概要

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Published: January 20, 2015
doi:

概要

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Abstract

调查使用病毒介导的基因转移到阻止或逆转听力丧失在很大程度上被转移到外周听觉系统。很少有研究探讨基因转移到中央听觉系统。脑干的背侧耳蜗核(DCN),它包含了听觉通路的二阶神经元,是用于基因转移的潜在位点。在这个协议中,对于鼠DCN经由后颅窝的方法直接和最大曝光技术被证实。这种方法允许对急性或存活手术。继DCN的直接可视化,一台主机的实验是可能的,包括注射视蛋白进入耳蜗核和随后刺激的由光纤耦合到蓝色光的激光。其他神经生理学实验,如电刺激和神经注射器描也是可行的。visualiza的水平化和刺激的持续时间可达到使这种方法适用于广泛的实验。

Introduction

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam®2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9

Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14

Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20

The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

Protocol

注:所有的实验程序按照马萨诸塞州眼的动物护理和使用委员会及耳科医院和哈佛医学院,这遵​​循国家动物保健指南,包括公共卫生服务政策上的人文关怀和使用实验动物,在执行ILAR指南,以及动物福利法。上市实验步骤如下左DCN的细节曝光。使用无菌器械,同时执行的生存术。 1.主开颅手术和背耳蜗核曝光麻醉通过腹腔内给药麻醉小鼠,年龄8-12周,体重18-24克,用甲苯?…

Representative Results

部分小脑吸入演示进入耳蜗核 后的皮肤和肌肉覆盖在颅骨被除去,颅骨表面的地标,例如冠状和LAMDA缝合线,表明开颅的大致定位。下面开颅咬骨钳,小脑可视化。小脑的小部分仔细吸出表明该CN,然后可将其注入( 图1)的可视化。 二级耳蜗核暴露的刺激与蓝光激光器 继DCN,与视蛋白的基因转移,和潜伏…

Discussion

本文介绍了DCN在操纵中枢听觉系统的小鼠模型直接可视化的技术。直接可视化的概括方法提供了主要的替代显著的优势,这是立体的方法。为主,DCN的直接可视化允许对脑干部位的立即确认,而立体定向的方法不能提供直接可视化。在于必要延长潜伏期,如在病毒介导的基因转移的情况下的实验中,存在用于低感染效率的潜力,如果注射液“射门”时,目标位置。另外,直接可视化的方法允许用?…

開示

The authors have nothing to disclose.

Acknowledgements

资金来源:这项工作是由基金会资助贝尔塔雷利(DJL),一个MED-EL补助(DJL),以及健康教育资助DC01089国家研究院​​(MCB)的支持。

Materials

Name of the Material / Equipment Company Catalog Number
Stereotaxic holder Stoelting 51500
Homeothermic blanket Harvard 507214
Scalpel blade #11 Fine Surgical Tools 10011-00
Iris scissor Fine Surgical Tools 14084-08
5 French suction Symmetry Surgical 2777914
Dental Points Henry Schein 100-8170
Bone rongeur Fine Surgical Tools 16020-14
10 µl Hamilton syringe Hamilton  7633-01
34 gauge, needle Hamilton  207434
Rongeurs Fine Surgical Tools 16021-14

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記事を引用
Kozin, E. D., Darrow, K. N., Hight, A. E., Lehmann, A. E., Kaplan, A. B., Brown, M. C., Lee, D. J. Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway. J. Vis. Exp. (95), e52426, doi:10.3791/52426 (2015).

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