the contralateral cochlear nucleus as well as descending projections from binaural nuclei. Evidence suggests that some of these commissural and efferent projections are excitatory, whereas others are inhibitory. The goals of this study were to investigate the nature and effects of these inputs in the DCN by measuring DCN principal cell (type IV unit) responses to a variety of contralateral monaural and binaural stimuli. As expected, the results of contralateral stimulation demonstrate a mixture of excitatory and inhibitory influences, although inhibitory effects predominate. Most type IV units are weakly, if at all, inhibited by tones but are strongly inhibited by broadband noise (BBN). The inhibition evoked by BBN is also low threshold and short latency. This inhibition is abolished and excitation is revealed when strychnine, a glycine-receptor antagonist, is applied to the DCN; application of bicuculline, a GABA_A-receptor antagonist, has similar effects but does not block the onset of inhibition. Manipulations of discrete fiber bundles suggest that the inhibitory, but not excitatory, inputs to DCN principal cells enter the DCN via its output pathway, and that the short latency inhibition is carried by commissural axons. Consistent with their respective monaural effects, responses to binaural tones as a function of interaural level difference are essentially the same as responses to ipsilateral tones, whereas binaural BBN responses decrease with increasing contralateral level. In comparison to monaural responses, binaural responses to virtual space stimuli show enhanced sensitivity to the elevation of a sound source in ipsilateral space but reduced sensitivity in contralateral space. These results show that the contralateral inputs to the DCN are functionally relevant in natural listening conditions, and that one role of these inputs is to enhance DCN processing of spectral sound localization cues produced by the pinna.