Virus-Mediated Gene Transfer Into the Mouse Dorsal Cochlear Nucleus for Light-Induced Auditory Stimulation

Abstract

Source: Kozin, E. D., et.al. Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway. J. Vis. Exp. (2015)

This video demonstrates virus-mediated gene transfer into the mouse dorsal cochlear nucleus (DCN) to enable light-induced auditory stimulation. A viral vector encoding opsin, a light-sensitive ion channel, is injected into the DCN following a craniotomy and cerebellar aspiration for direct access. The expressed opsins allow neurons to be activated by light, providing a model for studying auditory pathway responses using optogenetics.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Cerebellar Aspiration

1. Using a 5 French suction, aspirate the lateral-most portion of the left cerebellum overlying the DCN until the DCN is visual (Figure 1, Right Panel). Remove approximately 1/4 to 1/3 of the left cerebellum. The main landmark adjacent to the DCN is the ampulla of the superior semicircular canal.
   NOTE: Aspiration of the cerebellum is the key step of the protocol. Directed aspiration of the cerebellum is most successfully performed if it occurs in a single attempt and not with multiple passes of the suction as this will cause bleeding. To assist in aspiration, set the focal plane of the microscope at the expected depth of the CN, which will be slightly distal to the surface of the cerebellum. Setting the focal plane to the CN will improve visualization and ensure a sharp image.
2. Following aspiration, further bleeding, cerebrospinal fluid (CSF) build-up, and cerebellum displacement on the DCN are expected. Instill 0.5 cc of sterile saline quickly into the craniotomy to prevent blood coagulation. A combination of gentle dabbing with a dental point and suction may then be used to clear away a path for direct visualization of the DCN. Do not directly contact the surface of the DCN.

2. Pressure Microinjection for Virus-mediated Gene Transfer and Surgical Recovery

1. After the DCN is free of overlying blood and CSF and is clearly visible, make pressure microinjections into the DCN using a 10 µl Hamilton syringe over a 2 min period.
2. Introduce the needle with a micromanipulator until the tip is no longer visible under the surface of the DCN.
3. For optimal performance, use a 33 or 34 gauge needle (use a gastight syringe with the 34 gauge needle) with a shallow bevel, such as 45°, to minimize blunt trauma and localize the injection volume within the DCN, which is a thin and shallow brainstem structure (<300 µm thick).
   NOTE: Other microinjection instruments may be utilized. Ideally, the injection instrument should be flexible enough to allow for some slight bending if needed to directly target the DCN. Further, as the mouse moves slightly throughout the procedure due to respiration, the instrument should be robust enough to withstand minor amounts of movement.

3. Surgical Recovery

1. Immediately following injection, re-approximate the skin and allow the mouse to recover per standard recovery procedure. Remaining cerebellum and scar tissue will fill in the cavity caused by aspiration of the cerebellum. Constantly monitor animals until sufficient consciousness is regained to maintain sternal recumbency. Monitor heart rate, respiratory rate, as well as ability to feed.
   NOTE: No animal that has undergone surgery is returned to a cage with other animals until fully recovered.
2. If a mouse begins to walk in circles post-operatively (<5% of cases), typically 2-4 hr after closure of the skin incision, immediately sacrifice the mouse, as it would likely have a difficult time feeding. This side effect is likely due to aspiration of the cerebellum. The animal should be monitored for pain postoperatively, and appropriate narcotics should be given to ensure animal comfort based on institutional standards. Antibiotics may be necessary, if evidence of infection.

Results

Figure 1: Partial cerebellar aspiration demonstrates access to the dorsal cochlear nucleus. Left Panel: After the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotaxic holder	Stoelting	51500
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