This article describes a method for targeting corticospinal tract (CST) neurons in neonatal rats using viral injections. The technique allows for the analysis of CST neurons in the context of spinal cord injury.
Begin with an anesthetized neonatal rat with a surgically exposed cervical spinal cord.
This rat has been injected with an anterograde virus encoding a designer receptor and a fluorescent protein the virus targets corticospinal tract (CST) neurons in the brain's somatomotor cortex.
Identify the midline of the spinal cord using the spinal artery.
Position a glass needle containing a retrograde virus, which encodes a recombinase enzyme, behind the targeted spinal lamina where CST axon terminals reside.
Lower the needle into the spinal cord and inject the virus.
Allow the virus to diffuse, then slowly withdraw the needle and suture the incision.
The virus enters the CST axon terminal and travels retrogradely to the cell body, where it expresses the recombinase enzyme.
This enzyme activates the transgene delivered by the anterograde virus, allowing expression of the designer receptor and fluorescent protein only in doubly infected neurons. This method allows targeted analysis of CST neurons in spinal cord injury.
For direct injections into the spinal cord, use the tip of the glass injection needle to approximate the midline of the spinal cord by identifying the spinal artery.
Next, place the needle just posterior to the C5 lamina at the approximate midline and use this as the reference point. Then, using the micro manipulator, move the needle laterally to the right by 0.3 millimeters and lower the needle until it just touches the surface of the spinal cord.
From this depth, plunge the needle 0.6 millimeters into the spinal cord. Inject one microliter of the retro adeno-associated virus 2 carrying the Cre recombinase gene at 250 nanoliters per minute. Then wait for two minutes for the virus to diffuse into the spinal cord before slowly withdrawing the needle.
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