We acknowledge funding support by K23 AT008477 (Kong), NIH K23 DA031808 (Johnson), and the Chris Redlich Endowment in Pain Research (Mackey, Dixon).

1. Temporal Summation Protocol
<ol>
	<li>Equipment
	<ol>
		<li>Heat pulse delivery and location:
		<ol>
			<li>Used a circular thermode with a diameter of 2.9 cm. This thermode has a microprocessor driven, automated control system, and a peltier element that enables rapid temperature change. Place thermode on the thenar eminence of the subject and use a glove to secure the placement.</li>
		</ol>
		</li>
		<li>Computer Interface:
		<ol>
			<li>Interface the thermode with a software program on a laptop that precisely controls the temperature delivery. Preprogram the appropriate heat stimuli for each stage of the protocol (training 1 and 2 only, as the optimization and final trials follow the same pulse structure as training 2) according to specifications listed in Table 1.</li>
			<li>Execute the delivery of the heat pulses by selecting the heat stimulus program appropriate for each stage on the software, adjust the temperatures and hit &#34;start&#34; on the user interface.</li>
		</ol>
		</li>
		<li>Pain rating device:
		<ol>
			<li>Use the Computerized Visual Analog Scale (CoVAS). 
			NOTE: The CoVAS is a box with a mobile lever on a horizontal bar that represents the visual analog scale (VAS). It is an accessory to the main thermode.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Training
	<ol>
		<li>Define the visual analog scale (VAS) on the CoVAS:
		<ol>
			<li>Present the CoVAS to the participant and define the anchoring points on the VAS: 0 means no pain and 10 means the worst pain imaginable. Ascertain that the participant is comfortable moving the lever left and right with ease.</li>
		</ol>
		</li>
		<li>Define second pain:
		<ol>
			<li>Instruct the participant to focus on second pain from a heat pulse. Define second pain as &#34;a slow, burning, achy pain that takes place about one second after the heat pulse.&#34;</li>
		</ol>
		</li>
		<li>Rate SECOND pain from a single heat pulse (training trial 1):
		<ol>
			<li>Secure the thermode on the participant&#39;s thenar eminence. Instruct the participant to use CoVAS to rate second pain from each single pulse delivered through the thermode.</li>
			<li>Begin the pre-programed test on the laptop by hitting the &#34;start&#34; button on the software menu. Each heat pulse lasts 0.5 second and has an inter-stimulus interval of 10 s (see Fig. 1). Sequentially increase the baseline and peak temperatures of the heat pulse per Table 2.</li>
			<li>Observe closely the participants&#39; rating of second pain. As soon as the participant rates the second pain as more than 2/10, stop the training trial 1 by pressing the stop button on the software. Remove the thermode and record the final heat pulse parameters (i.e. baseline and peak temperatures).</li>
		</ol>
		</li>
		<li>Rate pain continuously from a TS trial (training trial 2):
		<ol>
			<li>Secure the thermode on the opposite hand. Inform the participant that they will receive a series of 10 rapid pulses. Instruct the participant to rate second pain only, and not focus on the faster heat pain sensation that immediately accompanies each pulse (first pain). Explain to the participant that the second pain may increase, decrease, or remain the same between each pulse.</li>
			<li>Begin the pre-programmed test on the laptop. Use the final baseline and peak temperatures recorded from step 1.2.3 above. Keep the pulse width at 0.5 s and the inter-stimulus interval at 2 s (Fig. 2). Switch hand and repeat this training trial if necessary until the participant is comfortable rating second pain continuously in response to rapid heat pulses.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Optimization
	<ol>
		<li>Define P1, Pmax and TSE:
		<ol>
			<li>Define P1 as the pain rating at approximately 2 s after the delivery of the peak temperature from the first pulse (See Fig. 3).11,21 
			NOTE: TSE is the estimated difference between the pain P1 and the maximum pain (Pmax).</li>
		</ol>
		</li>
		<li>Adjusting pulse temperature. Aim to achieve TSE between 30 and 70/100 by sequentially adjusting the peak and baseline temperature according to Figure 4. 
		NOTE: See Fig. 4 for optimization algorithm.</li>
		<li>Secure thermode: Using a surgical glove, secure the thermode to the participant&#39;s thenar eminence. Switch hand between each trial.</li>
		<li>Deliver heat pulses: Instruct the participant to only rate their second pain from the heat pulses. Confirm that the participant is ready to begin and then start the pre-programed test on laptop.</li>
		<li>Iterations: Repeat steps 1.3.2 -1.3.4 until the participant&#39;s pain rating at P1 is &#60;50/100 VAS and their TSE is between 30/100 and 70/100 VAS. Conduct no more than 5 optimization trials and ensure that there is a 3 min rest between each trial. Take the parameters from the very last trial as the final parameters.</li>
	</ol>
	</li>
	<li>Final Trials
	<ol>
		<li>Rest for 5 min between the optimization trial and the final trials. Repeat 1.3.3 and 1.3.4. using the final parameters from step 1.3.5 above. Have the participant rest for 3 min and then repeat again.</li>
	</ol>
	</li>
</ol>
2. Conditioned Pain Modulation Protocol
<ol>
	<li>Overview and equipment:
	<ol>
		<li>Test stimulus: 30 s contact heat (delivered by the same thermode as above) calibrated to 6 out of 10 pain (Heat-6). See 2.2 for details.</li>
		<li>Conditioning stimulus:
		<ol>
			<li>Construct the cold-water bath using a clear plastic box with a perforated dividing wall, and filling it with ice and water on one side and just water on the other. Gently agitate then insert thermometer to water-only side to ensure temperature is stable at 10 &#176;C.</li>
			<li>If unable to obtain stable temperature, attach a hydro pump to the side with ice to maintain stable temperature by circulating water constantly. Turn on the pump and make sure water side temperature is stable at 10 &#176;C.</li>
		</ol>
		</li>
		<li>Timing and computing CPM:
		<ol>
			<li>Apply the test stimulus to the participant once before and once during the last 30 s of the conditioning cold bath. Compute CPM as the difference in the pain level of the Heat-6 stimulus applied before and during the last 30 s of the cold bath.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calibrating for Heat-6:
	<ol>
		<li>Estimating Heat-6 via heat ramp:
		<ol>
			<li>Secure the thermode to the thenar eminence of the non-dominant hand of the participant. Starting at 32 &#176;C, increase the temperature at a rate of 0.3 &#176;C per second. 
			NOTE: A slow ramp rate is used to preferentially activate C-fibers20 which are responsible for CPM.12</li>
			<li>Stop whenever the participant reaches thermal tolerance or at 51 &#176;C maximum. Instruct the participant to rate the pain continuously during the heat ramp. Repeat 3 times and compute the average temperature where the pain rating is 6/10.</li>
		</ol>
		</li>
		<li>Fine thresholding: Apply the Heat-6 and instruct the participant to rate the pain. If the rating is between 5 and 7/10, proceed to 2.3.2. If it is &#62;7 or &#60;5, apply a series of 30 s thermal heat stimuli to the participant (alternating hands between each stimuli), ranging from 44 &#176;C to 49 &#176;C and changing in increments of 0.5-1 &#176;C, until the rating is between 5-7, allowing a 30 s rest between each stimulus.</li>
		<li>Finalizing Heat-6 and rating of the pre-conditioning, test stimulus:
		<ol>
			<li>End the fine thresholding once the participant&#39;s pain rating is between 5 and 7/10. NOTE: Use this final, within-range pain as the rating of the pre-conditioning testing stimulus for the computation of CPM. Also, record the temperature of the final Heat-6 that resulted in this rating.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>CPM protocol
	<ol>
		<li>Pre-condition test stimulus as per step 2.2.3.</li>
		<li>Conditioning stimulus:
		<ol>
			<li>Apply the cold-water bath at 10 &#176;C to the contralateral foot for 2 min. Instruct the participant to rate the pain from the cold-water bath at 0, 30, and 90 s.</li>
		</ol>
		</li>
		<li>Repeating the test stimulus during conditioning stimulus:
		<ol>
			<li>Apply the final Heat-6 stimulus from fine thresholding (2.2.3) to the same hand again during the last 30 s of the cold-water bath. Instruct the participant to rate the pain from Heat-6 at the end of the 30-second heat stimulus, which is also the end of the 120 s cold bath. 
			NOTE: CPM is computed as the difference in the pain rating of the testing stimulus reported before and during the cold bath.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Pizzo, P. A., Clark, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alleviating+suffering+101--pain+relief+in+the+United+States.">Alleviating suffering 101--pain relief in the United States.</a> N Engl J Med. 366 (3), 197-199 (2012).</li><li>Backonja, M. 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=Value+of+quantitative+sensory+testing+in+neurological+and+pain+disorders:+NeuPSIG+consensus.">Value of quantitative sensory testing in neurological and pain disorders: NeuPSIG consensus.</a> Pain. 154 (9), 1807-1819 (2013).</li><li>Arendt-Nielsen, L., Yarnitsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+clinical+applications+of+quantitative+sensory+testing+applied+to+skin,+muscles+and+visera.">Experimental and clinical applications of quantitative sensory testing applied to skin, muscles and visera.</a> J Pain. 10 (6), 556-572 (2009).</li><li>Cruz-Almeida, Y., Fillingim, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+quantitative+sensory+testing+move+us+closer+to+mechanism-based+pain+management?.">Can quantitative sensory testing move us closer to mechanism-based pain management?.</a> Pain Med. 15 (1), 61-72 (2014).</li><li>Curatolo, M., Arendt-Nielsen, L., Petersen-Felix, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence,+mechanisms,+and+clinical+implications+of+central+hypersensitivity+in+chronic+pain+after+whiplash+injury.">Evidence, mechanisms, and clinical implications of central hypersensitivity in chronic pain after whiplash injury.</a> Clin J Pain. 20 (6), 469-476 (2004).</li><li>Kong, J. T., Schnyer, R. N., Johnson, K. A., Mackey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+central+mechanisms+of+acupuncture+analgesia+using+dynamic+quantitative+sensory+testing:+a+review.">Understanding central mechanisms of acupuncture analgesia using dynamic quantitative sensory testing: a review.</a> Evid Based Complement Alternat Med. 2013, 187182 (2013).</li><li>Price, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+second+pain+and+flexion+reflexes+indicative+of+prolonged+central+summation.">Characteristics of second pain and flexion reflexes indicative of prolonged central summation.</a> Exp. Neurol. 37 (2), 371-387 (1972).</li><li>Price, D. D., Hu, J. W., Dubner, R., Gracely, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+suppression+of+first+pain+and+central+summation+of+second+pain+evoked+by+noxious+heat+pulses.">Peripheral suppression of first pain and central summation of second pain evoked by noxious heat pulses.</a> Pain. 3 (1), 57-68 (1977).</li><li>Arendt-Nielsen, L., Petersen-Felix, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wind-up+and+neuroplasticity:+is+there+a+correlation+to+clinical+pain?.">Wind-up and neuroplasticity: is there a correlation to clinical pain?.</a> Eur J Anaesthesiol Suppl. 10, 1-7 (1995).</li><li>Eide, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wind-up+and+the+NMDA+receptor+complex+from+a+clinical+perspective.">Wind-up and the NMDA receptor complex from a clinical perspective.</a> Eur J Pain. 4 (1), 5-15 (2000).</li><li>Kong, J. T., Johnson, K. A., Balise, R. R., Mackey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test-retest+reliability+of+thermal+temporal+summation+using+an+individualized+protocol.">Test-retest reliability of thermal temporal summation using an individualized protocol.</a> J Pain. 14 (1), 79-88 (2013).</li><li>Le Bars, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+whole+body+receptive+field+of+dorsal+horn+multireceptive+neurones.">The whole body receptive field of dorsal horn multireceptive neurones.</a> Brain Res Brain Res Rev. 40 (1-3), 29-44 (2002).</li><li>Pud, D., Granovsky, Y., Yarnitsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methodology+of+experimentally+induced+diffuse+noxious+inhibitory+control+(DNIC)-like+effect+in+humans.">The methodology of experimentally induced diffuse noxious inhibitory control (DNIC)-like effect in humans.</a> Pain. 144 (1-2), 16-19 (2009).</li><li>Yarnitsky, 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=Recommendations+on+terminology+and+practice+of+psychophysical+DNIC+testing.">Recommendations on terminology and practice of psychophysical DNIC testing.</a> Eur J Pain. 14 (4), 339 (2010).</li><li>Anderson, R. J., 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+summation+of+second+pain:+Variability+in+responses+to+a+fixed+protocol.">Temporal summation of second pain: Variability in responses to a fixed protocol.</a> Eur J Pain. , (2012).</li><li>Wilson, H., Carvalho, B., Granot, M., Landau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+stability+of+conditioned+pain+modulation+in+healthy+women+over+four+menstrual+cycles+at+the+follicular+and+luteal+phases.">Temporal stability of conditioned pain modulation in healthy women over four menstrual cycles at the follicular and luteal phases.</a> Pain. 154 (12), 2633-2638 (2013).</li><li>Backonja, M. 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=Quantitative+sensory+testing+in+measurement+of+neuropathic+pain+phenomena+and+other+sensory+abnormalities.">Quantitative sensory testing in measurement of neuropathic pain phenomena and other sensory abnormalities.</a> Clin J Pain. 25 (7), 641-647 (2009).</li><li>Price, D. D., Dubner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+first+and+second+pain+in+the+peripheral+and+central+nervous+systems.">Mechanisms of first and second pain in the peripheral and central nervous systems.</a> J Invest Dermatol. 69 (1), 167-171 (1977).</li><li>Van Hees, J., Gybels, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nociceptor+activity+in+human+nerve+during+painful+and+non+painful+skin+stimulation.">nociceptor activity in human nerve during painful and non painful skin stimulation.</a> J Neurol Neurosurg Psychiatry. 44 (7), 600-607 (1981).</li><li>Treede, R. D., Meyer, R. A., Raja, S. N., Campbell, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+two+different+heat+transduction+mechanisms+in+nociceptive+primary+afferents+innervating+monkey+skin.">Evidence for two different heat transduction mechanisms in nociceptive primary afferents innervating monkey skin.</a> J Physiol. 483 (Pt 3), 747-758 (1995).</li><li>Mauderli, A. P., Vierck, C. J., Cannon, R. L., Rodrigues, A., Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+skin+temperature+and+temporal+summation+of+heat+and+cold+pain.">Relationships between skin temperature and temporal summation of heat and cold pain.</a> J Neurophysiol. 90 (1), 100-109 (2003).</li><li>Granot, M., Granovsky, Y., Sprecher, E., Nir, R. R., Yarnitsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+heat-evoked+temporal+summation:+tonic+versus+repetitive-phasic+stimulation.">Contact heat-evoked temporal summation: tonic versus repetitive-phasic stimulation.</a> Pain. 122 (3), 295-305 (2006).</li><li>Eisenberg, E., Midbari, A., Haddad, M., Pud, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+the+analgesic+effect+to+oxycodone+by+'static'+and+'dynamic'+quantitative+sensory+testing+in+healthy+subjects.">Predicting the analgesic effect to oxycodone by 'static' and 'dynamic' quantitative sensory testing in healthy subjects.</a> Pain. 151 (1), 104-109 (2010).</li><li>Granovsky, Y., Yarnitsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+pain+medicine:+the+clinical+value+of+psychophysical+assessment+of+pain+modulation+profile.">Personalized pain medicine: the clinical value of psychophysical assessment of pain modulation profile.</a> Rambam Maimonides Med J. 4 (4), e0024 (2013).</li><li>Yarnitsky, D., Granot, M., Nahman-Averbuch, H., Khamaisi, M., Granovsky, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+pain+modulation+predicts+duloxetine+efficacy+in+painful+diabetic+neuropathy.">Conditioned pain modulation predicts duloxetine efficacy in painful diabetic neuropathy.</a> Pain. 153 (6), 1193-1198 (2012).</li><li>van Wijk, G., Veldhuijzen, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective+on+diffuse+noxious+inhibitory+controls+as+a+model+of+endogenous+pain+modulation+in+clinical+pain+syndromes.">Perspective on diffuse noxious inhibitory controls as a model of endogenous pain modulation in clinical pain syndromes.</a> J Pain. 11 (5), 408-419 (2010).</li><li>Granot, 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=Determinants+of+endogenous+analgesia+magnitude+in+a+diffuse+noxious+inhibitory+control+(DNIC)+paradigm:+do+conditioning+stimulus+painfulness,+gender+and+personality+variables+matter?.">Determinants of endogenous analgesia magnitude in a diffuse noxious inhibitory control (DNIC) paradigm: do conditioning stimulus painfulness, gender and personality variables matter?.</a> Pain. 136 (1-2), 142-149 (2008).</li><li>Raphael, K. G., Janal, M. N., Anathan, S., Cook, D. B., Staud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+summation+of+heat+pain+in+temporomandibular+disorder+patients.">Temporal summation of heat pain in temporomandibular disorder patients.</a> J Orofac Pain. 23 (1), 54-64 (2009).</li><li>Robinson, M. E., Bialosky, J. E., Bishop, M. D., Price, D. D., George, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supra-threshold+scaling,+temporal+summation,+and+after-sensation:+relationships+to+each+other+and+anxiety/fear.">Supra-threshold scaling, temporal summation, and after-sensation: relationships to each other and anxiety/fear.</a> J Pain Res. 3, 25-32 (2010).</li><li>Bernaba, M., Johnson, K. A., Kong, J. T., Mackey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+pain+modulation+is+minimally+influenced+by+cognitive+evaluation+or+imagery+of+the+conditioning+stimulus.">Conditioned pain modulation is minimally influenced by cognitive evaluation or imagery of the conditioning stimulus.</a> J Pain Res. 7, 689-697 (2014).</li></ol>

The authors have no conflicts of interests or any financial interests to disclose.

Critical Steps within the Protocol
The TS protocol includes the following in key steps in chronological order: multi-step training (using the visual analog scale to rate pain, rating of second pain from a single heat pulse, and rating second pain from rapid heat pulse trains); optimization of pulse temperatures; obtain TS in 2-3 trials with the optimized temperatures. As with most psychophysical measures, participant training is extremely critical to ensure that pain ratings are consistent across trials and are as accurate as possible. The optimization step is equally important, where both the baseline and peak pulse temperatures are adjusted such that the rating of the first heat pulse is less than 5/10, and the approximated TS is between 3 to 7.
The key steps of CPM include training of pain rating on visual analog scale, obtaining Heat-6 from slow heat ramps, confirming Heat-6 and fine thresholding if necessary, applying a cold bath to contralateral distal extremity and reapplying confirmed Heat-6 during the last 30 s of cold bath. Similar to the TS protocol, both the training and the individualization of the heat stimulus (Heat-6) are critical in the CPM protocol. Additionally, from experience as well as from the literature, repeating the Heat-6 stimulus during the last 30 s of the cold bath is critical and yields a greater magnitude of CPM compared to applying the heat stimulus after the cold bath.26 However, given that some individuals cannot tolerate the full 2 min of cold pressor at 10 degree Celsius, it might be reasonable to consider applying the testing stimulus immediately after the completion of the conditioning stimulus to standardize data collection across all individuals.
Modifications and Troubleshooting
The most common problem with the TS protocol is the inability to obtain TS, which can be due to 3 main causes. First, and most commonly, the pain rating from the first heat pulse may be so strong that it overwhelms the perception of any increase in pain with subsequent pulses (TS). The best way to minimize this problem is to follow the protocol and sequentially decrease the baseline and peak stimulus temperature until the pain rating of the first pulse is less than 5 (out of 10) before optimizing the magnitude of TS. The second cause, opposite to the first one, is when the participant perceives no pain whatsoever at the end of the 10 pulses even at the highest temperature settings. In such situations, one may consider increasing the baseline pulse temperature by 1 or 2&#160;&#176;C. Occasionally, an individual may simply have a hard time discerning and rating second pain, possibly due to both peripheral and central factors. Without reliable perception of second pain, it is very difficult to capture TS. In such situations, we find the best set of the temperatures that an individual can tolerate and record TS as zero.
The most common barriers to a successful CPM protocol are the instability of Heat-6 and the inability to tolerate a cold bath (10&#160;&#176;C) for 2min. Use the fine thresholding in the current protocol to address the first problem by adjusting heat stimulus temperature step-wise until the pain rating is between 5 and 7. For the second issue, note that the literature suggests the inhibitory effect from the conditioning stimulus is saturable.27 As such, even if a person cannot keep his or her foot in cold bath for 2 min, a sufficient CPM effect should occur with this intensely painful cold stimulus. Modify the protocol to record the duration of the foot submerged in cold water bath and deliver the heat stimulus immediately after the participant withdraw his or her foot from the cold bath. CPM is then calculated as the pain rating of the heat stimulus before subtracted by the pain rating of the heat stimulus applied immediately after the cold bath (not during, as the general protocol indicates).
Limitations of Technique
This method is not without limitations. First, despite our best effort, we were not able to elicit TS and CPM in every individual (missed 1 participant in TS and 1 in CPM, respectively). This, in part, may be due to the large between-individual variability in these parameters.5,15,16,28,29 However, the success rate was 94%, which was better than the 50-60% success rate quoted from the literature.22,28 Second, researchers should take caution when interpreting between-individual differences in TS generated by this method since we use different heat pulse temperatures to generate TS in each individual. Therefore, when comparing TS in a cross-sectional sample, one should consider both the differences in the magnitude of TS and in the temperatures used to generate it. The individualized TS method is best suited for longitudinal studies where the focus is on the changes in the same individual overtime. The same concern does not apply to the individualized CPM because the same conditioning stimulus is used for all individuals and only the change in the pain perception of individualized Heat-6 is recorded and not the raw score of Heat-6 pain. Although this method allows broad capturing of TS and CPM, it does take more time compared to methods where universal parameters are used. Finally, this technique requires an experienced operator and advanced heat testing machines, both of which are not practical for immediate adaption to busy clinical settings. We encourage future efforts to simplify the methods.
Significance of the Technique with Respect to Existing /Alternative Methods
Our method of individualizing TS and CPM parameters aim to remove influence of floor and ceiling effect due to variations in peripheral heat sensitivity. The methods presented improved on previous methods published by our group with the goals of both broader capturing and time efficiency.11,30 The advantage of individualizing TS and CPM is the ability to capture the state of ascending and descending pain processing in a broad range of individuals, thereby allowing the use of these parameters as a reasonable outcome measure for longitudinal studies.
Future Applications or Directions after Mastering the Technique
Future studies should focus on additional modifications to save time, collecting of TS and CPM data on large populations to characterize the range of these parameters in individuals who are pain free vs those with chronic pain, and on the correlation of the diversity in the TS and CPM response to specific physiologic processes in addition to windup and DNIC.

The International Association for the Study of Pain (IASP) defines pain as &#34;an unpleasant sensory and emotional experience.&#34; Chronic pain refers to pain that persists beyond 6 months. Chronic pain is a significant problem in the United States, affecting more than 100 million American adults at a cost upwards of $635 billion per annum.1 Due to the subjective nature of pain, it is difficult for researchers and clinicians to objectively measure a person&#39;s painful experience(s), therefore making it challenging to assess and treat pain. For this reason, it is important that we develop standardized tests to quantify pain as objectively as possible. One such test is QST, where standardized sensory stimuli are administered to and rated by the test subject.2 There are static and dynamic QST. The former typically assesses the sensory thresholds to or the rating of a single stimulus, while the latter assesses the response to a number of stimuli.3 Recently, dynamic QST has gained increasing attention because it offers the opportunity to probe the central processing of incoming nociceptive signals.4,5,6
Two key components of dynamic QST are temporal summation (TS) and conditioned pain modulation (CPM). Temporal summation refers to the increased perception of pain from repetitive, noxious stimuli. TS is a behavioral correlate of wind-up, the phenomenon where spinal secondary neurons display increased firing due to repetitive c-fiber input.7,8 Generally, TS can be induced by using various noxious stimuli, such as heat, electricity, and tactile methods (i.e. pressure or pinprick), provided that the frequency of the repetitive stimulus is greater than 0.3Hz, the natural frequency of c-fibers.9,10 Many researchers use repetitive heat pulses to generate TS because of the ease in producing and standardizing the noxious heat stimuli.11
CPM refers to the phenomenon of &#34;pain inhibits pain,&#34; where the presence of a second noxious stimulus decreases the pain perception from an initial noxious stimulus.12 The initial noxious stimulus, which is measured before and after (or during) the application of the second stimulus, is referred to as the test stimulus. The test stimulus can be thermal, electrical or tactile. Here thermal stimulus is often used as the testing stimulus because of its ease in adjustment and standardization.13 The second stimulus, called the conditioning stimulus, typically consists of a cold or hot water bath applied to a distal extremity.13 CPM is the behavioral correlate of Diffuse Noxious Inhibitory Control (DNIC), a physiological phenomenon where input from peripheral c-fibers results in diffuse inhibition from the brainstem of all incoming stimuli mediated by c-fiber from heterotopic fields.12,14
While TS and CPM both have the potential to reflect states and changes in central pain processing, limitations exist in both.4,5 For example, because there is a large variability in individuals&#39; sensitivity to heat, the application of a universal stimulus can result in lack of TS in up to 50% of individuals tested.11,15 Similarly, a thermal test stimulus often results in widely different pain ratings that may render CPM test impossible due to floor or ceiling effects.16 Therefore, to capture TS and CPM broadly, a protocol that adjusts the heat stimulus to the individual is needed. For TS, we adjust the heat pulse temperatures so that they can generate adequate increase in pain rating with each successive pulse; while for the CPM, we adjust the thermal test stimulus to moderately painful (6 out 10) for each individual so that adequate pain ratings may still exist after the application of a conditioning stimulus.
For all psychophysical tests, training of the participant in the proper rating of painful stimuli is crucial to the accuracy and reproducibility of these behavioral tests.17 This is particularly relevant for TS when multiple stimuli are presented at a rapid rate and, in the case of CPM, when two different stimuli are applied simultaneously to the participant. Furthermore, for TS from heat pulses, it is especially important to train the participant to rate the c-fiber mediated second pain (slow, burning, usually comes on about 1 second after the heat pulse) and not the first pain (mediated by A-delta fibers and come on immediately with the heat pulse).18,19 This is less an issue in CPM as the stimuli there are much longer (&#62;30 s) and C-fiber mediated sensation would dominate the noxious perception in these situations.19,20 In the protocol below, we will go over proper training of participants in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medoc Pathway CHEPS system</td>
			<td>Medoc Advanced Medical Systems&#160;&#160;</td>
			<td></td>
			<td>This system includes the machine to generate contact heat (Pathway), the thermode capable of rapid temperature change (CHEPS), and the Medoc software.&#160;</td>
		</tr>
		<tr>
			<td>CoVAS accessory hardware with the CHEPS systems</td>
			<td>Medoc Advanced Medical Systems&#160;&#160;</td>
			<td></td>
			<td>This device is a Medoc accessory that allows real-time pain rating by the participant.&#160;</td>
		</tr>
		<tr>
			<td>Laptop Computer</td>
			<td>Lenovo</td>
			<td></td>
			<td>This is the computer that runs the Medoc software and communicates with the Pathway machine.&#160;</td>
		</tr>
		<tr>
			<td>Glove</td>
			<td>Kimberly-Clark</td>
			<td></td>
			<td>Gloves are used to secure the thermode on the participant&#39;s thenar eminence.&#160;</td>
		</tr>
		<tr>
			<td>Clear plastic box with a perforated dividing wall - filled with Ice and water</td>
			<td></td>
			<td></td>
			<td>This box provides the cold water bath for the CPM task.&#160;</td>
		</tr>
		<tr>
			<td>Aquarium pump&#160;</td>
			<td>Aquarium Systems Micro-Jet pump MC 450</td>
			<td></td>
			<td>This pump circulates water, to maintain stable, even temperature in the cold water bath.&#160;</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Exergen Temporal Scanner, model TAT 2000&#160;</td>
			<td></td>
			<td>To monitor constantly the temperature of the water bath.&#160;</td>
		</tr>
		<tr>
			<td>Stop Watch</td>
			<td>Any handheld stop watch or stop watch built into a smartphone</td>
			<td></td>
			<td>To prompt the participant to rate pain at specific time pointds during the CPM task.&#160;</td>
		</tr>
	</tbody>
</table>

dynamic quantitative sensory testing to characterize central pain processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

In an on-going clinical trial where we performed deep phenotyping of patients with chronic axial low back pain, we included dynamic QST as an integral part of the assessment. Table 3 below summarizes baseline data from the first 15 patients where the exact TS and CPM protocol above was used. Note that the data includes patient #19 because not all 19 recruited patients showed up for their evaluation due to scheduling conflicts and other circumstance. Figure 5 visually displays the TS and CPM data side-by-side to reveal patterns of central pain processing changes in these patients.
As shown above, using an individualized optimization protocol, we obtained TS at the thenar eminence with an average of 2.7 on a 0-10 VAS scale. We were able to obtain TS in all 19 participants except for Participant 1, who was not able to reliably identify second pain without being overwhelmed by sensation from the first pain. Participant 13 needed to leave early thus did not undergo the CPM task. Otherwise all 18 participants demonstrated CPM, the average of which is about 3.1. We were successful in achieving some degree of TS and CPM in most of the subjects; and the magnitude of the TS and CPM is consistent with that from the literature.13,15,22
Furthermore, as demonstrated in Figure 4, the simultaneous measurement of TS and CPM can lead to insight on a patient&#39;s profile in central pain processing. To put it in simplified terms, high TS may indicate abnormally augmented ascending facilitation while a low CPM suggests impaired descending inhibition of nociceptive transmission. For example, Participant 10, showed high TS of 7.7 (out of 10), while Participant 18 demonstrated an essentially absent CPM (-0.5/10). Several pioneer studies have shown that such different profiles of central pain processing may predict different rates of development of chronic pain after surgery, and different responses to drugs that act on specific central pain pathways.4,23,24,25 In this example here, it is reasonable to speculate that Participant 10 might respond to Gabapentin, a calcium channel blocker while Participant 18, who has impaired CPM might respond to Duloxetine, a serotonin-norepinephrine reuptake inhibitor. Clearly more data and studies are needed to verify the hypotheses. The interventional trial is ongoing and will assess the response of these back-pain patients to verum and sham electroacupuncture. The individualized QST method presented here would allow us to accurately and longitudinally track the changes in TS and CPM in as many individuals as possible.
<img alt="Figure 1" src="/files/ftp_upload/54452/54452fig1.jpg" /> 
Figure 1. Single Heat Pulses Used in Training Trial 1. Each heat pule lasts 0.5 s and is 10 s apart from the next heat pulse. The baseline and peak temperatures of each pulse gradually increase according to Table 2. As soon as the participant perceives second pain from any of the pulses, the endpoint of training trial 1 is reached and the temperature settings of the pulse that leads to perception of second pain are recorded and used in Training Trial 2. <a href="http://cloudflare.jove.com/files/ftp_upload/54452/54452fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54452/54452fig2.jpg" /> 
Figure 2. Heat Pulse Train Used in Training Trial 2, Optimization and Final TS Trials. Ten heat pulses, each 0.5 s long, and 2 s apart, is delivered in a standard TS trial.
<img alt="Figure 3" src="/files/ftp_upload/54452/54452fig3.jpg" /> 
Figure 3. Defining P1, Pmax and TSE. P1: rating of the second pain from the first pulse; Pmax: maximum rating of second pain of the entire 10-pulse train; TSE: estimated magnitude of temporal summation. <a href="http://cloudflare.jove.com/files/ftp_upload/54452/54452fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54452/54452fig4.jpg" /> 
Figure 4. Algorithms to Individually Optimize Temporal Summation. The objective here is to find baseline and peak pulse temperatures that result in estimated temporal summation (TSE) between 3 and 7 out 10 VAS. If TSE is below goal, increase peak and baseline temperatures by 1 &#176;C sequentially. If TSE is above goal, decrease baseline and peak temperatures by 1 &#176;C sequentially. Before the above algorithm, make sure pain rating of the first heat pulse is &#8804; 5 by decreasing the baseline and peak pulse temperatures by small (0.5-1 &#176;C) increments. <a href="http://cloudflare.jove.com/files/ftp_upload/54452/54452fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54452/54452fig5.jpg" /> 
Figure 5. TS and CPM Profiles in Patients from an Ongoing Clinical Trial. Baseline measurement of TS and CPM in the first 19 patients with chronic low back pain from an ongoing clinical trial. The various patterns of relative TS and CPM magnitude reveals potential differences in central pain processing. <a href="http://cloudflare.jove.com/files/ftp_upload/54452/54452fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Trial</td>
			<td># of pulses</td>
			<td>Stimulus Duration</td>
			<td>Peak to peak ISI</td>
			<td>Ramp Rate</td>
			<td>Baseline Temp</td>
			<td>Max Temp</td>
		</tr>
		<tr>
			<td>Training Trial 1</td>
			<td>1 pulse</td>
			<td>0.5 s</td>
			<td>10 s</td>
			<td>40 &#176;C/s</td>
			<td>Variable (see Table 2)</td>
			<td>Variable (see Table 2)</td>
		</tr>
		<tr>
			<td>Training Trial 2</td>
			<td>10 pulses</td>
			<td>0.5 s</td>
			<td>2 s</td>
			<td>40 &#176;C/s</td>
			<td>from Training Trial 1</td>
			<td>from Training Trial 1</td>
		</tr>
		<tr>
			<td>Optimization Trials</td>
			<td>10 pulses</td>
			<td>0.5 s</td>
			<td>2 s</td>
			<td>40 &#176;C/s</td>
			<td>start with temps from Training Trial 1</td>
			<td>start with temps from Training Trial 1</td>
		</tr>
		<tr>
			<td>Final Trials&#160;</td>
			<td>10 pulses</td>
			<td>0.5 s</td>
			<td>2 s</td>
			<td>40 &#176;C/s</td>
			<td>Individualized</td>
			<td>Individualized&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Specifications of Heat Stimuli Used in the TS Protocol (including the training trials).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pulse Number</td>
			<td>Baseline Temperature (&#176;C)</td>
			<td>Peak Temperature (&#176;C)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>36</td>
			<td>45</td>
		</tr>
		<tr>
			<td>2</td>
			<td>36</td>
			<td>46</td>
		</tr>
		<tr>
			<td>3</td>
			<td>37</td>
			<td>47</td>
		</tr>
		<tr>
			<td>4</td>
			<td>38</td>
			<td>48</td>
		</tr>
		<tr>
			<td>5</td>
			<td>39</td>
			<td>49</td>
		</tr>
		<tr>
			<td>6</td>
			<td>40</td>
			<td>50</td>
		</tr>
		<tr>
			<td>7</td>
			<td>41</td>
			<td>51</td>
		</tr>
		<tr>
			<td>8</td>
			<td>42</td>
			<td>51</td>
		</tr>
		<tr>
			<td>9</td>
			<td>43</td>
			<td>51</td>
		</tr>
		<tr>
			<td>10</td>
			<td>44</td>
			<td>51</td>
		</tr>
	</tbody>
</table>
Table 2: Temperature Settings Used to Capture Second Pain in Training Trial 1. Refer to Figure 1 for shape of single heat pulses delivered with these temperature parameters.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Subj ID</td>
			<td>Base T</td>
			<td>Peak T</td>
			<td>TS</td>
			<td>Heat-6</td>
			<td>CPM</td>
		</tr>
		<tr>
			<td></td>
			<td>Celsius</td>
			<td>Celsius</td>
			<td>VAS</td>
			<td>Celsius</td>
			<td>VAS</td>
		</tr>
		<tr>
			<td>1</td>
			<td></td>
			<td></td>
			<td>0</td>
			<td>44</td>
			<td>4.4</td>
		</tr>
		<tr>
			<td>2</td>
			<td>41</td>
			<td>51</td>
			<td>0.43</td>
			<td>47.6</td>
			<td>4.717</td>
		</tr>
		<tr>
			<td>3</td>
			<td>40</td>
			<td>50</td>
			<td>0.5</td>
			<td>44.7</td>
			<td>4.42</td>
		</tr>
		<tr>
			<td>4</td>
			<td>38</td>
			<td>49</td>
			<td>0.45</td>
			<td>44</td>
			<td>4.355</td>
		</tr>
		<tr>
			<td>5</td>
			<td>42</td>
			<td>51</td>
			<td>3.287</td>
			<td>44</td>
			<td>4.0713</td>
		</tr>
		<tr>
			<td>6</td>
			<td>44</td>
			<td>51</td>
			<td>0.415</td>
			<td>45.5</td>
			<td>4.5085</td>
		</tr>
		<tr>
			<td>7</td>
			<td>44</td>
			<td>51</td>
			<td>4.2495</td>
			<td>45.5</td>
			<td>4.12505</td>
		</tr>
		<tr>
			<td>8</td>
			<td>41</td>
			<td>51</td>
			<td>2.575</td>
			<td>45</td>
			<td>4.2425</td>
		</tr>
		<tr>
			<td>9</td>
			<td>44</td>
			<td>51</td>
			<td>3.435</td>
			<td>48</td>
			<td>4.4565</td>
		</tr>
		<tr>
			<td>10</td>
			<td>43</td>
			<td>51</td>
			<td>7.713</td>
			<td>44</td>
			<td>3.6287</td>
		</tr>
		<tr>
			<td>11</td>
			<td>39</td>
			<td>51</td>
			<td>4.59</td>
			<td>42.5</td>
			<td>3.791</td>
		</tr>
		<tr>
			<td>12</td>
			<td>39</td>
			<td>49</td>
			<td>5.395</td>
			<td>43.3</td>
			<td>3.7905</td>
		</tr>
		<tr>
			<td>13</td>
			<td>44</td>
			<td>51</td>
			<td>2.165</td>
			<td></td>
			<td>0</td>
		</tr>
		<tr>
			<td>14</td>
			<td>39</td>
			<td>49</td>
			<td>2.55</td>
			<td>44.1</td>
			<td>4.155</td>
		</tr>
		<tr>
			<td>15</td>
			<td>40</td>
			<td>51</td>
			<td>4.0635</td>
			<td>44</td>
			<td>3.99365</td>
		</tr>
		<tr>
			<td>16</td>
			<td>42</td>
			<td>51</td>
			<td>3.45665</td>
			<td>45.5</td>
			<td>4.204335</td>
		</tr>
		<tr>
			<td>17</td>
			<td>40</td>
			<td>51</td>
			<td>0.953</td>
			<td>46.1</td>
			<td>4.5147</td>
		</tr>
		<tr>
			<td>18</td>
			<td>37.5</td>
			<td>49</td>
			<td>0.21</td>
			<td>45.5</td>
			<td>4.529</td>
		</tr>
		<tr>
			<td>19</td>
			<td>40</td>
			<td>51</td>
			<td>2.2135</td>
			<td>46.6</td>
			<td>4.43865</td>
		</tr>
	</tbody>
</table>
Table 3. Results of TS and CPM from an Ongoing Clinical Trial. See Figure 4 for the TS and CPM from the same participants graphed side by side to reveal pain modulation profiles.

Watch this Scientific Journal Video about Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing at JoVE.com

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

dynamic-quantitative-sensory-testing-to-characterize-central-pain

Research

JoVE Journal

Neuroscience

16.8K Views.  Stanford University School of Medicine. By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

Video: Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs&#39; sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.
Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs&#39; behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...