Widespread Transduction of Mouse Neocortical Neurons by Subarachnoid Injection of AAV2

Our research is focused on developing the method that allows viral transduction of large volume of neural tissue in the mouse neocortex. Typically, suspension of viral particles is injected directly into the brain parenchyma. Here the area transfection is determined by the diffusion of the viral particles in the brain tissue, which have a limited range.

We inject AAV2 viral vector viruses into the subarachnoid space via the brain surface. We found that subarachnoid administration of the virus led to almost fourfold increase in the infection area compared with intraparenchymal injection. The new technique provides widespread transduction of neocortical neurons in adult mice, and helps to preserve brain tissue for subsequent optical or electrophysiological recordings of neuronal activity.

Our injection method allows selective expression of the transgene in a large population layer five pyramidal neurons, even when using a strong non-selective promoter. However, the mechanism of such selectivity is not fully understood. To begin, secure the front teeth of an anesthetized mouse in the tooth bar, and mount the animal mask.

Carefully position the mouse on the ear bars. Shave the head from the eyes to behind the ears, then swab the shaved surface with 70%ethanol, followed by a 5%alcohol solution of iodine. Apply sterile 4%lidocaine solution topically, and inject 0.02 milliliters of dexamethasone subcutaneously to reduce pain and inflammation.

With a sterile scalpel and scissors, make a four to five millimeter midline incision starting between the ears to open the scalp. Expand the incision using scissors to avoid damage to the skull. Swab the skull surface with 3%hydrogen peroxide to visualize bregma and lambda.

Immediately stop the reaction with 0.9%sodium chloride saline. Then use a bone scraper to remove tissue from the skull surface. Mount the pre-prepared motorized injector with a Hamilton syringe on the stereotaxic arm.

Illuminate the exposed skull using a surgical light source and focus the microscope onto the bregma. Looking through the microscope, manipulate the stereotactic arm to align the needle tip directly over bregma. Align bregma and lambda horizontally using the needle tip.

Return to bregma and record the coordinates. Move the needle to the calculated coordinates. Lower it to the new position and mark it.

Select the site for viral injection near the target while considering viral spread. Now, use a sterile dental burr of 0.5 to 0.8 millimeters diameter to create a small craniotomy while viewing through the microscope. Use pressurized air to blow away bone dust.

Bathe the hole with sterile saline and remove the excess with a cotton swab. Then use a 27-gauge needle with a hook-shaped tip or fine tweezers to remove remaining bone, taking care not to damage the dura. Cover the skull with a sterile saline-moistened paper towel.

Then, place a sterile transparent film on top of the skull surface. Move the stereotaxic arm back and align the micro syringe needle above the transparent film. Then dispense excess oil until two microliters remain.

Pipette the viral injection volume and another two microliters of virus onto the transparent film. Looking through the microscope, lower the needle tip into the center of the virus drop. Now load the virus into the micro syringe with a motorized injector.

Remove the moistened paper from the skull and dry the area with a cotton swab. Reposition the stereotaxic arm and needle above the insertion site. Next, dispense a drop of virus to check for needle clogging.

Make a small slit in the dura using a 30-gauge needle with a hook-shaped tip. Lower the syringe needle to the dura and calculate insertion depth based on the surface contact point. Insert the needle slowly into the cortex to a depth of 300 micrometers, wait for two to three minutes, retract to 200 micrometers and wait another two to three minutes.

Begin viral injection at 0.06 microliters per minute for a total of one microliter. After infusion, keep the needle at the target site for 10 minutes to allow virus dispersion. Then retract the needle slowly to avoid backflow.

Dispense a drop of virus to confirm needle is not clogged. Close the scalp incision using 5-0 absorbable or non-absorbable sutures. Subarachnoid administration of a AAV2 led to a significantly larger area of neuronal infection compared to traditional intracortical injection, with nearly fourfold expansion in both mediolateral and rostrocaudal directions.

Subarachnoid virus injection resulted in strong green fluorescent labeling of neurons in cortical layers two, three, and five, while layers four and six showed no labeled cell bodies but displayed visible dendrites and axons respectively. Fluorescent particles that were added to the injected virus suspension during subarachnoid injection were found along the brain surface, indicating virus spread via cerebrospinal fluid, with broader surface coverage than neuronal labeling. Large surface distribution of green fluorescence in frontal sections confirmed widespread cortical infection following subarachnoid injection, in contrast to the small localized infection seen with standard injection.

In super granular layers, a substantial fraction of transduced neurons co-expressed parvalbumin or calbindin, while no co-labeled interneurons were detected in layer five, and all infected cells were represented exclusively by pyramidal neurons.