Name: Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning (Video) | JoVE Journal
Uploaded: 2015-08-21T14:00:00.000+00:00
Duration: 11 min 2 s
Description: Learn vagus nerve stimulation to enhance extinction of conditioned fear by inducing synaptic plasticity in forebrain networks. This method enables precise timing of neurotransmitter release for improved therapeutic strategies.

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5 minutes

This research was supported by the National Institute of Mental Health MH 086960-01A1 (Christa K. McIntyre).

All procedures described in this protocol are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and they were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.
1. Construction of VNS Cuffs
<ol>
	<li>Create a drilling tool by sawing off the sharp end of a 22 &#189; G needle.</li>
	<li>Run the now blunt end of the 22 &#189; G needle over a metal file several times to flatten it. Hold the needle at a 45&#186; angle to the file and run it several times over the file while rotating it. This will cause the metal to become thinner and furl inward. CAUTION: Insert the tip of the scalpel into the cut end of the needle and rotate with some downward force to unfurl the metal.</li>
	<li>Use magnifying binoculars for the remaining steps in section 1.</li>
	<li>Using a scalpel (10 or 15 blade) cut 4 mm segments of tubing.</li>
	<li>Place one 4 mm segment over a small drill bit or other similarly shaped tool. This is to hold the tubing in place while it is being manipulated.</li>
	<li>Drill 4 holes into the tubing (Figure 1A). Holes should make the four points of a 2 mm by 2 mm square and should be clean with no rough edges. 
	NOTE: The drilling tool needs to be resharpened (step 1.2) every 2 or 3 holes. Difficulty with this step is almost certainly due to a drilling tool that is not sharp enough.</li>
	<li>With the tubing still on the drill bit, use a scalpel to cut the tubing lengthwise between the holes so that two holes end up on either side of the cut (Figure 1B).</li>
	<li>Using a sewing needle and suture thread, pass suture through the holes to create the rigging for the ultimate placement of the cuff around the vagus nerve. Start with the needle inside the cuff and pass it through one of the holes, then go back through the adjacent hole from the outside (Figure 1C). Tie the thread together ~2 cm from the plastic so that the tubing and thread make a triangle. Allow ~8 cm of thread after the knot and trim. Repeat the process for the holes on the opposite side of the cut. The tubing is now ready to be wired.</li>
	<li>Prepare wires for cuffs.
	<ol>
		<li>Cut platinum iridium wire into 70 mm segments.</li>
		<li>Using the finest tip for the jewelry torch, create a sharp flame with blue center that is as refined as possible and use it to strip ~1 cm of the plastic coating from the wire. Apply the blue center of the flame to stripped end of the wire to create a small ball. Apply the blue center of the flame to several points of the stripped portion to fuse the seven strands together. The wire in these spots will appear to kink.</li>
		<li>On the opposite end of the wire, apply the blue center of the flame to the end to create a small ball. Minimize stripping the plastic on this end.</li>
	</ol>
	</li>
	<li>Wire the cuff (Figure 1D). 
	NOTE: Steps in section 1.10 must be done under magnifying binoculars.
	<ol>
		<li>Tape the cuff down using the threads so that it is oriented with the cut running horizontally. Pull the threads tight so that the cuff is pulled open and then tape down the threads.</li>
		<li>Grasp the balled end of the stripped side of the prepared wire with #5 forceps and push through the bottom right hole. Pull the forceps out of the hole, leaving the end of the wire in the middle of the cuff.</li>
		<li>Re-grasp the balled end of the stripped wire (now in the middle of the cuff) and push it through the top right hole. Pull the forceps out of the hole, leaving the wire passed completely through the cuff and loose on the top side of the cuff.</li>
		<li>Re-grasp the balled end of the stripped wire (now outside the cuff on the top side) and push it back through the top right hole from the inside. Continue doing so until the wire is securely in place: test by tugging on the opposite end of the wire. 
		NOTE: During this process it is critical to differentiate the stripped/insulated portions of the wire. The wire that is ultimately positioned in the &#8216;trough&#8217; of the cuff must be stripped, but everything below the bottom holes (outside the cuff on the bottom end) must be insulated. This ensures delivery of current only to the vagus nerve.&#160;</li>
		<li>Grasp the balled end of the insulated side and push it through the bottom right hole from the inside of the cuff, looping it around once.</li>
		<li>Repeat steps 1.10.2 &#8211; 1.10.5 on the left side of the cuff so that two wires end up fixed to the tubing, one on the right side and one on the left side.</li>
		<li>Place a gold pin into the arm of the helping hand with the hole facing up. Fill the hole with flux.</li>
		<li>Solder the insulated end of the wire (now attached to the cuff) into the pin.</li>
		<li>Allow the solder to cool, and then melt it again. This ensures a good connection between the end of the wire and the inside of the pin. Apply more solder if necessary. Repeat for second wire.</li>
		<li>With the leads running to the right, mark the top of the cuff with permanent marker. Also mark the gold pin attached to the top lead.</li>
	</ol>
	</li>
</ol>
2. Construction of Headcap for VNS Input Site
<ol>
	<li>Cut 30 mm segments of 26 AWG copper wire. Strip a small portion on each end.</li>
	<li>Cut the narrow end off loose gold pins and solder the stripped end of the wire to the cut end of the gold pin. Create two wire/pin compounds for each desired input site. Place a connector into the helping hands and solder the wire end of a wire/pin compound to the each of the two fluxed teeth of the connector (Figure 2G).</li>
	<li>Mark one of the wires with sharpie. During the surgery, position the implant with the marked wire rostral to the unmarked wire.</li>
</ol>
3. VNS Surgery
<ol>
	<li>Create custom glass tools for handling of vagus nerve during surgery.
	<ol>
		<li>Use borosilicate glass to pull a micropipette so that it has a long tapered tip. If no pipette puller is available, break the glass to create a longer edge.</li>
		<li>Hold the non-tapered end of the pipette with a thick cloth (to prevent burns) and press the tapered/broken end into a smooth fire-resistant surface while applying the blue flame from the jewelry torch to the tapered end. The glass will bend as it is pressed into the surface. Apply flame until a hook or J shape forms.</li>
	</ol>
	</li>
	<li>VNS surgical procedures
	<ol>
		<li>Gather and sterilize all tools. Prepare a sanitary, heated surgical area.</li>
		<li>Anesthetize animal with ketamine/xylazine (85 mg/kg, 5mg/kg, IP). Assess the depth of the anesthetic plane by monitoring the animal&#8217;s vocalizations and withdrawal-reflexes in response to toe and/or tail pinching.</li>
		<li>Shave the top of head and left side of animal&#8217;s neck. Protect the animal&#8217;s eyes with mineral oil or eye ointment. Apply iodine cleansing solution with gauze and then alcohol with gauze to the shaved areas. Repeat once.</li>
		<li>Inject 0.05 ml marcaine subcutaneously on the top of head and allow the bolus to disperse while placing the animal into the stereotaxic instrument.</li>
		<li>Use a scalpel to make an incision in the skin on the skull to expose both lambda and bregma. Prepare a path for the cuff by using blunt forceps to tunnel subcutaneously from the incision site down the left side in front of the ear to the left side of the neck.</li>
		<li>Pull the incision site open with hemostats. Using cotton swabs, apply hydrogen peroxide to the exposed skull to remove any remaining tissue.</li>
		<li>Using a scalpel, drill two shallow starter holes in the skull to place anchor screws. They should be placed far enough apart to allow room for the implant, but not too close to the surrounding tissue. Avoid placing screws directly on the midline.&#160;
		<ol>
			<li>With forceps and screwdriver, drive a bone screw into both holes. The screws should be tight in the holes, with the caps 2 - 3 mm above the surface of the skull to allow room for acrylic to fill in under and around the screws.</li>
		</ol>
		</li>
		<li>Fill the space under, around, and between the screws with a small amount of acrylic, avoiding the surrounding tissue. Then place a larger amount of acrylic in the middle of the skull between the two screws.</li>
		<li>Grab the implant so the marked wire is oriented rostral to the unmarked wire and quickly place the implant in the acrylic, taking care not to get acrylic into the gold pins, the clasp area, or the input site on top. Once positioned allow to set ~5 min until dry. This can also be done using the arm of the stereotaxic for support. Fill in any cracks or gaps between the implant and skull with a low viscosity blend of acrylic. Allow to dry.</li>
		<li>Use magnifying binoculars for the remaining steps in section 3.2.</li>
		<li>Remove the animal from the stereotaxic instrument. Place the animal on its right side, rotating it slightly toward the ventral position.</li>
		<li>Make a small incision approximately over the left jugular vein. The jaw bone and clavicle should be roughly equidistant to the incision site. Widen the incision using blunt dissection until the muscle layer is reached. The sternomastoid, sternohyoid, and omohyoid muscles should be visible. Use muscle retractors to keep the site open.</li>
		<li>Continue blunt dissecting along the natural furrows between the muscles. Look for the pulsing of the carotid artery. Heading through the muscle toward the pulsing will reveal the carotid artery. Pull the muscles back with the muscle retractor. The sheath containing the carotid artery also contains the vagus nerve. Carefully blunt dissect the sheath with the scissors.</li>
		<li>Identify the vagus nerve. It is the largest nerve in the carotid sheath and is usually to the animal&#8217;s left side of the artery but can be found on any side. Switch to the custom glass tools and separate the vagus nerve from the carotid artery. The nerve should be free of any other tissue for at least 5 mm.</li>
		<li>From the incision on the head, using the threads on the side of the cuff opposite the leads, pull the cuff through the previously made subcutaneous tunnel with forceps or small hemostats. Push it through the tissue into the incision site on the neck.</li>
		<li>Gently lift the nerve up using the glass tools and push the threads on the side of the cuff opposite the leads under the nerve. Pull the threads all the way through, taking care not to rub against the nerve. The cuff should be immediately adjacent to the nerve.</li>
		<li>Make sure the cuff is oriented with the marked &#8216;top&#8217; side superior. Drop the nerve into the middle of the cuff. The nerve should now lie across both wires in the trough of the cuff (Figure 2B). Tie the threads together to close the cuff.</li>
		<li>On the headcap, plug the pins attached to the cuff into the gold pins on the stimulation input site. Plug the marked pin into the gold pin attached to the most anterior tooth on the simulation input site.</li>
		<li>To verify that the cuff properly stimulates the vagus nerve, perform a cessation of breathing test by, connecting the stimulator to the stimulation input site on the headcap and running stimulation (0.2 mA, 60 Hz, up to 10 sec). Breathing should briefly stop and heart rate should drop, verifying cuff function.</li>
		<li>Secure the pins on the stimulation input site with acrylic. Cover the wires and verify that exposed pins and wires do not lead to short circuits. Use acrylic to smooth over any bumps or to fill in any gaps. Allow to dry.</li>
		<li>Suture closed both incision sites. Inject 0.05 ml marcaine subcutaneously near the neck incision site. Apply antibiotic ointment to incision sites. Optionally, leave a male connector in place on the stimulation input site to prevent damage or obstruction during the healing process.</li>
		<li>Treat with antibiotic and follow standard post-operative care including appropriate analgesia. Return animals to animal housing facility after they regain mobility. Allow 5 days for recovery. To ensure maximal longevity and function of the headcap, house animals singly for the remainder of the experiment. 
		NOTE: Sham-VNS rats undergo the same surgery, however the circuit is designed to short at the level of the headcap (i.e., a headcap is implanted and the vagus nerve is separated from the carotid artery, but no electrode cuff is placed around the nerve).</li>
	</ol>
	</li>
</ol>
4. Auditory Fear Conditioning
NOTE: This fear conditioning protocol is more intensive than most21 because the goal of these experiments is to enhance extinction. With mild fear conditioning that is easily extinguished, a floor effect can obscure this enhancement.
<ol>
	<li>House animals on a 12 hr&#160;light/dark cycle with ad libitum access to food and water. Handle animals daily during recovery from surgery.</li>
	<li>Set up the conditioning and testing apparatus, consisting of an operant box housed in a sound-attenuated chamber (Figure 2C). The operant box has clear plastic walls, 20 x 20 x 20 cm, and has a stainless steel grid floor which is connected to a foot shock generator. Use a white house-light to illuminate the chamber for video recording. Use a 9 kHz, 85 dB SPL tone as the conditioned stimulus.</li>
	<li>Record behavior using a digital camera located inside the chamber, above the operant box. View and monitor the session on a computer located outside the behavior room. Save videos for later analysis.</li>
	<li>Wipe chambers with 70% ethanol before and after each session to eliminate olfactory cues.</li>
	<li>Fear-condition the rats for 2 days (Figure 3A). Confirm that the rats are not innately afraid of the tone by presenting 5 tones (9 khz, 85 db, 30 sec) on the first day. Ensure that the freezing levels are negligible.
	<ol>
		<li>Follow the initial tone presentations with 8 tone-footshock (1 sec, 0.5 mA) pairings on each of 2 consecutive days. Repeat the tone-footshock pairings again on the second day. Vary the inter-stimulus-interval (ISI) between 2 and 4 min, averaging 3 min for every trial. Randomize the point at which the shock occurs during the tone.</li>
	</ol>
	</li>
	<li>On the third day, test the strength of the tone/shock association.&#160;Play 4 tones with an ISI of 3, 4, or 5 min (4 min average) in the absence of footshocks and record the animals&#8217; freezing behavior during the tone presentations and during the inter-stimulus-intervals as measures of the conditioned fear response (CFR).</li>
	<li>On day 4, begin extinction training with VNS or Sham VNS.
	<ol>
		<li>Plug the rats into the stimulator by inserting the male connectors from the stimulator into the stimulation input site. Place animals into the chamber (Figure 2A, 2C). Set the stimulator to 0.4 mA, 500 &#181;s pulse width at 30 Hz. Set stimulation to a total duration of 30.15 sec, starting 150 msec before the onset of the tone. Play animals 4 tones (as in step 4.6) and pair each tone presentation with VNS or Sham VNS.</li>
	</ol>
	</li>
	<li>Periodically test the electrical integrity of cuff and input site using an oscilloscope.
	<ol>
		<li>Plug the animal in as normal and split the output from the stimulator to the oscilloscope.</li>
		<li>Set the range on the oscilloscope as -20 V to +20 V and run stimulation. The waveform of the 30 Hz stimulation should be visible on the oscilloscope. Stimulations exceeding 10 V in size indicate high impedance and an improperly functioning cuff or connection at the stimulation input site.</li>
	</ol>
	</li>
	<li>To test the effect of VNS on extinction training run a second CFR test (as in step 4.6) on day 5. Record the time spent freezing during tone presentations and compare to baseline freezing recorded during the first CFR test (step 4.6).</li>
	<li>Analyze the videos using an independent observer who is blind to the treatment conditions. Measure time spent freezing during tone presentations using a stopwatch. Freezing is defined as complete immobility, during which the rat exhibits rapid respiration, lowered head, and spread paws22. Analysis of freezing behavior can be split into two phases: during tone presentation and during the inter-stimulus interval.&#160;</li>
</ol>
5. In Vivo Recordings of Evoked Field Potentials
Note: This step is optional. Evoked field potentials (EFPs) are recorded 24 hr&#160;after tests of reinstatement (Day 5) in isoflurane-anesthetized rats mounted in a stereotaxic apparatus, following standard procedures23,24.
<ol>
	<li>Gather and sterilize all tools.</li>
	<li>Induce anesthesia with isoflurane (5% in 100% oxygen, flowrate 1l/min) in a clear plastic chamber. Assess the depth of the anesthetic plane by monitoring the animal&#8217;s vocalizations and withdrawal-reflexes in response to toe and/or tail pinching. Use mineral oil or eye ointment to protect eyes.</li>
	<li>Inject 0.05 ml marcaine subcutaneously on the top of head and allow the bolus to disperse. Use a scalpel and hemostats to remove the headcap from the skull. Use as little force as possible to avoid obscuring bregma.</li>
	<li>Place the animal in the stereotaxic instrument. Use a scalpel to widen the incision to expose both lambda and bregma. Maintain anesthetic plane with isoflurane (3% in 100% oxygen, flowrate 1 L/min) via a nosecone.</li>
	<li>Drill holes into the skull above the infralimbic prefrontal cortex (IL) and the basolateral amygdala (BLA). Lower a glass microelectrodes (2M KCl; 1-2 MOhms resistance) into the BLA (D/V: 7.2, A/P: 2.7, M/L: 4.9 from bregma) and a stimulation electrode into the IL region of the medial prefrontal cortex (D/V: 4.6, A/P: 3.0, M/L: 0.7 from bregma) (Figure 4A).</li>
	<li>Stimulate the IL to evoke EFPs in the BLA. Data shown in Figure 4 was acquired with the following settings: A stimulation pulse of 0.3 msec duration, using a stimulation intensity that corresponded to 40% of the minimum current intensity that evoked a maximum field response (based on an input&#8211;output curve determined before collection of baseline data), delivered every 15 sec.</li>
	<li>Collect baseline data for a minimum of 10 - 15 min before inducing synaptic plasticity. 
	NOTE: The protocol used to evoke synaptic plasticity will vary with the requirements of the experiments and must be carefully selected by each experimenter. Data in Figure 4C shows changes in the EFP following 3 bursts of 100 pulses at 50 Hz (2 sec), with 20 sec inter-burst intervals at the minimum current intensity that evoked the maximum field response.</li>
	<li>Measure the amplitude of the EFP as the difference between the mean of a 5 msec window before the stimulation artifact and the mean of a 5 msec window around 20 - 25 msec after the stimulation artifact, corresponding to the negative peak of the field potential. Normalize data to baseline and set the average of a 10 min baseline as 100%. Use the averaged EFP amplitudes of another 10 min period after plasticity induction (e.g., 40 - 50 min post induction) to assess long-term changes in EFP amplitude.</li>
	<li>After the end of the recording decapitate the anesthetized animal and extract the brain. Prepare tissue for histological verification of electrode placement.</li>
</ol>

The authors have no competing interests or conflicts.

We present here a protocol that is used to facilitate extinction of conditioned fear during a single session of exposure to conditioned cues19 and to modulate plasticity in the pathway between the infralimbic cortex and the basolateral amygdala that may mediate extinction learning20. A crucial step for the success of this protocol is the proper delivery of VNS during extinction training. Therefore, special care should be given to the construction of the cuff electrodes and the placement of the cuff ...

Classical fear conditioning provides a widely used animal model to study the biological basis of anxiety disorders. During fear conditioning, an aversive stimulus (the unconditioned stimulus, US, e.g., a footshock) is presented in conjunction with a neutral stimulus, such as a tone and/or a context (the conditioned stimulus; CS). During fear conditioning, associations between the CS and the US are formed. Eventually the presentation of the CS alone elicits a fear response (the conditioned response; CR). In fear extinction, the CS is presented repeatedly in the absence of the US, causing the CR to gradually diminish1. Thus, extinction of conditioned fear is an active process in which fearful behavioral responses to neutral stimuli are attenuated when they no longer predict aversive outcomes. Extinction of conditioned responses requires consolidation of new memories that compete with learned associations. A hallmark of anxiety disorders is impaired extinction2-4. Thus, extinction of conditioned fear in animal models serves as an important paradigm both for inhibitory learning and as a model of behavior therapy for human anxiety disorders5,6.
Because there is close correspondence between fear and human anxiety, it is thought that these studies can provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder and will help to develop strategies to treat them. An important goal of preclinical research is to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate extinction learning. Vagus nerve stimulation (VNS) is a minimally invasive neuroprosthetic approach that might be used to provide tight temporal and circuit-specific modulation of brain areas and synapses engaged in an ongoing task. A series of recent studies from Michael Kilgard&#39;s group at The University of Texas at Dallas have shown that pairing VNS with discrete sensory or motor stimuli (e.g., a tone or a lever pull) is highly effective in promoting cortical plasticity to treat tinnitus7, or to overcome motor deficits following stroke8-10. In addition, non-contingent VNS that occurs within a short time-window after learning similarly promotes cortical plasticity and enhances memory consolidation in rats and in humans11-13.
Considering the role of the vagus nerve in the parasympathetic pathway, it is not surprising that it could participate in modulating memories and synaptic plasticity. Highly emotional events tend to produce stronger memories than non-emotional memories. This is likely due to the influence of stress hormones on memory consolidation. Posttraining administration of the stress hormone adrenaline enhances memory consolidation in human and non-human animals, but adrenaline does not cross the blood-brain-barrier14, 15. Therefore, stress-induced adrenaline release must impact the brain indirectly to enhance memory consolidation. Strong evidence suggests that the vagus nerve may be the link between circulating adrenaline and the brain. Miyashita and Williams16 found that systemic administration of adrenaline increased vagal nerve firing, and increased levels of norepinephrine in the amygdala17. Systemic administration of adrenaline does not enhance memory consolidation when &#946;-adrenergic receptors are blocked in the amygdala18 suggesting that the vagus nerve plays a role in the pathway that turns emotionally arousing experiences into long-term memories.
Thus, pairing VNS with training has the potential to enhance the brain changes that support memory consolidation and exposure to conditioned cues in the absence of reinforcement enhances the consolidation of extinction learning in rats19,20. Here we describe the use of VNS as a tool to promote cortical plasticity and facilitate extinction of a conditioned fear response.

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vagus nerve stimulation as a tool to induce plasticity in pathways relevant for extinction learning

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

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

This section illustrates examples of results that can be obtained by using VNS in combination with extinction learning to reduce the expression of the conditioned fear response in rats. For Days 1 and 2 (Auditory Fear Conditioning), rats were trained on an auditory fear conditioning task in which footshocks were paired with a tone. On Day 3 (Pre Treatment Test), tones were presented in the absence of footshocks to measure freezing levels and infer conditioned fear response acquisition. On Day 4 (Treatment) rats received ...

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

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.

vagus-nerve-stimulation-as-tool-to-induce-plasticity-pathways

Research

JoVE Journal

Behavior

Source: Sanchez, C. A., et al. Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats. J. Vis. Exp. (2020).This video demonstrates vagus nerve stimulation in an awake rat. A rat with an implanted chronic peripheral nerve cuff electrode and trained in a lever press task is connected to a stimulus generator via an implanted head cap. The vagus nerve is stimulated immediately after each successful lever press, resulting in the expansion of the motor map.

Vagus Nerve Stimulation via Chronically Implanted Peripheral Nerve Cuff Electrodes in Rats

Source: Childs, J. E., et al. Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning. J. Vis. Exp. (2015)This video demonstrates the method of vagus nerve stimulation (VNS) in a rat model to reduce freezing behavior. Fear-conditioned rats receive VNS during sound re-exposure, leading to reduced freezing and revealing its therapeutic potential.

Vagus Nerve Stimulation in a Rat Model to Reduce Freezing Behavior

Source: Moeller, S., et al., Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression. J. Vis. Exp. (2019)This video demonstrates a clinical protocol for Vagus Nerve Stimulation (VNS) as an adjunctive neurostimulation therapy for treatment-resistant depression (TRD).

Vagus Nerve Stimulation Therapy to Manage Treatment-Resistant Depression

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

The present protocol describes the surgical technique for implanting an electrode array onto the abdominal vagus nerve in rats, along with methods for chronic electrophysiology testing and stimulation using the implanted device.

Implantation Surgery for Abdominal Vagus Nerve Stimulation and Recording Studies in Awake Rats

Learn vagus nerve stimulation to enhance extinction of conditioned fear by inducing synaptic plasticity in forebrain networks. This method enables precise timing of neurotransmitter release for improved therapeutic strategies.

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

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