This article describes a method for targeting the dorsal cochlear nucleus (DCN) in mice using viral vectors. The procedure involves craniotomy, aspiration of cerebellar tissue, and precise injection of opsin-encoding viral vectors to facilitate light-induced stimulation of auditory pathways.
Begin with an anesthetized mouse with its skull exposed.
The skull features a craniotomy over the interparietal bone, left of the midline, and caudal to the lambdoid suture, with the dura removed to expose the cerebellum.
Aspirate the lateral left cerebellum to reveal the underlying dorsal cochlear nucleus or DCN, a brainstem structure that contains neurons processing auditory signals.
Add saline to prevent blood coagulation and clear residual fluid.
Position a microsyringe containing a viral vector encoding opsin, a light-sensitive ion channel.
Lower the needle into the DCN.
Inject the viral suspension at a controlled rate, then withdraw the needle.
Suture the incision and allow the mouse to recover.
The injected virus binds to DCN neuronal receptors and undergoes endocytosis.
Inside the neuronal nucleus, the viral DNA triggers gene expression, leading to opsin production.
The model is ready for light-induced stimulation of the auditory pathway.
Use a five French suction tube to aspirate the lateral most portion of the left cerebellum, overlying the DCN. To assit 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 ampulla 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.
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