The authors would like to thank Dr. Jhi-Joung Wang, who is the Vice Superintendent of Education at Chi-Mei Medical Center, and Dr. Chih-Chan Lin from the Laboratory Animal Center, Department of Medical Research, Chi-Mei Medical Center, 901 Zhonghua, Yongkang Dist., Tainan City 701, Taiwan, for providing the shooting venue. They would also like to thank Miss Ling-Yu Tang and Mr. Chung-Ham Wang from the Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan, for their valuable assistance in demonstrating the prototype system in real experiments with rats. The author gratefully acknowledges the support provided for this study by the Ministry of Science and Technology (MOST 104- 2218-E-167-001-) of Taiwan.

Ethics Statement: The experimental procedures were approved by the animal ethics committee of Southern Taiwan University of Science and Technology Laboratory Animal Center, National Science Council, Republic of China (Tainan, Taiwan).
1. Constructing the Running Wheel Structure
NOTE: All acrylic should be transparent.&#160;Wash the disassembled wheel with water, then use alcohol to wipe the rubber track and acrylic sheets after each use.&#160;&#160;
<ol>
	<li>Obtain an acrylic running wheel to be 55 cm in diameter and 15 cm in width. 
	NOTE: This wheel is larger than a traditional running wheel (diameter = 35 cm; width = 12 cm) (Figure 1A).</li>
	<li>Using a cutter, cut a quarter-circle opening into one side of the running wheel to act as an entrance and an exit as well for rats (Figure 1B). Place a layer of high-friction rubber track on the inside of the acrylic wheel (Figure 1B).</li>
	<li>Place an iron rod with bearings to connect the running wheel (Figure 1B). Place two acrylic triangular columns on either side of the running wheel to act as the support frame (Figure 1B).</li>
	<li>Attach a 1 mm thick semicircular, transparent acrylic sheet to the external sides of the two triangular columns using screws. Use this sheet to deploy the infrared sensors. Ensure that the acrylic sheets are approximately 3 cm away from each side of the running wheel.</li>
</ol>
2. Deploying the Infrared Sensors and Defining the Effective Exercise Area
NOTE: Take into account the running wheel size and the rat length in the design of an infrared system. A rat only triggers a single sensor at a time. In this experiment, rats are between 20 and 23 cm long.
<ol>
	<li>Drill a hole in the acrylic sheets every 45&#176; (arc interval = 21 cm), with the distance between two holes being roughly equivalent to the length of a test rat. Make the holes the same size as the infrared sensors (Figure 2A). 
	NOTE: For traditional MRWs, drill a hole every 70&#176; (arc interval = 21 cm, Figure 2B).</li>
	<li>During a PRW experiment, maintain the rats at a steady state of running between 0&#176; to 135&#176;. 
	NOTE: Thus define this area as the effective exercise area, while view all the other sections as the ineffective exercise areas. For traditional MRWs, define the effective exercise area as the portion between 0&#176; to 140&#176; (Figure 2B).</li>
</ol>
3. Driving the Running Wheel
<ol>
	<li>Use a brushless DC motor and a motor driver to drive the running wheel.</li>
	<li>Mount a 10 cm diameter rubber disk on the motor&#8217;s central axis (Figure 1B).</li>
	<li>Using the iron frame and springs to support the motor, connect the rubber disk from the motor&#8217;s central axis to the outer side of the running wheel. 
	NOTE: The springs must cooperate with the screws to allow dynamic motor height adjustments and to prevent rubber disk from being disconnected to the running wheel track because of loose springs.</li>
	<li>Operate the motor to drive the 10 cm diameter rubber disk using a microcontroller, and observe the wheel rotate due to the friction between the rubber disk and the runway of wheel, creating a motorized running wheel platform.</li>
	<li>Mount four infrared sensors sequentially between 0&#176; to 135&#176; (Figure 2A). 
	NOTE: For traditional MRWs, mount sensors between 0&#176; to 140&#176; (Figure 2B).</li>
	<li>Connect four pairs of infrared sensors mounted in both acrylic sheets to the microcontroller&#8217;s general pins using single core cables, thereby forming a positioning running wheel system.</li>
</ol>
4. Constructing an Adaptive Acceleration Curve
<ol>
	<li>Three days before the commencement of official 3 week exercise training, train rats by manually operating the running wheel. 
	NOTE: The goal is to allow the rats to become familiar with the running environment, and is to test whether each rat can endure running at 20 m/min.
	<ol>
		<li>During the manually operated training, gradually accelerate the running speed until a rat is unable to keep pace. When this occurs, decrease the speed until the rat regains a steady running pace, and then gradually increase the speed again until the rat reaches 20 m/min (dashed lines in Figure 3). The manual training involves seven rats to construct the training curves.</li>
	</ol>
	</li>
	<li>Using a numerical equation to fit the measured data on Day 3 of the manual testing, calculate the acceleration curves closest to the manual training (the curve with circles, Figure 3). Fit Equation 1 to raw data, where Cini = 8, Cfin = 20 and &#964; = 30 represent the initial velocity, the final velocity and the time constant, respectively). 
	NOTE: This equation adapts to a rat&#8217;s body condition. Therefore, refer to the calculated curve as an adaptive acceleration-training model. 
	<img alt="Equation 1" src="/files/ftp_upload/54354/54354eq1.jpg" />&#160; (1)</li>
	<li>Use Equation 1 for Week 1 of the formal training.</li>
	<li>For Weeks 2 and 3 of the training, adjust the parameters of Equation 1, that is to say, change A from 12 to 22, to allow the speed to reach 30 m/min.</li>
</ol>
5. Controlling the Software Program
NOTE: Exclusively develop a code for the microcontroller-based motor operation and for signal transmission from the infrared sensors to a computer for subsequent data analysis.
<ol>
	<li>Use C programming language to write a software control program containing one main program and two interrupt-service routines for the timer in the microcontroller18.
	<ol>
		<li>Ensure that the main program initializes the microcontroller&#8217;s register and constructs an adaptive acceleration curve model in the microcontroller&#8217;s memory.</li>
		<li>Use the interrupt-service routine of Timer 0 to activate the adaptive acceleration curve and calculate the entire training duration.</li>
		<li>Use the interrupt-service routine of Timer 1 to extract the signal data from the infrared sensors and to transfer the data to the computer.</li>
		<li>Use the main program to record the position of 0o to adjust the speed of running wheel.</li>
	</ol>
	</li>
	<li>Once the receiving IR sensor at 0&#176; is triggered, interpret it as a fall incidence, which is accumulated by the main program. The moment the occurrence times of fall incidences hits the 10% threshold of the number of the rat position detections, deaccelerate the running wheel automatically as a safety measure for trained rats.&#160;NOTE: The speed of running wheel is reduced until the rat can return to the safe zone (0o to 135o) and maintain a stable running state for the safety measure.</li>
</ol>
6. Operating the Positioning Running Wheel System
<ol>
	<li>Turn on the microcontroller and wait for an operator to press the button to start each week&#8217;s training model.
	<ol>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 1. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 20 m/min, and automatically stops after 30 min.</li>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 2. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 30 m/min, and automatically stops after 30 min.</li>
		<li>Press the &#8220;Start&#8221; button to begin the training model for Week 3. 
		NOTE: The motor automatically accelerates based on the adaptive acceleration curve until it reaches 30 m/min, and automatically stops after 60 min. 
		NOTE: Throughout the entire training process, transmit the signal data received from the infrared sensors to a computer wirelessly.</li>
	</ol>
	</li>
	<li>Using a computer, analyze the location data to obtain an effective exercise measure for the entire exercise process (<img alt="Equation 2" src="/files/ftp_upload/54354/54354eq2.jpg" />). See Equation 2. 
	<img alt="Equation 3" src="/files/ftp_upload/54354/54354eq3.jpg" />&#160; (2) 
	NOTE: EEE, EED and IED represent the effective exercise measure, the effective and ineffective exercise durations, respectively.</li>
</ol>
7. Training the Rats
<ol>
	<li>Randomly divide adult male Sprague-Dawley rats into five groups (n = 9 for each group): the sham, control, treadmill, MRW and PRW groups.</li>
	<li>Conduct a 3 week exercise training for the three exercise groups, i.e. the treadmill, MRW and PRW groups, while do not for the sham and the control groups. 
	NOTE: The 3 week exercise training for each exercise group is 20 m/min for 30 min during Week 1, 30 m/min for 30 min during Week 2 and 30 m/min for 60 min during Week 3.</li>
</ol>
8. Animal and Stroke Model
<ol>
	<li>As stated in Sec. 7.1, randomly divide all the involved adult male Sprague-Dawley rats, weighing between 250-280 g, into 5 groups.</li>
	<li>Weigh all the animals to ensure accurate drug dosage calculations. Anesthetize the rats with sodium pentobarbital (25 mg/kg, intraperitoneally [i.p.]) and a mixture containing ketamine (4.4 mg/ kg, intramuscularly [i.m.]), atropine (0.02633 mg/kg, [i.m.]) and xylazine (6.77 mg/kg, [i.m.]).
	<ol>
		<li>Assess the anesthetic depth by monitoring the breathing rate (normal 70-115 breaths/min), rhythm, depth of respiration, mucous membrane color and regular testing of reflexes, e.g. toe pinch, tail pinch, eyelid/eyelash and palpebral.</li>
	</ol>
	</li>
	<li>Insert temperature probes into the rectum, and maintain the rectal temperatures between 37 to 37.5 &#176;C using separate heating lamps.</li>
	<li>Induce focal ischemia, transient middle cerebral artery occlusion (MCAo) by inserting a filament into the internal carotid artery to occlude the orifice of the middle cerebral artery via external carotid artery approach19.
	<ol>
		<li>Perform the same operating procedures on the sham-operated animals, while do not insert a filament into the internal carotid artery. Maintain focal cerebral ischemia for 1 hr, remove the filament, close the incision, and then leave 1 cm of the nylon suture protruding, which could be withdrawn to allow reperfusion.</li>
	</ol>
	</li>
	<li>Administer subcutaneous (s.c.) injections of an analgesic (buprenorphine (0.05 mg/kg, s.c.)), into the animals for analgesia twice a day for 3 days.</li>
</ol>
9. Assessing Neural Damage
<ol>
	<li>Evaluate neurological and motor functions, respectively, by the neurological severity score (mNSS)20 and an inclined plane test21. 
	NOTE: mNSS is a composite of the motor (muscle status, abnormal movement), sensory (visual, tactile and proprioceptive) and reflex tests. Give one point for failure to perform a task. Rate neurological function on a scale of 0-18 (normal score = 0; maximal deficit score = 18).</li>
	<li>Assess all the rats in terms of behavioral performance the day before and daily over a time span of 7 days after the surgery.</li>
	<li>Measure the hind limb grip strength of the rat using an inclined plane.
	<ol>
		<li>Place rats on the inclined climbing apparatus on a daily basis, and acclimatize the rats to the apparatus and the testing conditions 1 week before testing.</li>
		<li>Place each rat on the apparatus and encourage the rat to climb the platform until the top of the apparatus during the acclimatization period.</li>
		<li>Place the rat at the top of the apparatus with the head downward during testing. Ensure that the body axis of the rat stays along to a 20 x 20 cm2 rubber ribbed surface on the inclined plane starting at an angle of 25&#176;.</li>
		<li>Increase the angle dynamically using a ball screw connected with a stepper motor to determine the maximal angle at which an animal could hold to the plane. Increase the angle of the inclined plane gradually until the mouse failed to hold on the inclined plane, and then detect a sliding down event. The baseline degree of inclined plane is 25&#176; in the beginning.</li>
		<li>Ask two observers (unaware of what treatment the rats had been given), to independently examine and score all the behavioral tests, i.e. the mean of the left- and right-side maximal angles.</li>
	</ol>
	</li>
	<li>Sacri&#64257;ce all the animals on day 7 after MCAo. Perfuse the hearts of the animals under deep anesthesia (sodium pentobarbital 100 mg/kg, i.p.) with saline22. Remove and then immerse the brain tissue in cold saline for 5 min, which is then sliced into 2.0 mm coronal sections using a tissue slicer22.</li>
	<li>Submerge the fresh brain sections into 2, 3, 5-triphenyltetrazolium chloride (TTC) at 37 &#176;C for 30 min, then transfer the slices to 5% formaldehyde solution for &#64257;xation at 4 &#176;C for 24 hr. Place the stained brain slices on a plexiglas holder.</li>
	<li>Photograph the TTC-stained slices with a calibrated scale using a CCD camera linked to a personal computer loaded with image processing software. Use a semi-automated image analysis system as well to estimate the infarcted area (mm2) from each TTC-stained brain slice23.</li>
	<li>Calculate the total infarct volume for each slice by summation of infarcted areas of all brain slices. Mark the unstained area (ischemic brain) separately on each side of 2 mm thick slices, then calculate the infarct volume and the mean value.</li>
	<li>Calculate the corrected infarct volume (CIV) as 
	CIV= {LT- (RT- RI)} <img alt="Equation 4" src="/files/ftp_upload/54354/54354eq4.jpg" /> d (3) 
	NOTE: Where LT and RT denote the areas of the left and right hemispheres in mm2, respectively, RI is the infarcted area in mm2, and d = 2 mm is the slice thickness.</li>
</ol>

<ol>
	<li>Mayo, N. E., Wood-Dauphinee, S., Cote, R., Durcan, L., Carlton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity,+participation,+and+quality+of+life+6+months+poststroke.">Activity, participation, and quality of life 6 months poststroke.</a> Arch Phys Med Rehabil. 83 (8), 1035-1042 (2002).</li><li>Duncan, P. W., Goldstein, L. B., Horner, R. D., Landsman, P. B., Samsa, G. P., Matchar, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+motor+recovery+of+upper+and+lower-extremities+after+stroke.">Similar motor recovery of upper and lower-extremities after stroke.</a> Stroke. 25 (6), 1181-1188 (1994).</li><li>Raichlen, D. A., Gordon, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+exercise+capacity+and+brain+size+in+mammals.">Relationship between exercise capacity and brain size in mammals.</a> PLoS One. 6 (6), (2011).</li><li>Trejo, J. L., Carro, E., Torres-Aleman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+insulin-like+growth+factor+I+mediates+exercise-induced+increases+in+the+number+of+new+neurons+in+the+adult+hippocampus.">Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus.</a> J Neurosci. 21 (5), 1628-1634 (2001).</li><li>Zhang, F., Wu, Y., Jia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+preconditioning+and+brain+ischemic+tolerance.">Exercise preconditioning and brain ischemic tolerance.</a> Neuroscience. 177, 170-176 (2011).</li><li>Wang, R. Y., Yang, Y. R., Yu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+treadmill+training+on+infarction+in+rats.">Protective effects of treadmill training on infarction in rats.</a> Brain Res. 922 (1), 140-143 (2001).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+pre-conditioning+reduces+brain+damage+in+ischemic+rats+that+may+be+associated+with+regional+angiogenesis+and+cellular+overexpression+of+neurotrophin.">Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin.</a> Neuroscience. 124 (3), 583-591 (2004).</li><li>Li, J., Luan, X. D., Clark, J. C., Rafols, J. A., Ding, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+against+transient+cerebral+ischemia+by+exercise+pre-conditioning+in+rats.">Neuroprotection against transient cerebral ischemia by exercise pre-conditioning in rats.</a> Brain Res. 26 (4), 404-408 (2004).</li><li>Leasure, J. L., Jones, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+and+voluntary+exercise+differentially+affect+brain+and+behavior.">Forced and voluntary exercise differentially affect brain and behavior.</a> Neuroscience. 156 (3), 456-465 (2008).</li><li>Chen, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Forced+running+wheel+system+with+a+microcontroller+that+provides+high-intensity+exercise+training+in+an+animal+ischemic+stroke+model.">A Forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model.</a> Braz J Med Biol Res. 47 (10), 858-868 (2014).</li><li>Chen, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+infrared-sensing+running+wheel+systems+with+an+effective+exercise+activity+indicator.">Improved infrared-sensing running wheel systems with an effective exercise activity indicator.</a> PLoS One. 10 (4), (2015).</li><li>Fantegrossi, W. E., Xiao, W. R., Zimmerman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technology+for+modulating+locomotor+activity+as+an+operant+response+in+the+mouse:+Implications+for+neuroscience+studies+involving+&#34;exercise&#34;+in+rodents.">Novel technology for modulating locomotor activity as an operant response in the mouse: Implications for neuroscience studies involving &#34;exercise&#34; in rodents.</a> J Neurosci Methods. 212 (2), 338-343 (2013).</li><li>Hayes, K., 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=Forced,+not+voluntary,+exercise+effectively+induces+neuroprotection+in+stroke.">Forced, not voluntary, exercise effectively induces neuroprotection in stroke.</a> Acta Neuropathol. 115 (3), 289-296 (2008).</li><li>Arida, R. M., Scorza, C. A., da Silva, A. V., Scorza, F. A., Cavalheiro, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+spontaneous+versus+forced+exercise+in+rats+on+the+staining+of+parvalbumin-positive+neurons+in+the+hippocampal+formation.">Differential effects of spontaneous versus forced exercise in rats on the staining of parvalbumin-positive neurons in the hippocampal formation.</a> Neurosci Lett. 364 (3), 135-138 (2004).</li><li>Waters, R. P., 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=Selection+for+aerobic+capacity+affects+corticosterone,+monoamines+and+wheel-running+activity.">Selection for aerobic capacity affects corticosterone, monoamines and wheel-running activity.</a> Physiol Behav. (4-5), 1044-1054 (2008).</li><li>Ke, Z., Yip, S. P., Li, L., Zheng, X. -. X., Tong, K. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+voluntary,+involuntary,+and+forced+exercises+on+brain-derived+neurotrophic+factor+and+motor+function+recovery:+A+rat+brain+ischemia+model.">The effects of voluntary, involuntary, and forced exercises on brain-derived neurotrophic factor and motor function recovery: A rat brain ischemia model.</a> PLoS One. 6 (2), (2011).</li><li>Caton, S. 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=Low-carbohydrate+high-fat+diets+in+combination+with+daily+exercise+in+rats:+Effects+on+body+weight+regulation,+body+composition+and+exercise+capacity.">Low-carbohydrate high-fat diets in combination with daily exercise in rats: Effects on body weight regulation, body composition and exercise capacity.</a> Physiol Behav. 106 (2), 185-192 (2012).</li><li>. C8051F330/1/2/3/4/5 datasheet Available from: <a target="_blank" href="https://www.silabs.com/Support%20Documents/TechnicalDocs/C8051F33x.pdf">https://www.silabs.com/Support%20Documents/TechnicalDocs/C8051F33x.pdf</a> (2006)</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Chen, J. L., 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=Therapeutic+benefit+of+intravenous+administration+of+bone+marrow+stromal+cells+after+cerebral+ischemia+in+rats.">Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats.</a> Stroke. 32 (4), 1005-1011 (2001).</li><li>Chang, M. -. W., Young, M. -. S., Lin, M. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inclined+plane+system+with+microcontroller+to+determine+limb+motor+function+of+laboratory+animals.">An inclined plane system with microcontroller to determine limb motor function of laboratory animals.</a> J Neurosci Methods. 168 (1), 186-194 (2008).</li><li>Gartshore, G., Patterson, J., Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+ischemia+and+reperfusion+on+the+course+of+brain+tissue+swelling+and+blood-brain+barrier+permeability+in+a+rodent+model+of+transient+focal+cerebral+ischemia.">Influence of ischemia and reperfusion on the course of brain tissue swelling and blood-brain barrier permeability in a rodent model of transient focal cerebral ischemia.</a> Exp Neurol. 147 (2), 353-360 (1997).</li><li>Chen, F., 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=Rodent+stroke+induced+by+photochemical+occlusion+of+proximal+middle+cerebral+artery:+Evolution+monitored+with+MR+imaging+and+histopathology.">Rodent stroke induced by photochemical occlusion of proximal middle cerebral artery: Evolution monitored with MR imaging and histopathology.</a> Eur J Radiol. 63 (1), 68-75 (2007).</li><li>Almenning, I., Rieber-Mohn, A., Lundgren, K. M., Lovvik, T. S., Garnaes, K. K., Moholdt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high+intensity+interval+training+and+strength+training+on+metabolic,+cardiovascular+and+hormonal+outcomes+in+women+with+polycystic+ovary+syndrome:+a+pilot+study.">Effects of high intensity interval training and strength training on metabolic, cardiovascular and hormonal outcomes in women with polycystic ovary syndrome: a pilot study.</a> PLoS One. 10 (9), (2015).</li><li>Costigan, S. A., Eather, N., Plotnikoff, R. C., Taaffe, D. R., Lubans, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+interval+training+for+improving+health-related+fitness+in+adolescents:+a+systematic+review+and+meta-analysis.">High-intensity interval training for improving health-related fitness in adolescents: a systematic review and meta-analysis.</a> Br J Sports Med. 49 (19), (2015).</li></ol>

The authors have nothing to disclose.

This protocol describes a highly effective running wheel system for&#160;reducing the severity of the effects of the stroke&#160;in animals. As a rat-friendly testbed, this platform is designed as well in such a way that a stable running speed can be maintained by rats throughout a running process by means of a predetermined adaptive acceleration curve. In typical training systems, preset training speeds and durations are set manually. Once an exercise commences, a preset speed is reached very shortly. In this context, i...

Strokes exist continuously as a financial burden to countries globally, leaving countless patients physically and mentally disabled1,2. There is clinical evidence to suggest that regular exercise can improve nerve regeneration and strengthen neural connections3,4, and it is also shown that exercise can decrease the risk of suffering ischemic strokes5. With either a treadmill or a running wheel as an exercise training system, rodents, such as rats, serve as a proxy for humans for testing the effectiveness of exercises in a vast majority of clinical experiments6-8. A training system normally involves training a rat for a certain period of time, during which a rat runs at a certain speed. Therefore, the training intensity is generally calculated according to the exercise speed and duration6-8. The same approach is applied to estimate the amount of exercise required for neurophysiological protection. However, the experimental exercises are sometimes found to be ineffective, such as when a rat stumbles, falls, or grabs the rails once they are unable to catch up with the running wheel speed9-11. Needless to say, incidents of ineffective exercise significantly reduce the exercise benefit. Even though there is no any universally accepted approach currently to quantify the effective exercises for reducing brain damage, the level of effective exercises still stands as an objective appraisal for clinical researchers to illustrate the benefits of exercise in the discipline of neurophysiology.
There exist a number of limitations on commercially available animal mobility systems used in today&#39;s brain damage reduction&#160;experiments12. In a treadmill case, rats are forced to run by means of electric shocks, inducing tremendous psychological stress on the animals and thereby interference in the final neurophysiological test results8,13,14. Running wheels can be categorized into two types, namely voluntary and forced. Voluntary running wheels allow rats to run naturally, creating excessive variability due of the differences in the rats&#39; physical traits and abilities15, while motorized running wheels (MRWs) employ a motor to turn the wheel, forcing rats to run. Despite also being a form of forced training, MRWs imposes less psychological stress on rats than treadmills13,16,17. However, experiments using MRWs have reported that rats sometimes interrupt the exercise by grabbing the rails on the wheel track and refusing to run at speeds exceeding 20 m/min9. These examples show that animal mobility systems currently available have an inherent disadvantage that inhibits effective exercising. For objective rat training purposes, the development of a highly effective training system but with low interference is therefore viewed as an urgent issue for neurophysiological exercise experiments.
This study presents a highly effective running wheel system for experiments on&#160;reducing the severity of the effects of the stroke11. In addition to a reduced number of interference factors during a training process, this system detects the running position of a rat using infrared sensors embedded in the wheel, thereby achieving a more reliable estimate of effective exercise activity. The psychological stress imposed by traditional treadmills and the frequent exercise interruptions in MRWs both skew the objectivity of the resulting exercise estimates. A positioning running wheel (PRW) system presented in this study is developed in an attempt to minimize the unwanted interference while providing a reliable training model for quantifying effective exercise.

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brushless DC motor</td>
			<td style="width:195px;">Oriental Motor</td>
			<td style="width:247px;">BLEM512-GFS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motor driver</td>
			<td>Oriental Motor</td>
			<td>BLED12A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Motor reducer</td>
			<td>Oriental Motor</td>
			<td style="width:247px;">GFS5G20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Speedometer</td>
			<td>Oriental Motor</td>
			<td style="width:247px;">OPX-2A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Treadmill</td>
			<td style="width:195px;">Columbus Instruments</td>
			<td style="width:247px;">Exer-6M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infrared transmitter&#160;</td>
			<td style="width:195px;">Seeed Studio</td>
			<td>TSAL6200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infrared Receiver</td>
			<td style="width:195px;">Seeed Studio</td>
			<td>TSOP382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcontroller</td>
			<td>Silicon Labs</td>
			<td>C8051F330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCD camera</td>
			<td>Canon Inc.</td>
			<td>EOS 450D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image processing software</td>
			<td>Adobe Systems Incorporated</td>
			<td>ADOBE Photoshop CS5 12.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image analysis</td>
			<td>Media Cybernetics</td>
			<td>Pro Plus 4.50.29</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pentobarbital</td>
			<td style="width:195px;">Sigma-Aldrich (Saint Louis, MO, USA)</td>
			<td style="width:247px;">SIGMA P-3761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:195px;">Pfizer (Kent, UK)</td>
			<td style="width:247px;">&#160;1867-66-9</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Atropine</td>
			<td style="width:195px;">Taiwan Biotech Co., Ltd. (Taoyuan, Taiwan)</td>
			<td style="width:247px;">A03BA01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Xylazine</td>
			<td style="width:195px;">Sigma-Aldrich (Saint Louis, MO, USA)</td>
			<td style="width:247px;">SIGMA X1126</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Buprenorphine</td>
			<td style="width:195px;">Sigma-Aldrich (Saint Louis, MO, USA)</td>
			<td style="width:247px;">B9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia</td>
			<td>Sigma Chemical</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

an innovative running wheel-based mechanism for improved rat training performance

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity for reducing the severity of the effects of the stroke&#160;in rats. This system provides more effective animal exercise training than commercially available systems such as treadmills and motorized running wheels (MRWs). In contrast to an MRW that can only achieve speeds below 20 m/min, rats are permitted to run at a stable speed of 30 m/min on a more spacious and high-density rubber running track supported by a 15 cm wide acrylic wheel with a diameter of 55 cm in this work. Using a predefined adaptive acceleration curve, the system not only reduces the operator error but also trains the rats to run persistently until a specified intensity is reached. As a way to evaluate the exercise effectiveness, real-time position of a rat is detected by four pairs of infrared sensors deployed on the running wheel. Once an adaptive acceleration curve is initiated using a microcontroller, the data obtained by the infrared sensors are automatically recorded and analyzed in a computer. For comparison purposes, 3 week training is conducted on rats using a treadmill, an MRW and a PRW. After surgically inducing middle cerebral artery occlusion (MCAo), modified neurological severity scores (mNSS) and an inclined plane test were conducted to assess the neurological damages to the rats. PRW is experimentally validated as the most effective among such animal mobility systems. Furthermore, an exercise effectiveness measure, based on rat position analysis, showed that there is a high negative correlation between the effective exercise and the infarct volume, and can be employed to quantify a rat training in any type of brain damage reduction&#160;experiments.

This section is devoted to comparisons, made 1 week after surgery, on the mNSS scores, incline plane test results and brain infarct volumes among five groups. Figure 4A and 4B present the average mNSS scores and the average of incline plane test results, respectively. The PRW group appears as the best in terms of mNSS improvement. The significant differences between PRW and MRW and between treadmill and PRW clearly suggest that the PRW protects against st...

Watch this Scientific Journal Video about An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance at JoVE.com

An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol&#39;s potential for stroke prevention experiments.

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity ...

an-innovative-running-wheel-based-mechanism-for-improved-rat-training

Universidad San Sebastian

Research

JoVE Journal

Behavior

8.8K Views.  National Chin-Yi University of Technology. This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol's potential for stroke prevention experiments.

Video: An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

Vision Training Methods for Sports Concussion Mitigation and Management

There is emerging evidence supporting the use vision training, including light board training tools, as a concussion baseline and neuro-diagnostic tool and potentially as a supportive component to concussion prevention strategies. This paper is focused on providing detailed methods for select vision training tools and reporting normative data for comparison when vision training is a part of a sports management program. The overall program includes standard vision training methods including tachistoscope, Brock&#8217;s string, and strobe glasses, as well as specialized light board training algorithms. Stereopsis is measured as a means to monitor vision training affects. In addition, quantitative results for vision training methods as well as baseline and post-testing *A and Reaction Test measures with progressive scores are reported. Collegiate athletes consistently improve after six weeks of training in their stereopsis, *A and Reaction Test scores. When vision training is initiated as a team wide exercise, the incidence of concussion decreases in players who participate in training compared to players who do not receive the vision training. Vision training produces functional and performance changes that, when monitored, can be used to assess the success of the vision training and can be initiated as part of a sports medical intervention for concussion prevention.

This paper describes a protocol to conduct, quantitatively monitor, and assess the success of vision training initiated as part of a sports medical management program including intervention for concussion prevention and performance enhancement.

There is emerging evidence supporting the use vision training, including light board training tools, as a concussion baseline and neuro-diagnostic tool ...

Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular function include transcranial magnetic stimulation, electromyography, and three-dimensional motion capture. The advent of readily available and cost-effective virtual reality solutions has expanded the capabilities of researchers in recreating &#8220;real-world&#8221; environments and movements in a laboratory setting. Naturalistic movement analysis will not only garner a greater understanding of motor control in healthy individuals, but also permit the design of experiments and rehabilitation strategies that target specific motor impairments (e.g. stroke). The combined use of these tools will lead to increasingly deeper understanding of neural mechanisms of motor control. A key requirement when combining these data acquisition systems is fine temporal correspondence between the various data streams. This protocol describes a multifunctional system&#8217;s overall connectivity, intersystem signaling, and the temporal synchronization of recorded data. Synchronization of the component systems is primarily accomplished through the use of a customizable circuit, readily made with off the shelf components and minimal electronics assembly skills.

Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback.

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

A Vibrotactile Feedback Device for Seated Balance Assessment and Training

Postural perturbations, motion tracking, and sensory feedback are modern techniques used to challenge, assess, and train upright sitting, respectively. The goal of the developed protocol is to construct and operate a sitting platform that can be passively destabilized while an inertial measurement unit quantifies its motion and vibrating elements deliver tactile feedback to the user. Interchangeable seat attachments alter the stability level of the device to safely challenge sitting balance. A built-in microcontroller allows fine-tuning of the feedback parameters to augment sensory function. Posturographic measures, typical of balance assessment protocols, summarize the motion signals acquired during timed balance trials. No dynamic sitting protocol to date provides variable challenge, quantification, and sensory feedback free of laboratory constraints. Our results demonstrate that non-disabled users of the device exhibit significant changes in posturographic measures when balance difficulty is altered or vibrational feedback provided. The portable, versatile device has potential applications in rehabilitation (following skeletal, muscular, or neurological injury), training (for sports or spatial awareness), entertainment (via virtual or augmented reality), and research (of sitting-related disorders).

A sitting platform has been developed and assembled that passively destabilizes sitting posture in humans. During the user&#39;s stabilizing task, an inertial measurement unit records the device&#39;s motion, and vibrating elements deliver performance-based feedback to the seat. The portable, versatile device may be used in rehabilitation, assessment, and training paradigms.

Postural perturbations, motion tracking, and sensory feedback are modern techniques used to challenge, assess, and train upright sitting, respectively. ...

Training Dogs for Awake, Unrestrained Functional Magnetic Resonance Imaging

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.

Magnetic resonance imaging (MRI) on unrestrained awake dogs is a new method with several advantages over imaging with physical or chemical restraint. This protocol introduces a cost-effective training method that minimizes training in the MRI environment, which can be expensive, and maximizes the subject pool available for canine functional MRI.

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for ...

Working Memory Training for Older Participants: A Control Group Training Regimen and Initial Intellectual Functioning Assessment

The efficacy of cognitive training interventions is recently highly debated. There is no consensus on what kind of training regimen is the most effective. Also, individual characteristics as predictors of training outcome are still being investigated. In this article, we show the attempt to address this issue by examining not only the impact of working memory (WM) training on cognitive effectiveness in older adults but also the influence of the initial WM capacity (WMC) on the training&#39;s outcome. We describe in detail how to perform 5 weeks of an adaptive dual n-back training with an active control group (memory quiz). We are focusing here on technical aspects of the training as well as on the initial assessment of participants&#39; WMC. The evaluation of pre and post training performance of other cognitive dimensions was based on the results of tests of memory updating, inhibition, attention shifting, short-term memory (STM) and reasoning. We have found that the initial level of WMC predicts the efficiency of the n-back training intervention. We have also noticed the post training improvement in almost all aspects of cognitive functioning we measured, but those effects were mostly intervention independent.

A cognitive training intervention in elderly population together with the assessment of the pre training cognitive abilities is presented. We show two versions of training - experimental and active control - and demonstrate their effects on the array of cognitive tests.

The efficacy of cognitive training interventions is recently highly debated. There is no consensus on what kind of training regimen is the most effective. ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...