Research reported in this publication was supported by funding from the National Institutes of Health National Center of Neuromodulation for Rehabilitation, NIH/NICHD Grant Number P2CHD086844 which was awarded to the Medical University of South Carolina. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or NICHD.

This experimental protocol illustrates a typical taVNS set-up for use in a laboratory or clinical setting in which we target stimulating the anterior wall of the auditory canal (tragus) in a supine posture with an 8mm diameter round metal electrode. These methods can be mimicked for alternative active treatment sites by simply changing electrode position to the cymba concha. All methods and procedures have been IRB approved by the Human Research Protection Program (HRPP) at City College New York.
1. Materials
<ol>
	<li>Ensure all materials required to administer taVNS are prepared (Figure 3). The taVNS stimulator may be either a battery driven device that meets local safety regulations or powered from a conventional electrical outlet with built-in safety mechanisms that prevent unintended electrical surges. A constant current (current controlled) stimulator with a maximum output of 5 mA is required.</li>
	<li>For taVNS, use stimulation electrodes made of a round conductive metal (tin, Ag/AgCl, gold) combined with a conductive medium such as electrolyte gel or conductive paste (see table of materials). Alternatively, use conductive electrodes made with flexible conductive carbon electrodes and conductive gel that may or may not be adhesive. Never place electrodes directly on the skin without a conductive medium, as this may pose unnecessary risk to the participant and can cause discomfort or pain.</li>
	<li>Use computer running script software (see Table of Materials) that is programmed and used to control the stimulator and initiate stimulation with specific parameters. These parameters include current intensity (mA), pulse width (&#956;s), frequency (Hz), duty cycle (On/Off time, s), session duration (min).</li>
	<li>Use alcohol preparation pads (70% isopropyl alcohol) to prepare the skin surface before attaching electrodes to the ear. This removes surface oils from the skin surface and reduces the resistance of the skin, ensuring stimulation is delivered at safe power levels.</li>
</ol>
2. Ear Targeting and Skin Preparation
<ol>
	<li>Use the following general inclusion criteria for conducting taVNS in the research setting: Age 18&#8211;70, no facial or ear pain, no recent ear trauma, no metal implants including pacemakers, not pregnant.</li>
	<li>In the experiments involving healthy participants in a laboratory setting, use the following exclusion criteria: personal or family history of seizure, mood, or cardiovascular disorders, dependence on alcohol or recent illicit drug use, on any pharmacological agents known to increase seizure risk.</li>
	<li>Seat the participant on a comfortable bed or chair in a supine or other relaxed position with legs elevated and head supported.</li>
	<li>Inspect the left ear of the participant. Ensure no jewelry is attached and all make-up and lotion are removed. Confirm there are no skin-related contraindications at the site of stimulation, including sun burn, cuts, lesions, open sores.</li>
	<li>Find the stimulation target, landmarked by the anterior wall of the outer ear canal externally by finding the tragus. Stimulation will be delivered to the portion of the ear canal directly behind the tragus (Figure 4).</li>
	<li>Use an alcohol prep pad to gently scrub the target site, both internally and externally, to decrease skin resistance and increase conductance.</li>
</ol>
3. Electrode Preparation and Placement
<ol>
	<li>If using non-disposable electrodes, visually inspect electrodes to ensure clean, corrosion-free surface is exposed. Ensure that electrodes are disinfected to prevent spread of bacteria between subjects. This can be done using alcohol or sterilization wipes to scrub the electrodes. If using disposable electrodes, skip to step 3.2.</li>
	<li>Spread a thin layer of conductive paste to the surface of the electrode evenly. This will distribute electricity to the stimulation site. For an 8 mm diameter round electrode, a pea-sized amount of paste is sufficient. Spread the paste using a narrow wooden applicator to form a thin layer &#60;1 mm of paste on both electrodes.</li>
	<li>Connect electrode cables to the stimulation device while the device is turned off and verify the polarity of the electrodes (red/positive electrode: anode, black/negative electrode: cathode). This is an important detail as targeting is polarity specific &#8212; the anode (red/positive terminal) is the electrode placed inside the ear canal and targeting the anterior wall of the outer ear canal. The cathode (black/negative terminal) sits on the outside of the ear attached to the tragus. For sham stimulation, the anode is placed on the anterior side of the ear.</li>
	<li>Clip the spring electrode onto the tragus with the anode making contact with the anterior wall of the outer ear canal and the cathode contacting the anterior part of the tragus. 
	NOTE: If conducting sham stimulation, clip the electrode onto the earlobe (active control). Alternatively, sham stimulation may be delivered by attaching stimulation clips to active site and delivering no electrical current (passive control).</li>
	<li>As subjects will feel the pressure of the electrodes clipped to their ear, ensure this pressure is not uncomfortable or disruptive to regional blood flow as demonstrated by pale white skin at clip site or physical pain sensed by the subject. After this point, determine perceptual threshold (PT) which will be described in the next procedural step.</li>
</ol>
4. Determination of Perceptual Threshold (PT)
NOTE: Perceptual threshold is a critical value used to determine the power of taVNS stimulation. This value is defined as the minimum amount of electricity required to perceive electrical stimulation on the skin described as a pricking or tingling sensation.
<ol>
	<li>Determine the PT using a simple step-up and step-down binary parametric search. First turn on the stimulator and set the output to 3 mA. Deliver a 1 second train of taVNS stimulation at desired pulse width (typically 250&#8211;500 &#956;s) and frequency (25 Hz, can vary based on application).</li>
	<li>Ask the subject whether they felt the stimulation. Sensation is generally reported as a &#34;tickle&#34; or &#34;pricking&#34; sensation.
	<ol>
		<li>If YES, turn down stimulation intensity by 50% and repeat step 4.2. If NO, increase stimulation intensity by 50% and repeat step 4.2.</li>
	</ol>
	</li>
	<li>Repeat the process described in step 4.2 until recording a minimum of 4 &#34;YES&#34; responses in which the 4th YES response must come after a NO. The intensity (in mA) of the PT will the value at which the subject says their fourth YES response to.</li>
	<li>Use the example PT threshold finding is listed in Table 1 to assist in PT determination.</li>
</ol>
5. Delivering Stimulation
<ol>
	<li>Once the subject is comfortable stimulation electrodes with properly attached to the desired target, and the perceptual threshold determined at the desired pulse width and frequency, begin the stimulation.</li>
	<li>Use a computer running a pulse generating GUI (e.g., stimDesigner, freeware included with this manuscript) connected to a data acquisition unit (DAQ) to drive the stimulation system. The software should output TTL pulses as programmable settings (Figure 5). The TTL pulses will be sent via a BNC cable to the stimulator &#34;trigger in&#34; port. This interface software/stimulator interface allows modulation of frequency, duty cycle (on/off time), and session duration (Figure 6). The GUI used is attached as a free, open-source resource with this manuscript.
	<ol>
		<li>Ensure that stimulation is delivered at super-threshold levels, such as 200% of PT8,9. For example, if the PT was determined to be 0.8 mA, stimulation will be delivered at 1.6 mA.</li>
		<li>Ensure that the guidelines for duty cycles are followed when conducting long stimulation sessions. Typical duty cycles have 30&#8211;60 s &#34;on&#34; periods and 60&#8211;120 s &#34;off&#34; periods, or 20&#8211;50% duty cycles.</li>
		<li>Vary the length of stimulation session (total time). Studies suggest that 30-60 min stimulation sessions at a 25% duty cycle is safe and free of any acute side effects or adverse events. These sessions can be repeated with 12&#8211;24 h between sessions safely. 
		NOTE: taVNS safety is unclear for longer periods of stimulation sessions, larger percentage duty cycles (&#62;40%), accelerated paradigms, and higher stimulation current doses.</li>
	</ol>
	</li>
</ol>
6. After taVNS
<ol>
	<li>When stimulation is completed, record objective data regarding the stimulation discomfort and side effects. Although taVNS, like implantable VNS, has limited safety concerns8,28, monitor and record sensation, discomfort, and any adverse events on a rating from 0&#8211;1029.</li>
	<li>Remove the stimulation electrode from the ear and clean residual conductive paste from the subject&#39;s ear using an alcohol prep pad.</li>
	<li>Use alcohol to clean and disinfect the stimulation electrode immediately upon removing from the subject&#39;s ear.</li>
	<li>Inspect the ear for redness or irritation at the stimulation site and record any observations.</li>
</ol>

The authors have nothing to disclose.

As in all novel modalities, all the described steps are critical in the safe administration of taVNS. Of ultimate concern is subject safety, which includes not only mitigating risks before taVNS via proper screening, but also monitoring subjects during stimulation for discomfort, pain, or adverse events. Here are the three most important consideration for administering taVNS. Screening for taVNS contraindications - contraindications are as follows: any current or past history of cardiovascular disorders, facial or ear pain, recent ear trauma, metal implants above the level of the neck. For proper subject skin preparation, removing any surface oils, dirt, or makeup from the surface of the skin with alcohol helps with conductivity of the electrodes, reduces stimulation voltage required to drive the stimulator, and ultimately results in a more tolerable and safe stimulation session. It is encouraged to use a stimulator and electrodes meeting Low Output Transcranial Electrical Stimulation (LOTES) guidelines32. LOTES sets guidelines and industry standards for electrical stimulators that are built for stimulation of the head and neck and it is encouraged for groups to read this document before building their own systems. It is recommended to use either a FDA-cleared plug-in stimulator (see Table of Materials), or a low voltage (&#60;50 V), battery powered, constant current stimulator with appropriate safety measures built-in to avoid unintended over-delivery of current to the stimulation site. Ensure that electrodes are manufactured and assembled for specific use in taVNS. Ensure that the current manufacturing and engineering guidelines are followed as a reference if lab-made customized systems are used.
One consideration for taVNS is to ensure that the voltage output of the constant current stimulator can surmount the resistance of the skin and deliver the current required for stimulation. Ohm&#39;s law (V=IR) demonstrates the relationship between current (I) and skin resistance (R). A minimum of a 20 V tabletop stimulator is recommended to avoid an underpowered system. Heat generated from the scalp or environment may degrade the conductive paste. If this occurs, it is recommended to stop stimulation and re-prep skin and electrodes with new conductive paste.
A limitation of taVNS is the vast parameter space. It is unknown as to which is more important &#8212; pulse width or frequency. There is a lacking data in recent taVNS trials that answer such questions. The various behavioral effects are derived from a variety of pulse widths, frequencies and stimulation currents13,33,34,35,36,37,38,39.
At this time, it is suggested that the 500 &#181;s pulse width to be the most biologically active9. With respect to frequency, it has been demonstrated that 25 Hz is an effective frequency, although current investigations into optimal such as higher frequencies (&#62;25 Hz), bilateral stimulation (left and right ears), and investigational burst paradigms are being conducted. Studies exploring different parameters of stimulation, alternative stimulation sites, and duty cycle optimization are needed to advance and refine the taVNS method.
taVNS is a promising non-invasive alternative to conventional VNS. taVNS provides an inexpensive (&#60;$5,000 in the demonstrated experimental setup, cost heavily dependent on type of stimulator used) and straightforward method that can be used to translate positive findings in animal models exploring the use of VNS on a variety of disorders, noninvasively modulate the autonomic nervous system, and potentially miniaturized and optimized for at-home neuromodulation for the treatment of neuropsychiatric and other disorders.
The future potential and possible applications of taVNS are vast. taVNS can serve as a promising adjunct or standalone treatment for neuropsychiatric disorders such as depression and epilepsy, taVNS-paired rehabilitation training to restore or accelerate learning of a behavior40, decrease inflammatory response41,42, and can potentially be used to enhance performance and autonomic function8,10.

Cranial nerve X, better known as the vagus nerve, is a large nerve tract that originates in the brainstem of the central nervous system and travels throughout the periphery, targeting every major organ in the thorax and abdomen (Figure 1)1. Vagus nerve stimulation (VNS) involves surgical implantation of bipolar electrodes around the left cervical branch of the vagus nerve. Electrical pulses are delivered to the vagus nerve via an implanted pulse generator (IPG) surgically implanted in the chest2. Although VNS is currently FDA-approved for epilepsy, refractory depression, and chronic obesity, it is a costly procedure requiring a hospital visit and surgery. Long-term safety of VNS is well established, and the majority of safety considerations regard current intensity related side effects (hoarse voice, throat pain) without serious stimulation-related adverse effects over the past 25 years of its clinical use3.
Recently, a noninvasive form of VNS known as transcutaneous auricular vagus nerve stimulation (taVNS) has emerged4. taVNS delivers electrical stimulation to the auricular branch of the vagus nerve (ABVN), an easily accessible target that innervates the human ear5. Over the last decade, several groups have demonstrated the safety and tolerability of this method6,7,8, including central and peripheral nervous system effects9,10, and behavioral effects7,11,12,13 in neuropsychiatric populations. taVNS is also being explored in individuals as a promising enhancer of cognitive14,15 and social functioning16,17,18. As taVNS is becoming established, it offers the ability for researchers and clinicians to rapidly translate the promising VNS research that has been described in various disorders ranging from neurological and psychological trauma19,20,21, addiction22, inflammation23, and tinnitus24,25.
In principle, taVNS is methodologically similar to conventionally administered transcutaneous electrical nerve stimulation (TENS) used to treat musculoskeletal pain disorders26. The difference is that taVNS is delivered to specific anatomical ear targets that are believed to be innervated by the ABVN5. The field is still determining optimal stimulation targets27, although the two most common placements are the anterior wall of the outer ear canal (tragus) and the cymba conchae. Sham stimulation may be conducted by stimulating the earlobe of the ear, an area believed to have minimal ABVN innervation (Figure 2). Alternatively, sham may be delivered via a passive control method in which electrodes are attached to active sites, but no stimulation is delivered. Stimulation parameters may vary between groups, however according to the literature, stimulation is delivered in a pulsatile fashion (pulse width: 250&#8211;500 &#956;s, frequency: 10&#8211;25 Hz) and delivered at an individualized constant current (&#60;5 mA). Stimulation current varies by individual and experimental protocol, with many groups exploring various intensities as a function of an individual perceptual threshold (PT). The PT is defined as the minimum amount of current eliciting a perceived sensation at the target site and is usually determined via parametric estimation by customized sequential testing (PEST) software described in this report.
taVNS is a safe technique that may be administered in the laboratory or clinical setting. Side-effects of taVNS are minimal, with skin irritation or redness being the most common side-effect. Most taVNS studies explore stimulation of the left ear, as it is believed to be safer, although data in a large trial (Badran et al. 2018) reveal that right-sided stimulation has no increases in the risk of adverse events. Due to the wealth of literature in unilateral-left side stimulation, we will illustrate the typical taVNS set-up for laboratory studies investigating the use of left-sided taVNS as an intervention.

<table border="0" cellpadding="0" cellspacing="0" style="width:728px;" width="728">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">70% Isopropyl Alcohol Wipes</td>
			<td style="width:88px;">Any</td>
			<td style="width:88px;">N/A</td>
			<td style="width:357px;">Any alcohol preparation pads used for skin in appropriate.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Constant Current Stimulator (Triggerable)</td>
			<td style="width:88px;">Soterix Medical</td>
			<td style="width:88px;">N/A</td>
			<td>Stimulator manufactured for custom use by Soterix Medical</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Disposable Conductive Electrodes</td>
			<td style="width:88px;">Custom Built</td>
			<td style="width:88px;">N/A</td>
			<td>Stimulation electrodes are custom built at the City College Neural Engineering Lab (Badran/Bikson)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Matlab Software w/ Stimulation GUI</td>
			<td style="width:88px;">MathWorks</td>
			<td style="width:88px;">N/A</td>
			<td>MATLAB used for programing pulse pattern</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Ten20 Conductive Paste</td>
			<td style="width:88px;">Weaver and Company</td>
			<td style="width:88px;">N/A</td>
			<td>Conductive paste used for administration of stimulation</td>
		</tr>
	</tbody>
</table>

laboratory administration of transcutaneous auricular vagus nerve stimulation (tavns): technique, targeting, and considerations

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus nerve stimulation (taVNS). Unlike cervically-implanted VNS, taVNS is an inexpensive and non-surgical method used to modulate the vagus system. taVNS is appealing as it allows for rapid translation of basic VNS research and serves as a safe, inexpensive, and portable neurostimulation system for the future treatment of central and peripheral disease. The background and rationale for taVNS is described, along with electrical and parametric considerations, proper ear targeting and attachment of stimulation electrodes, individual dosing via determination of perception threshold (PT), and safe administration of taVNS.

When proper skin preparation is conducted, perceptual thresholds are inversely correlated with stimulation pulse width. As pulse width increases, the perceptual threshold decreases (Figure 7). Initial studies by this group exploring the effect of pulse width on PT in healthy individuals (meeting inclusion/exclusion criteria listed above), determined that the combined overall (n=15, 7 female, mean age 26.5 &#177; 4.99) PT at 100 &#956;s = 3.92 &#177; 1.1 mA; 200 &#956;s = 2.24 &#177; 0.74 mA; 500 &#956;s = 1.24 &#177; 0.41 mA. These thresholds suggest that a constant current stimulator with capacity of delivering up to 5 mA of current is required for stimulation of 500 &#956;s pulse width parameters, and a minimum of a 10mA stimulator is required for lower pulse widths (Table 2). Fine tuning of the current is required, with increments of 0.1 mA are necessary for precise stimulation.
Delivering stimulation at 200% PT is tolerable and relatively pain free as demonstrated by pain numerical rating scales (NRS) scales9,30. The NRS scale is a rating system for pain from 0-10 in which individuals report pain or discomfort29. Both Active and Sham stimulation rate similarly low pain levels (NRS &#60;3 for all stimulation pulse widths. More specifically, the biologically-active pulse width of 500 &#956;s delivered at 25 Hz is reported on average to rate as Active = 1.98 &#177; 0.83, Sham = 2.17 &#177; 1.27 (n=25, 9 female, mean age 25.16 &#177; 4.16 years) (Table 3). Pain ratings for other parameters are no more painful than the 25 Hz parameter and the details can be found in the groups&#39; prior work30.
Safety and tolerability of 30 min to 1 hour sessions at a 20&#8211;50% duty cycle has been widely reported in the literature with some studies delivering multiple sessions in the same day spread 12&#8211;15 h apart12,31. No serious adverse events have been reported from 60 subjects participating in several series of experiments with subjects participating from 1 to 8 repeated visits spread a minimum of 24 hours apart.
taVNS, when administered as reported in this manuscript, has been demonstrated to modulate the autonomic nervous system, induce functional brain activity changes as measured by fMRI BOLD, and piloted to treat neuropsychiatric disorders and aid in rehabilitation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58984/58984fig1.jpg" /> 
Figure 1: Vagus Nerve Efferent Projections and Cross-section.&#160;(A)&#160;Efferent projections of the vagus nerve target every major organ on the body with wide effects on bodily function (B) Cross-section of the vagus nerve, demonstrating the inside anatomy of the nerve as a series of&#160;bundles of nerves all contained within one major pathway. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58984/58984fig2.jpg" /> 
Figure 2: taVNS Ear Targets. Targeting the ABVN can be accomplished by stimulating the anterior wall of the outer ear canal, landmarked notably by the tragus (A1), or cymba conchae (A2). Sham stimulation is administered to the earlobe (S). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58984/58984fig3.jpg" /> 
Figure 3: Key Components. The minimum required components for proper administration of taVNS are the following (A) ear stimulation electrodes, (B) conductive gel and alcohol prep pads, (C) computer capable of sending and receiving TTL pulses to a (D) constant current stimulator to trigger stimulation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58984/58984fig4.jpg" /> 
Figure 4: Example Setup. This photo shows an individual receiving taVNS of the left ear while in position to undergo an experimental paradigm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58984/58984fig5.jpg" /> 
Figure 5: Screenshot of the GUI used for stimulation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58984/58984fig6.jpg" /> 
Figure 6: Electrical Stimulation Waveform Manipulations. Direct square wave electrical current can be delivered at various parameters. This figure demonstrates key properties of the waveform that can be changed in order to achieve desired biologic effects. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58984/58984fig7.jpg" /> 
Figure 7: Perceptual Threshold Values at Increasing Pulse Widths. As pulse width increases, perceptual threshold (PT) decreases. Most healthy individuals will have a PT within 2 standard deviations (SD) of these mean values. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/58984fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58984/58984table1.jpg" /> 
Table 1: Example of How to Determine Perceptual Threshold (PT). This table shows an example sequence of Yes/No responses used to parametrically determine PT.
<img alt="Table 2" src="/files/ftp_upload/58984/58984table2.jpg" /> 
Table 2: Stimulation Current Levels. Values of stimulation current in mA (200% PT) for each pulse width (n=15).
<img alt="Table 3" src="/files/ftp_upload/58984/58984table3.jpg" /> 
Table 3: PT, Stimulation Current, and Pain Values for Suggested Stimulation Parameters. Values of stimulation current in mA (200% PT) for each pulse width (n=25).
<img alt="Supplemental File Figure" src="/files/ftp_upload/58984/58984supfig1.jpg" /> 
Supplemental File: Freeware GUI used in this protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58984/stimDesigner.zip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation taVNS: Technique, Targeting, and Considerations at JoVE.com

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

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

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus ...

laboratory-administration-transcutaneous-auricular-vagus-nerve

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38.1K Views.  City College of New York. A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

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

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State-Dependency Effects on TMS: A Look at Motive Phosphene Behavior

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Optogenetic Stimulation of the Auditory Nerve

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro