The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.
गिरफ्तारी या सुनवाई हानि रिवर्स करने के लिए वायरस की मध्यस्थता जीन स्थानांतरण के उपयोग में जांच काफी हद तक परिधीय श्रवण प्रणाली में चला गया है। कुछ अध्ययनों से केंद्रीय श्रवण प्रणाली के लिए जीन स्थानांतरण की जांच की है। श्रवण मार्ग के दूसरे क्रम न्यूरॉन्स होते हैं जो brainstem के पृष्ठीय कर्णावत नाभिक (DCN), जीन स्थानांतरण के लिए एक संभावित साइट है। इस प्रोटोकॉल में, एक पीछे खात दृष्टिकोण के माध्यम से murine DCN के प्रत्यक्ष और अधिक से अधिक लोगों तक पहुंचाने के लिए एक तकनीक का प्रदर्शन किया जाता है। यह दृष्टिकोण या तो तीव्र या अस्तित्व सर्जरी के लिए अनुमति देता है। DCN के प्रत्यक्ष दृश्य के बाद, प्रयोगों की एक मेजबान एक नीली बत्ती लेजर करने के लिए मिलकर एक ऑप्टिकल फाइबर से कर्णावत नाभिक और बाद में उत्तेजना में opsins इंजेक्शन सहित संभव हो रहे हैं। इस तरह बिजली की उत्तेजना और तंत्रिका इंजेक्टर ट्रेसिंग के रूप में अन्य Neurophysiology प्रयोगों भी संभव है। Visualiza का स्तरtion और उत्तेजना की अवधि में प्राप्त प्रयोगों की एक विस्तृत श्रृंखला के लिए इस दृष्टिकोण लागू कर सकते हैं।
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.
इस पत्र में केंद्रीय श्रवण प्रणाली के हेरफेर के लिए murine मॉडल में DCN के प्रत्यक्ष दृश्य की तकनीक का वर्णन है। प्रत्यक्ष दृश्य की उल्लिखित दृष्टिकोण stereotaxic दृष्टिकोण हैं जो मुख्य वैकल्पिक अधिक महत्वपूर्ण ?…
The authors have nothing to disclose.
अनुदान: यह काम एक फाउंडेशन Bertarelli अनुदान (DJL), एक मेड-ईएल अनुदान (DJL), और स्वास्थ्य अनुदान DC01089 का एक राष्ट्रीय संस्थान (एमसीबी) द्वारा समर्थित किया गया।
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 |