The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.
逮捕または難聴逆転するウイルス媒介遺伝子導入の使用の調査は、主に末梢聴覚系に追いやられています。いくつかの研究は、中枢聴覚系への遺伝子導入を検討した。聴覚路の二次ニューロンが含まれている脳幹の背側蝸牛神経核(DCN)は、遺伝子導入のための潜在的部位である。このプロトコルでは、後頭蓋窩アプローチを経由してネズミDCNの直接的かつ最大の露出のための技術が実証されている。このアプローチは、急性または生存のどちらかの手術が可能になります。 DCNの直接可視化に続いて、実験のホストは、青色光レーザに結合された光ファイバによる蝸牛神経核およびその後の刺激へのオプシンの注射を含む、可能である。そのような電気刺激と神経インジェクタトレーシングのような他の神経生理学実験は、また可能である。visualizaのレベルると、刺激の持続時間は、達成可能な実験の広範囲にこのアプローチを適用する。
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.
本稿では、中央聴覚系を操作するためのマウスモデルにおけるDCNの直接可視化の技術が記載されている。直接可視化の概略アプローチは、定位アプローチである主要な代替、上の大きな利点を提供する。定位のアプローチが直接可視化を与えないのに対し、主に、DCNの直接可視化は、脳幹の部位の即時確認することができます。注入は、ターゲットの場所を「ミス」した場合場合、ウイルス?…
The authors have nothing to disclose.
資金調達:この作品は、財団Bertarelli補助金(DJL)、MED-ELの助成金(DJL)、国立衛生研究所補助金のDC01089(MCB)によってサポートされていました。
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 |