Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

The overall goal of intraspinal delivery of recombinant adeno-associated viruses is to enable the histological labeling and functional manipulation of genetically defined neurons in the dorsal spinal cord. This method can help answer key questions in the pain field, such as which neuronal circuits are required for the perception of different pain modalities. The main advantage of this technique is that genetically labeled neurons can be manipulated in a specially restricted and temporally controlled fashion.

Locate the most caudal rib pair by palpating along the vertebral column of the anesthetized mouse. Then, make a 1.5 to 2.5 centimeter longitudinal cut into the skin, starting from just rostral to the most caudal rib pair. Lift the skin with forceps and detach it from the underlying muscle with scissors.

Throughout the surgery, keep the exposed tissues moist with sterile 0.9%sodium chloride. Using fine forceps and small scissors, make an incision into the next to thin membranous layer, right next to the midline, and cut it away from the spinous processes. Several anatomical landmark can aid in identifying the correct vertebrae to target.

Here, we describe three alternatives. Palpate along the vertebral column. The most caudal rib pair is located just rostral to the T13 vertebrae and the pelvic bone at hip level is at the level of the L6 vertebrae.

Alternatively, pull back the skin towards the tail to expose the iliac crest. At the same level, the most caudal pair of visible intetransverse ligaments joins the L6 spinal process. Count backwards in a caudal to rostral direction to identify the vertebrae of interest.

Otherwise, locate the spot where the tendon along the side of the vertebral column is whitest and most medial. The T13 vertebrae is located just rostral. The lumbar spinal cord segment L4 is located within this vertebrae.

Next, place the animal onto a cushion of rolled up tissues to elevate it to the spinal clamps of the stereotaxic frame. Align the clamps adjacent to the target vertebrae. Fix one clamp in position, then hold the vertebral column with Adson forceps while fixing the second clamp.

Confirm proper clamping by carefully pressing on the targeted vertebrae. Then, remove the paraspinous muscle above the vertebrae of interest. First, make an incision just medial to the tendons parallel to the vertebral column.

Then, make perpendicular incisions rostral and caudal to the target vertebrae. Next, use rongeurs to tear or cut away the paraspinous muscle above the vertebrae of interest. Use forceps as needed to remove tissue remaining on the vertebrae or above the dura in the intravertebral space.

The dorsal blood vessel should now be visible, marking the midline of the spinal cord. For unilateral injections, perform a partial laminectomy using a fine dentist drill equipped with a 0.5 millimeter spherical cutter to very carefully drill a hole into the middle of the target side of the vertebrae. Remove any remaining bone fragments with a 26 gauge beveled needle to expose the spinal cord.

Use the needle to perforate the dura in the intravertebral spaces rostral and caudal to the target vertebrae, approximately 300 microns lateral to the dorsal blood vessel. Then, perforate the dura underneath the drilled hole. At this point, cerebrospinal fluid should escape from the holes and the spinal cord should be slightly bulging out.

Prepare the injection syringe by first mounting the glass capillary onto a microliter syringe. Fasten the nut tightly to ensure a tight and secure fit. Fill the microliter syringe with sterile distilled water, then depress the plunger.

If the water is not easily expelled from the tip, the micropipette is likely blocked and should be exchanged. Mount the syringe onto a micromanipulator connected to an electronically controlled microinjector. Then, use the microinjector to draw up about one microliter of air to separate the water and virus.

Next, pipette a 2.5 microliter droplet of diluted virus solution onto a piece of paraffin film. Carefully move the tip of the micropipette into the droplet using the sterotaxic frame and draw it up into the micropipette. Then, press dispense until a drop of virus solution emerges at the tip.

Retract the syringe and use a pen to mark the micropipette with a scale to monitor the dispensed volume. Use the stereotaxic arm to move the tip of the micropipette over one of the holes in the dura and then down until a slight depression appears on the surface of the exposed spinal cord. To target the injection to the spinal dorsal horn, move the tip down to a depth of 500 microns in 100 micron increments and then up 200 microns to allow for mechanical stabilization of the tissue.

The final depth of the tip is 300 microns. After programming the pump to inject 300 nanoliters at an injection speed of 50 nanoliters per minute, press the start button to begin the infusion. After the injection is complete, check the scale on the micropipette to see whether the virus level has dropped.

Leave the micropipette in place for an additional three minutes to allow the pressure to equilibrate. After three minutes, slowly retract the micropipette and then repeat the procedure for the other two injection sites. After the final injection, remove the spinal clamps and the cushion below the mouse.

Close the incision layers with interrupted stitches, suturing the superficial tissue layers with absorbable sutures and the skin with non-absorbable sutures. Apply iodine disinfectant on the sutured wound. Terminate the anesthesia and leave the animal on the heat mat until it recovers before returning it to its home cage.

Spinal tissue at the injection site is to be examined at the end of the experiment. This is necessary to verify successful injection and transduction and to exclude excessive tissue damage as a result of the surgery. To illustrate the expression levels that can be obtained by intraspinal injection of recombinant AAV, a virus encoding the eGFP fluorescent protein was injected into the lumbar spinal cord of wild-type mice.

Three injections spaced approximately one millimeter apart produced a nearly continuous infection of lumbar spinal segments L3 to L5.Virus injection at a depth of 300 microns from the spinal surface leads to predominant infection of cells in the spinal cord dorsal horn. However, infected cells could also be found in the ventral horn. A cre dependent flex vector expressing eGFP was injected into the spinal cord of GlyT2:Cre transgenic mice.

This resulted in more restricted eGFP expression reflecting the distribution of GLYT2 positive neurons. Following this protocol will allow targeting of three consecutive spine segments. We found that this is sufficient to obtain robust behavior results.

This technique will allow studying the function of specific neuron and cell populations and sensory pathways.