Name: preparation of peripheral nerve stimulation electrodes for chronic implantation in rats
Uploaded: 2020-07-14T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats at JoVE.com

This work was funded by the University of Texas at Dallas and the UT Board of Regents. We thank Solomon Golding, Bilaal Hassan, Marghi Jani, and Ching-Tzu Tseng for technical assistance.

All procedures described in this protocol are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.
1. Stimulating cuff electrode fabrication
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	<li>Prepare the cuff tubing.
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		<li>Using a razor blade, cut a piece of polymer tubing 2.5 mm in length. Insert forceps tips or a paper clip through the tubing and use the blade to make a slit lengthwis...

<ol>
	<li>Koopman, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+inhibits+cytokine+production+and+attenuates+disease+severity+in+rheumatoid+arthritis.">Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2016).</li><li>Levine, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurostimulation+of+the+cholinergic+anti-inflammatory+pathway+ameliorates+disease+in+rat+collagen-induced+arthritis.">Neurostimulation of the cholinergic anti-inflammatory pathway ameliorates disease in rat collagen-induced arthritis.</a> PLoS One. , (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+vagus+nerve+stimulation+improves+autonomic+control+and+attenuates+systemic+inflammation+and+heart+failure+progression+in+a+canine+high-rate+pacing+model.">Chronic vagus nerve stimulation improves autonomic control and attenuates systemic inflammation and heart failure progression in a canine high-rate pacing model.</a> Circulation: Heart Failure. , (2009).</li><li>Ganzer, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed-loop+neuromodulation+restores+network+connectivity+and+motor+control+after+spinal+cord+injury.">Closed-loop neuromodulation restores network connectivity and motor control after spinal cord injury.</a> Elife. , (2018).</li><li>Hays, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+during+rehabilitative+training+enhances+recovery+of+forelimb+function+after+ischemic+stroke+in+aged+rats.">Vagus nerve stimulation during rehabilitative training enhances recovery of forelimb function after ischemic stroke in aged rats.</a> Neurobiology of Aging. , (2016).</li><li>Khodaparast, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+delivered+during+motor+rehabilitation+improves+recovery+in+a+rat+model+of+stroke.">Vagus nerve stimulation delivered during motor rehabilitation improves recovery in a rat model of stroke.</a> Neurorehabilitation and Neural Repair. , (2014).</li><li>Meyers, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+enhances+stable+plasticity+and+generalization+of+stroke+recovery.">Vagus nerve stimulation enhances stable plasticity and generalization of stroke recovery.</a> Stroke. , (2018).</li><li>Hays, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+during+rehabilitative+training+improves+functional+recovery+after+intracerebral+hemorrhage.">Vagus nerve stimulation during rehabilitative training improves functional recovery after intracerebral hemorrhage.</a> Stroke. , (2014).</li><li>Farrand, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+improves+locomotion+and+neuronal+populations+in+a+model+of+Parkinson's+disease.">Vagus nerve stimulation improves locomotion and neuronal populations in a model of Parkinson's disease.</a> Brain Stimulationation. , (2017).</li><li>Souza, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+reverses+the+extinction+impairments+in+a+model+of+PTSD+with+prolonged+and+repeated+trauma.">Vagus nerve stimulation reverses the extinction impairments in a model of PTSD with prolonged and repeated trauma.</a> Stress. , (2019).</li><li>Noble, L. J., Souza, R. R., McIntyre, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+as+a+tool+for+enhancing+extinction+in+exposure-based+therapies.">Vagus nerve stimulation as a tool for enhancing extinction in exposure-based therapies.</a> Psychopharmacology. , (2019).</li><li>Childs, J. E., Kim, S., Driskill, C. M., Hsiu, E., Kroener, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+during+extinction+learning+reduces+conditioned+place+preference+and+context-induced+reinstatement+of+cocaine+seeking.">Vagus nerve stimulation during extinction learning reduces conditioned place preference and context-induced reinstatement of cocaine seeking.</a> Brain Stimulationation. , (2019).</li><li>Pe&#241;a, D. F., Engineer, N. D., McIntyre, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+remission+of+conditioned+fear+expression+with+extinction+training+paired+with+vagus+nerve+stimulation.">Rapid remission of conditioned fear expression with extinction training paired with vagus nerve stimulation.</a> Biological Psychiatry. , (2013).</li><li>Childs, J. E., DeLeon, J., Nickel, E., Kroener, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation+reduces+cocaine+seeking+and+alters+plasticity+in+the+extinction+network.">Vagus nerve stimulation reduces cocaine seeking and alters plasticity in the extinction network.</a> Learning &amp; Memory. , (2017).</li><li>Engineer, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+plasticity+in+auditory+cortex+improves+neural+discrimination+of+speech+sounds.">Temporal plasticity in auditory cortex improves neural discrimination of speech sounds.</a> Brain Stimulationation. , (2017).</li><li>Rios, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+Construction+of+Rat+Nerve+Stimulation+Cuff+Electrodes.">Protocol for Construction of Rat Nerve Stimulation Cuff Electrodes.</a> Methods Protoc. , (2019).</li><li>Childs, J. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+and+serotonin+are+required+for+vagus+nerve+stimulation+directed+cortical+plasticity.">Norepinephrine and serotonin are required for vagus nerve stimulation directed cortical plasticity.</a> Exp. Neurol. , (2019).</li><li>Hulsey, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+of+Motor+Cortex+by+Vagus+Nerve+Stimulation+Requires+Cholinergic+Innervation.">Reorganization of Motor Cortex by Vagus Nerve Stimulation Requires Cholinergic Innervation.</a> Brain Stimulation. 9, 174-181 (2016).</li><li>Bouverot, P., Crance, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+Film+Multi-Electrode+Softening+Cuffs+for+Selective+Neuromodulation.">Thin Film Multi-Electrode Softening Cuffs for Selective Neuromodulation.</a> Scientific Reports. , (2018).</li><li>Thakur, R., Nair, A. R., Jin, A., Fridman, G. 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Here we describe a simple, low-cost approach for assembly of chronically implantable stimulating cuff electrodes for use in rodents, facilitating preclinical investigations of this emerging therapy. This simplified method requires no specialized training or equipment, and uses a small number of tools and supplies that are easily accessible to most research labs, reducing both the monetary and labor costs of device manufacture compared to other approaches16,26<sup...

Recently, the demand for chronically implantable cuff electrodes for stimulation of peripheral nerves has grown, as studies increasingly demonstrate the preclinical usefulness of this technique for the treatment of numerous inflammatory diseases1,2,3 and neurological disorders4,5,6,7,8,9,10,11,12,13,14,15. Chronic VNS, for example, has been shown to enhance neocortical plasticity in a variety of learning contexts, improving motor rehabilitation4,5,6,7,8, extinction learning10,11,12,13,14, and sensory discrimination15. Commercially available peripheral nerve cuff electrodes are often associated with extended times for order fulfillment and relatively high costs, which can limit their accessibility. Alternatively, protocols for &#8220;in-house&#8221; fabrication of chronically implantable cuff electrodes remain limited, and rodent anatomy presents particular challenges due to their small size. Current protocols for constructing cuff electrodes for chronic rodent experiments often require the use of complex equipment and techniques, as well as extensively trained personnel. In this protocol, we demonstrate a simplified approach to cuff electrode fabrication based on previously published and widely used methods16,17. We validate the functionality of our chronically implanted electrodes in rats by demonstrating that, at the time of cuff implantation around the left cervical vagus nerve, stimulation applied to the cuff electrodes successfully produced a cessation of breathing and drop in SpO2. Stimulation of afferent pulmonary receptor vagal fibers is known to engage the Hering-Breuer reflex, in which the inhibition of several respiratory nuclei in the brainstem results in the suppression inspiration18. Thus, cessation of breathing consistent with the Hering-Breuer reflex, and the resulting drop in SpO2, provide a straightforward test for proper electrode implantation and cuff function in anesthetized rats. To validate the long-term functionality of chronically implanted cuff electrodes, reflex responses were measured at the time of implantation and compared to the responses obtained in the same animals six weeks after implantation. A second group of rats was implanted with VNS cuff electrodes after behavioral training on a lever pressing task. In these rats, VNS paired with correct task performance produced reorganization of the cortical motor map, consistent with previously published studies19,20,21,22. At the time of motor cortical mapping under anesthesia, which occurred 5&#8211;10 weeks after device implantation, we further validated cuff function in VNS-treated animals by confirming that VNS successfully induced a cessation of breathing and a greater than 5% drop in SpO2.
The recently published protocols from Childs et al.17 and Rios et al.16 provide a well-validated starting point for a simplified cuff electrode fabrication approach, as this popular method has been utilized by multiple labs conducting chronic VNS studies in rodents1,2,3,4,5,6,7,8,9,10,11. The original method involves several high-precision steps for manipulating the fine microwires such that cuff electrode fabrication takes over an hour to complete, and extensive training to perform reliably. The simplified approach described here requires significantly fewer materials and tools and can be completed in under one hour by minimally trained personnel.

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		<tr height="21">
			<td height="21" style="height:21px;width:441px;">Biocompatible polyurethane-based polymer tubing, 0.080&#34; OD x 0.040&#34; ID</td>
			<td style="width:178px;">Braintree Scientific</td>
			<td style="width:174px;">MRE080 36 FT</td>
			<td style="width:542px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td style="width:178px;">AM Scopes</td>
			<td style="width:174px;">#SM-6T-FRL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Serrated Scissors, straight, 22mm cutting edge</td>
			<td style="width:178px;">Fine Science Tools</td>
			<td style="width:174px;">#14058-09</td>
			<td>for cutting Pt/Ir wire and suture thread</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps, #5 Dumont forceps, straight, 11 cm, 0.1 x 0.06 mm tip</td>
			<td style="width:178px;">Fine Science Tools</td>
			<td style="width:174px;">#11626-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps, ceramic tipped forceps, 0.3 mm x 30 mm tips</td>
			<td style="width:178px;">Electron Microscopy Sciences</td>
			<td style="width:174px;">#78127-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Pins, PCB Press Fit Socket</td>
			<td style="width:178px;">Mill-Max</td>
			<td style="width:174px;">#1001-0-15-15-30-27-04-0</td>
			<td>or similar small pins for connecting cuff leads to headcap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isobutane lighter</td>
			<td style="width:178px;">BIC</td>
			<td style="width:174px;">#LCP21-AST</td>
			<td>for de-insulating Pt/Ir wire</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Micro strip connector with latch, 4-pin</td>
			<td style="width:178px;">Omnetics</td>
			<td style="width:174px;">A24002-004 / PS1-04-SS-LT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette tip, 10 uL</td>
			<td style="width:178px;">VWR</td>
			<td>89079-464</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum-Iridium (90/10%) Wire, 0.001&#34; (diameter) x 9 strands, PTFE insulated</td>
			<td style="width:178px;">Sigmund Cohn</td>
			<td style="width:174px;">10IR9/49T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor Blade, Single Edge, Surgical Carbon Steel No.9</td>
			<td style="width:178px;">VWR</td>
			<td style="width:174px;">#55411-050</td>
			<td>for cutting MicroRenathane tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sewing needle, ca. 4.0 cm length x 0.7 mm diameter (size 6-7)</td>
			<td style="width:178px;">Singer</td>
			<td style="width:174px;">00276</td>
			<td>Smaller needle for threading Pt/Ir wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sewing needle, ca. 4.5 cm length x 0.8 mm diameter (size 2-3)</td>
			<td style="width:178px;">Singer</td>
			<td style="width:174px;">00276</td>
			<td>Larger needle for pinning cuff during assembly and for threading suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small foam board</td>
			<td style="width:178px;">Juvo+/Amazon</td>
			<td>B07C9637SJ</td>
			<td>for fabrication platform; our dimensions are ca. 2.5&#34; x 3.5&#34; x 1&#34; (L x W x H)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solder, multicore lead-free, 0.38mm diameter</td>
			<td style="width:178px;">Loctite/Multicore</td>
			<td style="width:174px;">#796037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering station</td>
			<td style="width:178px;">Weller</td>
			<td style="width:174px;">WES51</td>
			<td>or similar soldering iron compatible with long conical tips (this part has been discontinued)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering tip, long conical, 0.01&#34; / 0.4 mm</td>
			<td style="width:178px;">Weller</td>
			<td style="width:174px;">1UNF8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture, nonabsorbable braided silk ,size 6/0</td>
			<td style="width:178px;">Fine Science tools</td>
			<td style="width:174px;">#18020-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV (405 nm) spot light</td>
			<td style="width:178px;">Henkel/Loctite</td>
			<td style="width:174px;">#2182207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Light Cure Adhesive 25 ml</td>
			<td style="width:178px;">Henkel/Loctite</td>
			<td style="width:174px;">AA 3106</td>
			<td>or similar biocompatible UV cure adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wire wrapping wire, 30 AWG</td>
			<td style="width:178px;">Digikey</td>
			<td style="width:174px;">K396-ND</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of peripheral nerve stimulation electrodes for chronic implantation in rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Vagus nerve cuff electrodes and headcaps were chronically implanted in rats according to previously published surgical procedures17,19,20,21,22. Prior to implantation, impedance at 1 kHz was measured across the cuff leads with the cuff tubing submerged in saline (impedance = 1.2 &#177; 0.17 k&#937; [mean &#177; std]; N = 9). Only cuffs with impedances less tha...

Watch this Scientific Journal Video about Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats at JoVE.com

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

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.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Peripheral nerve stimulation is increasingly under investigation as a treatment for a variety of diseases including inflammatory diseases and neural disorders. Construction of chronically implantable peripheral nerve cuff electrodes for small laboratory animals can be time consuming and expensive. This protocol describes a simple, low cost approach for constructing chronically implantable peripheral nerve stimulating electrodes for use in rats.The method requires very few and easily accessible materials and supplies and a reduced number of hazardous or technically demanded steps compared to similar techniques which reduces both the cost of the device as well as the time required to train personnel and device assembly. This technique was designed to enable preclinical studies that test the effectiveness of chronic Vegas nerve stimulation during stroke recovery or other types of Metro dysfunction. Begin by preparing the cuff tubing.Use a razor blade to cut a 2.5 millimeter piece of polymer tubing. Then insert a paperclip or force tips through the tubing and make a lengthwise slip through the wall of the tubing on one side of the cuff. Remove the forceps from the tubing and insert a large sewing needle through the midline of the cuff.Place the needle into the foam board to pin the cuff in place during the remaining assembly steps. To place a suture for securing cuff closure during implantation insert a small sewing needle through the wall of the cuff on the midline approximately point five millimeters from the top slit on one side. Then insert two centimeters of a six zero suture through the eye of the needle and pull the needle through the wall of the tubing to thread the suture into the cuff.Puncture a second hole through the tubing wall approximately point five millimeters below the first hole along the midline of the cuff. Insert the suture through the eye of the needle and pull the needle through the tubing wall. Apply a small amount of UV cure adhesive to the short end of the suture extending from the lower hole and pull the longer suture end until the lower tail is nearly flush with the exterior wall of the tubing.Then cure the adhesive with a UV wand. Repeat this process to place another suture on the opposite side of the cuff. Next place the Platinum Iridium wire leads.Use the small sewing needle to make four holes in the cuff wall. Then insert the sewing needle again, this time working from exterior to interior through the first lead hole. Insert approximately point five centimeters of a 7.5 centimeter Platinum Iridium wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff wall.Adjust the wire so that approximately 4.5 centimeters extends on the exterior side of the cuff. Insert the needle through the first lead hole again then through the second lead hole. Insert point five centimeters of the shorter end of the wire through the eye of the needle and pull the needle through the tubing to thread the wire lead through the cuff walls.Repeat the process to thread the wire through the third and fourth lead holes. Using a butane lighter, carefully remove the insulation from five to six millimeters of the wires extending from the second and fourth lead holes Then gently pull on the end of the wire extending from the first hole until the uninsulated portion of the wire is flush with the hole. Repeat this with the other lead to align the uninsulated end of the wire threaded through the third and fourth lead holes.Apply a small amount of UV cure adhesive to the wire loops on the exterior side of the cuff at the first and third lead holes. Then use the UV wand to cure of the adhesive and secure the leads in place. Use a small pipette tip to push the uninsulated wire leads against the interior wall of the cuff.Pull the one millimeter tails of the wire flat against the exterior surface of the cuff, taking care not to short them together. Then apply a small amount of UV adhesive to just cover the two tails and cure it. Remove the large needle with the cuff assembly from the foam board.Insert a three centimeter length of a six zero suture through the eye of the needle and pull the needle through the tubing to thread the suture through the bottom of the cuff at the midpoint. Insert the small sewing needle through the same midline hole working from interior to exterior to avoid deformation, the tubing and the wire leads. Then insert the exterior tail of the suture through the eye of the needle and pull the needle through the cuff wall to create a loop of suture around the edge of the cuff.Create the second loop around the opposite end of the cuff by tying the ends of the suture and a half knot on the exterior side of the cuff. While holding the knot flat against the tubing, apply a small amount of UV adhesive to the half knot and cure it. Carefully cut the ends of the suture thread as close to the knot as possible.Then, use a butane lighter to remove the insulation from approximately three millimeters at the end of each of the Platinum Iridium wire leads. Solder the cup side of a gold pin to the uninsulated end of each lead. Before proceeding with implantation test the impedance of the assembled device to verify that the assembled cuff has an impedance of less than two kilo ohm at one kilohertz.After assembling the head cap and implanting the cuff electrodes according to manuscript directions, stimulate the vagus nerve while the rat is awake. Connect the rat to a stimulus generator via the implanted head cap and adjust the stimulation settings. For VNS induced reorganization of the motor cortical map, deliver a single train of 15 biphasic pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere at a frequency of 30 hertz.A stimulation train is delivered immediately after the detection of each successful lever press throughout 10, 30 minute training sessions. Use an oscilloscope to monitor successful delivery of current stimulation. This protocol, was used to chronically implant vagus nerve cuff electrodes and head caps in rats.During implantation, functional validation of all cuffs was performed by testing for a VNS induced drop in blood oxygen saturation. To evoke this response, a 10 second train of 30 hertz pulses, each with a width of 100 microseconds, and an amplitude of 800 micro ampere was delivered across the cuff leads. For all cuffs, we observed a VNS induced cessation of breathing for the duration of the 10 seconds stimulation, which was accompanied by a drop in blood oxygen saturation of at least 5%confirming cuff function and proper implantation.To test the long term functionality of the devices, rats were anesthetized six weeks after implantation, and VNS was again applied. For seven out of nine devices, the magnitude of stimulation evoked change in blood oxygen saturation, did not differ from that observed at initial implantation, suggesting continued excellent performance. The remaining two devices, increasing the stimulation amplitude to 1.6 milliampere was sufficient to evoke a reliable reduction in blood oxygen saturation of at least 5%suggesting that these devices continued to function.But the changes in impedance, nerve damage, or cuff orientation may have resulted in reduced performance. To further test the long term functionality of the chronically implanted devices, a second group of rats was trained on a simplified version of a skilled reaching lever press task. The rats were required to reach two centimeters outside the training booth to fully depress the lever and then to release it within two seconds.Consistent with prior studies demonstrating that vagus nerve stimulation drives expansion of task relevant motor map representations, VNS treated rats exhibited significantly larger proximal forelimb representations than Sham treated rats. This protocol can be easily adapted to accommodate chronic stimulation experiments in other laboratory species or at other peripheral nerve sites, including sciatic or sacral nerves. For example.

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