Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the 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

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser. Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible. The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

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 vectors^5,6, 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 intr....

Protocol

NOTE: All experimental procedures are performed in accordance with the Animal Care and Use Committee of the Massachusetts Eye and Ear Infirmary and Harvard Medical School, which follow national animal care guidelines, including Public Health Service Policy on Humane Care and Use of laboratory Animals, the ILAR Guide, and the Animal Welfare Act. Experimental procedures listed below detail exposure of the left DCN. Use sterile instruments while performing survival surgery.

1. Primary Craniotomy and Dorsal Cochlear Nucleus Exposure

1. Anesthesia
   1. Anesthetize a mouse, aged 8–12 weeks, weighing 18–24 g, with xylazine 20 mg/k....

Results

Partial Cerebellar Aspiration Demonstrates Access to the Cochlear Nucleus

After the skin and muscle overlying the skull are removed, skull surface landmarks, such as the coronal and lamda suture lines, demonstrate the approximate localization of the craniotomy. Following craniotomy with rongeurs, the cerebellum is visualized. Careful aspiration of the small portion of the cerebellum demonstrates visualization of the CN, which can then be injected (Figure 1).

Discussion

This paper describes the technique of direct visualization of the DCN in the murine model for manipulation of the central auditory system. The outlined approach of direct visualization provides significant advantages over the main alternative, which are stereotaxic approaches. Primarily, direct visualization of the DCN allows for immediate confirmation of the site of the brainstem, whereas stereotaxic approaches do not afford direct visualization. In experiments that necessitate prolonged incubation periods, as is the ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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