This article describes a method for using HiRet viral vectors to trace neuronal pathways in the spinal cord of rats. The technique involves microinjecting a viral vector that facilitates retrograde transport and allows for the expression of fluorescent proteins.
Take a microinjector with a microneedle containing a highly efficient retrograde transport or HiRet viral vector.
The glycoproteins in the HiRet virus envelope facilitate its entry into neurons via synapses. The virus also carries a gene encoding a fluorescent protein.
Take an anesthetized rat with an exposed lumbar spinal cord 1 to 4.
Using the midline of the spinal cord as a reference, position the needle above the injection site.
Lower the needle into the spinal cord.
Begin administering the virus at a controlled rate.
Allow sufficient time for viral diffusion before withdrawing the needle.
Neuronal receptors bind with viral glycoproteins, facilitating virus entry via the synapse and transport through the axon to the soma, a process called retrograde transport.
The viral gene gets integrated with the host genome, enabling the expression of fluorescent proteins.
These viruses also travel through connected neurons, enabling tracking of neuronal pathways.
Place the needle at the midline of the spinal cord and use the Vernier scale on the micromanipulator to direct the needle laterally 0.8 millimetres. Then lower the needle to the spinal cord until it is indenting, but not puncturing the dura. And use a quick twisting motion to puncture the dura with the needle until the needle tip has sunk to a depth of 1.5 millimetres.
Once the needle is in place, program the injector to deliver the virus at a rate of 400 nano liters per minute and confirm that virus is entering the spinal cord by observing the progress of the dye front. Once the injection is finished, allow the needle to rest in the spinal cord for two to five minutes to facilitate the diffusion of the virus, before slowly withdrawing the needle for positioning at the next injection site.
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