Direkte visualisering af den murine Dorsal Cochlear Nucleus for optogenetic Stimulering af Auditory Pathway
Summary
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
Undersøgelse af brug af virus-medieret genoverførsel til at arrestere eller vende høretab er stort set blevet forvist til det perifere auditive system. Kun få studier har undersøgt genoverførsel til det centrale auditive system. Den dorsale cochlear kerne (DCN) af hjernestammen, som indeholder anden ordens neuroner i den auditive vej, er et potentielt sted for genoverførsel. I denne protokol, er en teknik til direkte og maksimal eksponering af murine DCN via en posterior fossa tilgang demonstreret. Denne fremgangsmåde giver mulighed for enten akut eller overlevelse kirurgi. Efter direkte visualisering af DCN, er mulige med en masse af eksperimenter, herunder injektion af opsiner i cochlear kerne og efterfølgende stimulering med en optisk fiber koblet til et blåt lys laser. Andre neurofysiologi eksperimenter, såsom elektrisk stimulering og neurale injektor tracings er også mulig. Niveauet af visualization og varigheden af stimulation opnåelige gøre denne fremgangsmåde gælder for en bred vifte af eksperimenter.
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
Representative Results
Discussion
Dette papir beskriver teknikken af direkte visualisering af DCN i den murine model for manipulation af det centrale auditive system. Den skitserede fremgangsmåde med direkte visualisering giver betydelige fordele i forhold til de vigtigste alternativ, som er stereotaksiske tilgange. Primært direkte visualisering af DCN muliggør øjeblikkelig bekræftelse af stedet af hjernestammen, mens stereotaksiske fremgangsmåder ikke giver direkte visualisering. I forsøg, der nødvendiggør langvarig inkubationsperioder, …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Finansiering: Dette arbejde blev støttet af en fond Bertarelli tilskud (DJL), en MED-EL tilskud (DJL) og en National Institutes of Health Tilskud DC01089 (MCB).
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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