Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

The overall goal of this procedure is to facilitate extinction of conditioned fear by stimulation of the vagus nerve. This is accomplished by first constructing electrodes for the delivery of current to the vagus nerve. The next steps are to implant these electrodes around the mid cervical section of the vagus nerve, along with a head cap after a recovery period, animals next undergo auditory fear conditioning and extinction training with vagus nerve or sham stimulation.

The final step is to test the expression of conditioned fear and to assess the reduction in the condition freezing response. Ultimately, vagus nerve stimulation is used to enhance extinction learning by inducing plasticity in extinction networks. The main advantage of this technique over existing methods like systemic drug treatment, is the ability to precisely regulate the timing of the treatment in order to achieve network specific release of neurotransmitters that can be paired with ongoing behavior.

This method can help answer key questions in the field of emotional learning and memory, particularly how the release of stress hormones like epinephrine and glucocorticoids can influence the consolidation of long-term memories. The implications of this technique extend toward exposure therapies used to treat anxiety and trauma related disorders, as well as rehabilitation from addictions. Additionally, vagus nerve stimulation can be applied to other models of learning in synaptic plasticity such as motor learning after stroke or traumatic brain injury.

In preparation, build a drilling tool from a needle to start making the cuff slip four centimeters of micro renaine tubing onto a drill bed. Then use the needle drill to drill four holes, two millimeters apart, forming the corners of a square. The holes should be clean with no rough edges.

Next, cut the tubing lengthwise between the holes. Now using suture thread, build the rigging to secure the cuff to the vagus nerve on either side. Tie the sutures together about two centimeters from the plastic, so the tubing and thread make a triangle, extend each suture length about eight centimeters past the knot.

Next, cut. Insulated seven stranded platinum meridium wire into 70 millimeter segments. Now using a jewelry torch strip about a centimeter of insulation from one end of the wire.

Then use the flame to melt the platinum meridium where it is exposed. Using this technique used together the seven exposed strands of platinum meridium wire into a single stronger wire, melt the end of the wire into a ball on the opposite end of the fused wire segment. Melt the platinum meridium wire into a small ball with minimal stripping of the insulation.

Now tape down the threaded plastic cuff with a cut edge along the horizontal plane with a threads pulled tightly and the cuff outstretched. Using forceps, feed the exposed end of the wire preparation through the bottom right hole and pull it to the middle of the cuff. Then push the wire through the top right hole and loop the wire back over the cuff.

Continue by pushing the wire through the top right hole again, and then with a tug, tighten the wire and make sure that it is secure. It is critical that the wire positioned in the trough of the cuff is uninsulated while the wire outside the cuff on the bottom end remains insulated, so the delivered current only goes to the vagus nerve. Next, push the bald end through the bottom right hole from inside the cuff.

Thus completing a loop, place a wire on the left side of the cuff in exactly the same way on the end of the wire, not fix to the cuff. Solder the bald end of the wire to a gold pin. Let the solder cool and remelted ensuring a good connection.

Add more solder as needed. Now prepare the connectors for the head cap of VNS input site for each input site. Strip the ends of two 30 millimeter segments of 26 A WG copper wire and cut the narrow end off a loose gold pin in then solder together each pair of wires to a pin following.

This solder the loose end of the wires to the two flux teeth of a connector. Thus the connectors are made. After preparing the animal for surgery in a stereotaxic frame, use a scalpel to incise the skin over the skull and expose the lambda and bgma.

Using blunt forceps tunnel a path subcutaneously from the incision down the left side in front of the ear and to the left side of the neck. The path will be for the cuff. Next, using a scalpel open two small starter holes for the anchor screws.

Do not place these holes directly over the midline. Then with forceps and a screwdriver, drive in two bone screws leaving room for acrylic to fill in below the screws. Then fill in the space under a round and between the screws with acrylic, keep the acrylic off the surrounding tissues and apply extra acrylic between the screws.

Next, grip the implant so that the marked wire is rostral to the unmarked wire. And quickly position the implant into the acrylic. Do not get any acrylic on the gold pins, the clasp area or the input site.

Let this dry for five minutes and prepare to continue working under a stereoscope. Make a small incision over the left jugular vein directly between the jawbone and the clavicle. Widen the incision with a blunt dissection down to the muscles.

Expose the sternal mastoid, sternal hyoid, and omohyoid. Continue by bluntly dissecting along the furrows between the muscles to find the carotid artery. The sheath containing the carotid also contains the vagus nerve.

Using scissors, carefully dissect the sheath to expose a five millimeter length of the vagus nerve. Next from the head incision, use the threads on the side of the cuff opposite the leads to pull the cuff through the subcutaneous tunnel and into the neck incision site. Gently lift the nerve with glass tools and push the cuff threads under the nerve.

Be careful to avoid rubbing against the nerve. The top side of the cuff should be facing the animal's superior side. Now drop the nerve into the middle of the cuff and close the cuff on the head cap.

Attach the pins connected to the cuff to the pins on the stimulation input site to confirm that the cuff is properly positioned. Perform a cessation of breathing. Test breathing should stop briefly and the heart rate should also drop.

After confirming the cuff position, secure the pins to the stimulation input site with acrylic. Then close up the surgical site and treat the animal with Marcaine and topical antibiotics. Let the animal recover for five days without housemates.

Prior to running tests, set up the operant conditioning box under video camera surveillance. On the first day of behavioral training, present the tone five times for 30 seconds to demonstrate there is no innate fear of the tone. After the initial tone presentation, fear condition the rats on day one and two.

Play a 32nd tone with a randomly placed one second, 0.5 milliamp foot shock. Deliver eight tone shock pairings each day with three to five minutes between tone presentations. On the third day test the strength of the tone shock association.

Lay the tone alone four times, three to five minutes apart. Record the animal's freezing behavior on the fourth and fifth day. Perform the extinction training with vagus nerve stimulation or sham stimulation.

Plug the rat into the stimulator and place it in the operant conditioning box. 150 milliseconds. Before each tone, stimulate the vagus nerve for 30 seconds.

Perform four stimulation tone pairings with three to five minute gaps. Periodically check the electrical integrity of the VNS cuff and input site using an oscilloscope using the described protocol. Rats were trained on an auditory fear conditioning task with vagus nerve stimulation during extinction.

Animals that received a limited number of non reinforced exposures to the condition tone during the extinction phase show no reduction in condition fear. In contrast, BNS treated rats demonstrate a large reduction in freezing, similar to that seen in sham treated animals that received extended extinction training. BNS animals also showed reduced freezing behavior outside the presentation of the condition tone, suggesting that their extinction training generalized to the context.

Further investigation showed that pairing VNS with extinction training alters metaplastic in the pathway between the infra limbic cortex and the basal lateral amygdala. In anesthetized animals in sham stimulated animals that failed to extinguish the condition. Fear response reef burst stimulation of the infra limbic cortex induced long-term depression of the evoke local field in the basal lateral amygdala.

This synaptic depression was reversed in animals that exhibited significant extinction of the condition fear response. Moreover, administration of VNS during a single extinction session promoted induction of long-term potentiation. After watching this video, you should have a good understanding of how to use VNS in conjunction with behavioral training.

Once mastered VNS component making and surgery can be done in about three hours if it's performed properly. While attempting this procedure, it's important to remember to keep electrical connections intact.