Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

Convection enhanced delivery, or CED, of optogenetic viral vectors in non human primates will enable researchers to manipulate neural activity at a large scale to understand complex neural computations and behaviors. With CED we can achieve high optogenetic expression across large regions of the primate brain with only a few injections in a short amount of time compared to traditional methods. Magnetic resonance or MR compatible chamber implantation is performed using standard practice in non human primate surgeries, the procedure is described in detail in the text protocol and outlined briefly in this video.

Following animal sedation, positioning, and initial incision as described in the text protocol, create a circular craniotomy to cover the preplanned trajectories for injections using a trephine. Create an indentation at the center of the planned craniotomy sufficiently deep in the skull to anchor the trephine using the adjustable centering point of the trephine. Once the center has been made, lower the trephine onto the skull and rotate the trephine clockwise and counterclockwise while applying downward pressure until the bone cap can be removed with forceps.

After lifting the dura as detailed in the text protocol, cut the dura from the center to the edge of the craniotomy and continue along the edge with fine ophthalmic scissors. Mount the cylindrical custom made convection enhanced delivery MR compatible chamber to the skull on top of the craniotomy to provide cannula support during infusion such that the curvature of the chamber flange aligns well with the curvature of the skull. Secure the implants to the skull using either plastic screws and dental acrylic or a few titanium screws.

Soon after implanting the MR compatible chamber and inserting the cannula injection grid into the chamber, fill the grid with 0.9%saline for visualizing the injection locations via MR anatomical images. Also fill the chamber cavities with wet sterile absorbable gel foam to maintain the moisture of the brain. Cover the skin and the cylinder with a sterile antimicrobial incise drape to maintain the sterility of the cylinders during transport and MR infusions.

Place a vitamin E capsule to mark the top of the injection grid for positive identification. While the animal remains intubated, detach the endotracheal tube from the anesthesia circuit and reattach it to a portable MR compatible isoflurane machine. Transport the animal to the MRI scanner.

Acquire T1 images to calculate the distance from the top of the injection grid and the cortical surface. The vitamin E capsule is clearly visible in T1 images and should be used as a marker for the top of the injection grid. Acquire T2 images to determine the optimal cannula guides for each site based on the targeted site of infusion.

The cannula grid is filled with saline which is best visible in T2 images. Using the MR imaging software, scroll through the coronal and sagittal planes to find the target location of infusion. After thawing the viral vector for a few hours at room temperature, mix the viral vector with the MR contrast agent gadoteridol by pipette or vortex mixing.

Load the mixed virus into 0.2 milliliter high pressure IV tubing. Using high pressure IV tubing, connect a long extension line to an MR compatible three milliliter syringe and prime with saline. Connect the other end of the long extension tubing to the 0.2 milliliter IV tubing loaded with the virus.

Then attach the reflux resistant cannula with a one milliliter stepped tip to the distal end of this assembly with a clamp style catheter connector. Lastly set the syringe in an MR compatible pump. Using the obtained baseline anatomical MR images, select the cannula injection grid location and insertion depth needed to reach the target infusion site.

Mark the insertion depth on the cannula using sterile tape. Begin the infusion at a rate of one microliter per minute and visually validate the flow of the fluid in the infusion line by observing the ejection of fluid from the cannula tip. Insert the cannula manually through the injection grid to the target depth while maintaining flow in the infusion line as this will prevent the penetrated tissue from clogging the cannula during insertion.

Now acquire fast flash T1-weighted images for online monitoring of the viral vector infusion. After infusing approximately 10 microliters of the vector such that enough virus is infused to detect in the MR scanner, obtain MR images to verify correct cannula placement as evident by the observed spread of the virus. If the depth of the inserted cannula is incorrect, adjust the depth accordingly or slowly remove the cannula and reattempt the insertion.

Monitor the infusion via guidance of online MR images. Increase the infusion rate to five microliters per minute by one microliter per minute steps every few minutes. It is important to increase the infusion rate slowly and not to exceed five microliters per minute as high infusion rates could cause tissue damage.

After infusing approximately 40 microliters of the viral vector, begin reducing the infusion rate by one microliter per minute steps. Stop the infusion after approximately 50 microliters is injected. Leave the cannula in place for ten minutes.

Slowly remove the cannula from the brain and move to the next location. Cover the cylinder with a sterile drape at the end of injections before animal transport, then transport the animal back to the operating room. Histological analyses on serial coronal sections reveal large scale optogenetic expression around the infusion sites.

Shown here is the baseline coronal MR image and the spread of the contrast agent after the infusion for the same MR coronal slice. A coronal tissue section is shown from approximately the same. Peroxidase staining reflects expression of the yellow fluorescent protein or EYFP reporter.

Good alignment is observed between the area of EYFP expression, measured with surface epifluorescense and with histological staining. These include the region of vector spread estimated from MR images. White dots indicate injection sites and the entire black region represents the area exposed by the craniotomy.

Shown here is a low magnification image of the coronal sections stained with anti GFP antibody showing the medial lateral aspect of YFP expression in the somatosensory cortex. The black arrowhead indicates the location of the cannula track. The adjacent tissue is shown at greater magnification to show laminar distribution of the YFP positive cells.

Densely populated regions of YFP positive cells are located predominately in layers two through three and five through six. Further enlargement reveals typical pyramidal cells in layers two through three, layer five, and layer six. It is important to leave the cannula in place for 10 minutes after the flow has stopped.

This will ensure that the virus has percolated into the surrounding targeted neural tissue. CED is an efficient approach to achieve uniformly high expression levels across large brain regions in mammals. It has the potential to expand our understanding of neural circuits and connectivity.

After viral transduction by CED, subsequent optogenetic stimulation experiments can be paired with electrical recording and behavioral essays to reveal the complex circuit dynamics within the brain. This technique has been previously employed by Yazdan-Shahmorad and colleagues in 2018 in which optogenetic neural modulation was used to induce network wide connectivity changes in the sensory motor cortex.