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

The overall goal of this procedure is to surgically access the cochlear nucleus in a mouse model to enable optogenetics based experiments. This is achieved by first preparing the mouse for craniotomy and retracting the skin soft tissue and muscle overlying the scalp laterally. As a second step, a craniotomy is made on the left inter parietal bone, about two millimeters coddle to the lambda suture line.

This region overlies the cochlear nucleus. Next, using a five French suction tube aspirate the lateral most portion of the left cerebellum overlying the DCN. The result shows an unobstructed view of the DCN.

The main advantages of this technique over assisting methods such as stereotactic guidance, are to both attain direct visualization of the cochlear nucleus and to place a neuros simulator such as a light source or an electrode array onto the brain stem surface. This method can help us answer key questions in the fields of both optogenetics and auditory. No prosthesis, such as can gene transfer enable the auditory brainstem to be stimulated by light with sufficient facial and temporal resolution.

After anesthetizing a mouse with ketamine and xylazine via intraperitoneal administration, check the toe pinch withdrawal reflex, and monitor its heart rate and respiratory rate to confirm proper anesthesia. Then apply vet ointment to both eyes to prevent dryness. Next, shave the hair overlying the scalp to provide unobstructed access to the surgical site.

After that, place the mouse in a small animal stereotaxic holder. Adjust the snout clamp so that it is loose enough to allow for adequate respiration, but tight enough to completely immobilize the head of the mouse. Under a microscope, make a vertical incision through the skin, starting at the midline directly between the pinna and extending to the coddle portion of the occiput.

Then displace the skin laterally. Remove the muscles overlying the left parietal, inter parietal, and occipital bones of the skull with a scalpel or iris scissor. In the meantime, minimize bleeding by applying gentle pressure with a cotton tipped applicator for 10 to 15 seconds.

Next, identify the sagittal and lambda sutures lines. Using a pair of Ron jewels. Make a craniotomy on the left inter parietal bone about two millimeters coddle to the lambda suture line.

This region overlies the DCN following craniotomy. A thin layer of dura that overlies the cerebellum should be observed carefully remove it using a scalpel blade. Remove the coagulated blood with cotton tips or gently drip saline onto the cerebellum to clear the blood away.

Then use a five French suction tube to aspirate the lateral most portion of the left cerebellum, overlying the DCN to assist. In aspiration, set the focal plane of the microscope at the expected depth of the cochlear nucleus to improve visualization and ensure a sharp image. Remove approximately a quarter to one third of the left cerebellum.

The main landmark adjacent to the DCN is the AM of the superior semi-circular canal. Following aspiration, further bleeding, cerebral spinal fluid buildup and cerebellum displacement on the DCN will appear instill saline quickly into the craniotomy to prevent blood coagulation. Use a combination of gentle dabbing with a dental point and suction to clear a path for direct visualization of the DCN.

After the DCN is clearly visible and free of overlying blood and CSF, make a pressure micro injection of the vector into it with a five microliter gas tight Hamilton syringe. Use a 34 gauge needle with a shallow bevel to minimize blunt trauma and localize the injection volume within the DCN. Then slowly introduce the needle with a micro manipulator until the tip is no longer visible under the surface of the DCN.

Start injecting 1.5 microliters of the vector. After this is completed, slowly withdraw the needle, subsequently re approximate the skin and allow the mouse to recover per standard recovery procedure. After two to four weeks of healing and virus mediated gene transfer incubation, re anesthetize the vector injected mouse, then remove the scar tissue over the craniotomy site.

After that, identify a combination of remaining cerebellum and scar tissue overlying the DCN. Use a five French suction to aspirate the overlying cerebellum and scar tissue. Following aspiration.

Quickly apply saline into the craniotomy. To prevent blood ululation use a combination of gentle dental point dabbing and suction to clear the path for direct visualization of the DCN. The DCN is now accessible for the optogenetics based physiology experiments shown here is the placement of a laser fiber directly on the surface of the DCN, which allows for optical stimulation of the opsin infected DCNA blue light pulse series at the surface of the infected DCN results in an optically driven auditory brainstem response.

Using a micro manipulator, an optical fiber coupled to a blue light laser is placed directly onto the surface of the cochlear nucleus and turned on. Here we can identify pulsed blue light on the surface of the cochlear nucleus. Following this procedure, other methods such as electrically simulating nucleus can be performed, allowing us to directly compare not only the acoustically evoked, but also the electrically evoked neural responses with that evoked by optical simulation.

After watching this video, you should have a good understanding of how to prepare and perform the craniotomy and cerebellar aspiration with the ultimate goal of direct visualization and access to the cochlear nucleus.