Many gene therapeutic and neuroscientific studies involve the injections of AAV vectors into the brains of non-human primates. For these studies to be successful, the injection technique must be reliable. Our injection technique is instantaneous and accurate.

This procedure works well for injection in anesthetized or awake animals. Our homemade cannula is easy to handle and low-cost. Demonstrating the procedure will be Kevin Mai, an engineering technician from my laboratory.

To begin, blunt a 30-gauge 13-millimeter hypodermic needle tip with a disc grinder. Then, cut a piece of stainless steel tubing to a length depending on the depth of the target brain area. With a disc grinder, bevel one end of the cut tube and smooth the other.

Deburr the inside of the tube with a broach. Next, cut a piece of PTFE tubing to a length appropriate for the vector solution volume to be loaded. Flare both ends of the tube by inserting the blunted hypodermic needle.

Insert the blunted hypodermic needle approximately five millimeters into one end of the PTFE tube. Then insert the unbeveled end of the stainless steel tubing approximately five millimeters into the other end. Test the cannula by injecting filtered water through the hypodermic needle hub.

Confirm that water exits the beveled stainless steel tubing tip smoothly and that water does not leak from either junction. After testing the cannula, place it in an autoclave bag and sterilize it by autoclaving. Gently transfer the vector solution to a sterilized PCR tube with a P-20 pipetter, avoiding bubble formation.

Next, attach the cannula with the beveled tip facing down to a vertically-oriented stereotaxic holder. Then connect a one-milliliter luer lock syringe to the hypodermic needle hub of the cannula. Submerge the beveled tip into the vector solution.

Then, apply gentle negative pressure with the one-milliliter syringe to load the solution into the cannula while visually tracking the meniscus between the solution and air. Once the vector solution has been loaded, continue the gentle negative pressure until the solution reaches the needle hub. Then, remove the one-milliliter syringe and slowly inject colored mineral oil along the inside wall of the hypodermic needle hub, taking care to avoid air bubbles.

Attach the hypodermic needle hub to one of the two open ports of a three-way luer lock stopcock. Then, close the port. Fill a one-milliliter syringe with air and attach it to either of the other two ports.

Finally, close the remaining port of the stopcock to connect the syringe to the cannula. Attaching the syringe forces air into the stopcock, and shunting this air to the unoccupied port prevents it from pushing the vector solution back down the cannula. Slowly push the air from the syringe into the cannula.

Once the colored oil appears at the tip of the blunt needle in the PTFE tubing, check for air between the solution and the colored oil. Keep applying positive pressure until a drop of vector solution is visible at the beveled cannula tip. Then, close the stopcock to prevent the vector from exiting the cannula by gravity.

Once the vector solution has been loaded, affix the cannula to the stereotaxic manipulator. Then, to connect the stopcock to the electric air pump, take the pump tubing from the non-sterile assistant. Grasp the luer lock connector through the wall of a sterile sleeve.

Drop the collar of the sleeve, allowing it to extend along the tube by gravity. Then, affix the stopcock and tape the sleeve tightly around the luer lock connector. Next, test the air pump and cannula by setting the air pump to low pressure, before turning it on and increasing the pressure until the oil advances through the cannula and a drop of vector solution is visible at the cannula tip.

Tape a plastic ruler to the PTFE tubing to measure the movement of the meniscus during injection. Then, drive the cannula down with the stereotaxic manipulator until the tip reaches the surface and record the depth. Drive the cannula to the deepest site to be injected along the track.

The surface will dimple upon contact. To minimize mis-targeting due to tissue compression, drive the cannula down and overshoot the deepest injection site by 500 micrometers. Then, retract slowly.

Other alternatives are driving the cannula down slowly or driving it quickly and waiting at the bottom for one to five minutes. Once the cannula is positioned at the deepest injection site, use the electric air pump to inject 0.5 microliters of the vector solution over 10 to 30 seconds. Confirm injection flow by tracking the meniscus between the colored oil and the vector solution in the PTFE tube.

After waiting for one minute, retract the cannula to the next injection site along the track. After the final injection, leave the cannula in place for 10 minutes to avoid vector efflux. Then, retract the cannula and discard it in a biohazard sharps container.

Injection of AAV vectors carrying the channel rhodopsin II transgene in the left superior colliculus, followed by stimulation with continuous blue light at 40 milliwatts, produced a series of consecutive action potentials. One-millisecond light pulses failed to evoke action potentials at 40 milliwatts, but reliably evoked them at 160 milliwatts. Optical stimulation of the superior colliculus triggered by visually-guided saccades reduced saccade gain gradually.

The slowness of this change is consistent with optogenetically-induced plasticity. Delivery of yellow laser light to the oculomotor vermis after AAV injections in the nucleus reticularis tegmentae pontis suppressed mossy fiber activity, which is an input to the cerebellum. This suppression reduced the Purkinje cell activity, which is an output of the cerebellum.

During optogenetic suppression, the firing rate decreased and changed to a burst pores pattern, suggesting that the inhibited mossy fiber input to Purkinje cells influences the saccade deceleration phase by driving the second burst. Channel rhodopsin was expressed exclusively in Purkinje cells of the oculomotor vermis. Delivery of short light pulses increased simple spike activity of an isolated Purkinje cell.

A single 1.5-millisecond light pulse frequently evoked more than one simple spike. Optogenetic simple spike activation timed to occur during the saccade increased saccade amplitude, confirming the disinhibitory role of Purkinje cells on the oculomotor burst generator. Air bubbles in the cannula can disrupt the oil vector meniscus, complicate the determination of injection volumes, and prevent fine control of flow rate.

For these reasons, loading the vector solutions without air bubbles is critical for this procedure. Six to eight weeks after the injection, optogenetic manipulations can be performed and the brain tissue can be processed for histological analysis. This technique allows researchers to test many vectors in few animals, which, in turn, facilitates the development and validation of new vectors.

A particular area of current interest is in the development of vectors that will target specific neuronal types.

Summary

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