Summary

Directe visualisatie van het Muizen Dorsale Cochlear Nucleus voor optogenetische Stimulatie van de Auditieve Pathway

Published: January 20, 2015
doi:

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

Onderzoek naar het gebruik van virus-gemedieerde genoverdracht te arresteren of achteruit gehoorverlies is grotendeels verbannen naar de perifere auditieve systeem. Enkele studies hebben genoverdracht naar het centrale auditieve systeem onderzocht. De dorsale cochleaire nucleus (DCN) van de hersenstam, die tweede orde neuronen van het auditieve pad bevat, is een potentiële site voor genoverdracht. In dit protocol is een techniek voor het direct en maximale blootstelling van de muizen DCN via een fossa posterior aanpak is. Deze aanpak maakt het voor zowel acute of overleving chirurgie. Na directe visualisatie van het DCN, een reeks experimenten mogelijk, zoals injectie van opsins in de cochleaire kern en daaropvolgende stimulatie door een optische vezel gekoppeld met een blauw laserlicht. Andere neurofysiologie experimenten, zoals elektrische stimulatie en neurale injector traces zijn ook mogelijk. Het niveau van visualizatie en de duur van de stimulatie stand te brengen is deze benadering voor diverse experimenten.

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

OPMERKING: Alle experimentele procedures worden uitgevoerd in overeenstemming met de Animal Care en gebruik Comite van het Massachusetts Eye en Ear Infirmary en de Harvard Medical School, die de nationale richtlijnen verzorging van dieren te volgen, waaronder GGD Beleid inzake Humane zorg en het gebruik van proefdieren, de ILAR Guide, en de Animal Welfare Act. Opgesomd experimentele procedures hieronder detail blootstelling van de linker DCN. Gebruik steriele instrumenten tijdens het uitvoeren van de overleving chirurgie. <p class="jove…

Representative Results

Gedeeltelijke Cerebellar Aspiration toegang verschaft tot de Cochlear Nucleus Nadat de huid en spieren die over de schedel verwijderd, schedeloppervlak monumenten, zoals de coronale en Lamda hechtdraad lijnen tonen de benaderde lokalisatie van de craniotomie. Na craniotomie met rongeurs, wordt het cerebellum gevisualiseerd. Zorgvuldig aspireren van het kleine deel van het cerebellum toont visualisatie van de GN, die vervolgens kan worden geïnjecteerd (Figuur 1). </…

Discussion

Dit artikel beschrijft de techniek van directe visualisatie van het DCN in het muismodel manipulatie van het centrale auditieve systeem. De geschetste aanpak van directe visualisatie biedt aanzienlijke voordelen ten opzichte van de belangrijkste alternatief, dat stereotaxisch benaderingen zijn. In de eerste plaats, directe visualisatie van de DCN zorgt voor directe bevestiging van de site van de hersenstam, terwijl stereotaxisch benaderingen niet veroorloven directe visualisatie. In experimenten die incubatietijd noodza…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Financiering: Dit werk werd ondersteund door een stichting Bertarelli subsidie ​​(DJL), een MED-EL-subsidie ​​(DJL), en een National Institutes of Health Subsidies 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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Cite This Article
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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