This work is supported by R15 DK097644 and R15 DK108668. We thank Dr. Chaitanya K Gavini and Dr. Megan Rich for prior contributions and Dr. Stanley Dannemiller for ensuring our&#160;compliance with institutional animal use guidelines. A special thank you to Dr. Tim Bartness for providing the fundamental research necessary to build this method and its associated studies. Figures 1A,&#160;C,&#160;D&#160;and Figure 2A were created using Biorender.com.

These methods can be applied to both rat and mouse models and were performed with institutional approval (Kent State University, IACUC Approval #359 and #340 CN 12-04).&#160;Prior to the implementation of the protocol, animals should be housed in conformance with the Guide for the Care and Use of Laboratory Animals.
1. Preparing the transponder reader
NOTE: Prior to use, the transponder reader must be set; the following steps only include the setting changes necessary for this study. This portion of the protocol is directly associated with the DAS-8027-IUS portable readers; other reader models should follow instructions provided by the manual to achieve programming results.
<ol>
	<li>Set Audio Beep to OFF.
	<ol>
		<li>Turn on the device by pressing the SCAN button and wait for the lighting to appear on the OLED display. Press and hold the BACK/MENU button to get to the menu screen.</li>
		<li>Using the NEXT/ENTER button, scroll through the options until OPERATIONAL SETUP. Here, toggle the up or down arrows to turn YES and open the operational submenu.</li>
		<li>Using the NEXT/ENTER button, scroll to AUDIO BEEP. As the default setting is ON, toggle the up or down arrows and change the setting to OFF.</li>
		<li>Press the NEXT/ENTER button to save this setting change.</li>
	</ol>
	</li>
	<li>Set Vibrate Upon Read to ON.
	<ol>
		<li>Follow step 1.1&#160;through step 1.2 or complete the next step directly after step 1.4.</li>
		<li>Using the NEXT/ENTER button, scroll to VIBRATE UPON READ. As the default setting is OFF, toggle the up and down arrows and change the setting to ON to feel, via vibration, when the reading has been completed regardless of being able to view the screen.</li>
	</ol>
	</li>
</ol>
2. Program transponders
NOTE: Each implanted transponder should first be programmed with an animal identification (animal ID or transponder ID). This nomenclature can be used as secondary identification for the test subject (e.g., four digits for mouse strain abbreviation, location of transponder, and an additional three to four digits to indicate animal number). Programming can be completed days in advance of surgery while keeping the transponders sterile prior to surgery.
<ol>
	<li>Enter the ID Code on the transponder.
	<ol>
		<li>Apply a booster coil to the reader head&#8212;a specific accessory for model DAS 8027-IUS, which helps in the programming procedure.</li>
		<li>Using a gloved hand, place the transponder (within the applicator) into the booster coil.</li>
		<li>Turn on the device by pressing the SCAN button and wait for the OLED display to light up. Press and hold the BACK/MENU button to get to the menu screen.</li>
		<li>Using the NEXT/ENTER button, scroll through the options until WRITE TRANSPONDER ID. Here, toggle the up or down arrows to turn YES.</li>
		<li>Using the NEXT/ENTER button, toggle to ENTER ID CODE.</li>
		<li>Use the up and down arrow keys to scroll through numbers and letters. Press NEXT/ENTER after each character selection to move to the following character.</li>
		<li>When the ID code is complete, press SCAN to write the transponder.</li>
		<li>Remove the transponder from the booster coil and repeat as necessary. Check that the transponder reads temperature changes by warming the enclosed transponders between gloved hands and measuring using the temperature scanner. 
		&#8203;NOTE: AUTO MULTI WRITE and SEQUENTIAL COUNT settings can be set to ON to allow for multiple or sequential transponder programming during a session. Each transponder should be tested during programming.</li>
	</ol>
	</li>
</ol>
3. Prepare &#34;home cage balls&#34;
<ol>
	<li>Place 5 cm x 5 cm odorless/control towel into a tea ball.</li>
	<li>Place these home cage balls in new home cages after surgery to begin habituating the animal to the method in which the contextual stimuli will be presented during testing. Replace these home cage balls every 2 weeks.</li>
</ol>
4. Surgery and postoperative care
<ol>
	<li>Weigh and record the subjects&#39; pre-surgery body weight. Using an induction chamber, provide anesthesia (e.g., 2-5% isoflurane) to the animal.</li>
	<li>Using electric clippers, completely shave the hind limb. Administer analgesia (e.g., 5 mg/kg of ketoprofen, s.c.) in compliance with institutional guidelines. 
	NOTE: Additional analgesia may be required if this procedure is combined with other surgical methods.</li>
	<li>Clean the area with 70% alcohol (or commercially available sterile alcohol wipe) and povidone-iodine wash (or commercially available sterile, individually wrapped betadine swabs) alternating at least three times, ending with povidone-iodine.</li>
	<li>Return the animal to the induction chamber, and anesthetize the animal to surgical levels. Then, set up the mouse in a face mask for continued exposure to anesthesia. Apply neomycin ophthalmic ointment to the eyes of the animal to prevent dryness while under anesthesia. 
	NOTE: The procedure should not start until the mouse shows no evidence of pain reception (i.e., corneal reflex, tail pinch response, toe pinch reflex).</li>
	<li>Using only surgical scissors, make a shallow cut through the skin on the right hind limb.</li>
	<li>Moving parallel to the gastrocnemius, place the sharp edge of a preprogrammed and uncapped sterile transponder into the incision. Ensure that the green plunger faces up and is visible. Continue pushing the transponder applicator into the incision until the opening of the transponder applicator is no longer visible. 
	NOTE: Do not accidentally press the green plunger on the transponder applicator during step 4.6. Premature discharge of the transponder will lead to improper placement.</li>
	<li>Turn the applicator 180&#176;, resulting in the green plunger facing down toward the mouse&#39;s limb, no longer visible to the experimenter. Push the transponder applicator into the final location. Once in ideal placement, adjacent to or partially enclosed in the gastrocnemius, push the green plunger, allowing the applicator&#39;s pressure to guide the investigator&#39;s hand back away from the mouse.</li>
	<li>Using forceps, hold together the opened skin and place a wound clip with a sterile autoclip or sterile suture. If needed, use absorbable sutures prior to the sterile autoclip to close the fascia layer. Using the transponder-reader, check the temperature of the mouse muscle.</li>
	<li>Remove the mouse from anesthesia and place it in a clean home cage placed atop a water-circulating heating pad set to low for recovery. Ensure that the home cage includes a tea ball with an odorless towel to begin habituation. 
	NOTE: The mouse should awaken from surgery within 15 min. Food can be placed at the bottom of the cage for easy access during recovery days.</li>
	<li>Postoperative care
	<ol>
		<li>Record mouse weights and temperatures daily using a transponder-reader for at least 2 days after surgery or until mice regain or stabilize body weight.</li>
		<li>Administer non-narcotic analgesia (e.g., 5 mg/kg of ketoprofen, s.c.) once daily to the mice for at least 2 days post surgery, with additional doses provided as needed. 
		NOTE: Mice and rats should fully recover within 5-8 days of surgery and can undergo habituation and testing procedures.</li>
	</ol>
	</li>
</ol>
5. Testing preparation - home cage
<ol>
	<li>Constructing risers 
	NOTE: The below step is based on 194 mm x 181 mm x 398 mm mouse filter-topped cages. To fit larger cages (e.g., a rat home cage), the width will need to be adjusted.
	<ol>
		<li>Cut the PVC pipe with a ratcheting PVC cutter into eight sections and assemble following Figure 1C. This will give an open tabletop structure that can hold approximately four cages. Make the desired number of risers.</li>
	</ol>
	</li>
	<li>Room setup
	<ol>
		<li>Assign a location to each riser within the testing room. Separate the risers set to receive different contextual stimuli (i.e., odors) by a minimum of 2 m to avoid confounding variables. 
		NOTE: Each mouse should have an assigned testing location within the testing room and on the physical risers as much as is feasible to avoid developing associations between different locations and thermogenic stimuli.</li>
		<li>Using magnetic strips, attach surgical sheets or gowns across the risers, creating a visual barrier between the researcher and the test subjects. Set this barrier to minimize temperature changes resulting from mouse activity when viewing experimenters moving toward the cage or around the testing room.</li>
		<li>(Optional) Place mirrors on the surface below the risers to ease viewing of the cage bottom during testing. 
		NOTE: Risers can be sanitized through a cage wash system. Cloth or surgical sheets should be laundered prior to habituation and testing.</li>
	</ol>
	</li>
	<li>Tea ball preparation
	<ol>
		<li>Prepare tea balls with control and PO towels (approximately 5 cm x 5 cm). To avoid cross-contamination, prepare control-odor tea balls first. 
		NOTE: Predator-odor towels should be pathogen-tested prior to use. These towels should also be contained, and materials that interact with them should be immediately sanitized (i.e., cage wash), preventing exposure of the odor to other animals.</li>
	</ol>
	</li>
</ol>
6. Temperature testing - home cage
NOTE: Animals need to be habituated to the entire testing procedure, excluding experimental contextual, or pharmacological stimuli. This should be completed a minimum of 4x before testing.
<ol>
	<li>Transfer the animals to the prepared testing&#160;room. Place the animals in a preassigned location on the riser. This location should be the same throughout all habituation and testing procedures.</li>
	<li>Remove the &#34;home cage ball&#34; from the mouse home cage and re-cover the cages with a cloth or surgical sheet. Allow the mice to acclimate to the testing space for 1-2 h.</li>
	<li>After acclimation is complete, use the scanner to measure and record the baseline temperature of each subject. Avoid manipulating the cloth coverings during measurements. 
	NOTE: Pharmacological agents can be applied here. Wait time post injection or application can be added as needed before testing. Recording a secondary baseline directly before testing is recommended after the addition of a pharmacological agent to monitor the response to pharmacological stimuli. If odor response is not being tested, temperature measurements of the mice can begin directly after injection. Randomization should be employed when providing any stimuli.</li>
	<li>Uncover the cage and place the tea ball (control or PO) onto the floor of the home cage. Replace the cage lid and cloth covering.</li>
	<li>Begin the stopwatch. Measure the temperatures of the test subjects in the same order of tea ball placement. Record temperatures and clock time of measurements following the desired time points.</li>
	<li>When the experiment is complete, remove the treatment ball. Place the mice that received PO in a new home cage with the original &#34;home cage ball&#34;. Return the &#34;home cage ball&#34; to the cage of the mice that received control odor. Transfer the mice to the housing location. 
	&#8203;NOTE: The above procedure can be translated to rat models within appropriately sized cages. Adjustments to the measurements suggested in Figure 1C may be required to allow for better access to the bottom of the home cage.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64264/64264fig01.jpg" /> 
Figure 1: Transponders and home cage temperature testing. (A) Diagram of unilateral transponder placement for testing temperature in a mouse gastrocnemius. Once programmed and placed, the transponder-reader (DAS-8027-IUS, shown) can be used to measure temperature. (B) Left, photo of an open mesh stainless steel tea ball and a 5 cm x 5 cm towel. Right, enclosed tea ball, used to hold habituation and odor towels in home cage testing. (C) Schematic of risers constructed with PVC piping for home cage testing. (D) Workflow of home cage testing protocol. (E) Facility images of home cage testing area. Left, four mouse cages atop a riser. Magnetic strips are located on the adjacent wall, and magnets and surgical cloth are on the table. Right, covered mouse cages on risers. (A), (C), and (D) were created with Biorender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64264/64264fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Temperature testing - treadmill walking
<ol>
	<li>Assign each animal a treadmill as their assigned location for habituation and testing procedures.</li>
	<li>Prepare the treadmills for testing, ensuring that the shockers are functional. 
	NOTE: For treadmill walking, treadmills should be set at the lowest available pace that promotes continuous movement but not running for both habituation and testing. For the 1012M-2 Modular Enclosed Metabolic Treadmill, this is 5.2 m/min for mice and 7 m/min for rats. This pace may need to be adjusted based on the obesity of the subject.&#160;Shockers should be set to an intensity and repetition rate of 5.0.</li>
	<li>Habituation
	<ol>
		<li>Move the mice to the testing room. Allow mice 1-2 h to acclimate to the room transfer in their home cages.</li>
		<li>After acclimation, guide the mice to the opening of their assigned treadmill and close the treadmill. Start the belt, shocker, and stopwatch.</li>
		<li>Allow the mice to walk on the treadmills for 15 min, using shock stimulus as motivation for movement. Stop the test immediately if an animal remains on an active shocker for an extended period.</li>
		<li>After the test, remove the mice and return them to the home cages.</li>
		<li>Clean the treadmills using liquid detergent and water.</li>
	</ol>
	</li>
	<li>Testing
	<ol>
		<li>Move the mice to the testing room. Allow the mice 1-2 h to acclimate to the room transfer in their home cages.</li>
		<li>Measure and record baseline temperature prior to moving the mouse to the treadmill. 
		NOTE: For tests including pharmacological agents, apply or inject them here, following the schematic shown in Figure 2A. Wait time after injection can be added as needed before the mice are placed on the treadmill. Randomization should be employed when providing any stimuli.</li>
		<li>Place 5 cm x 5 cm squares of control or PO towels within the treadmill closest to the front of the treadmill. Adhere the towels to the ceiling of the treadmill or underneath for easy placement and removal.</li>
		<li>Guide the mice into the assigned treadmill. Turn on the treadmill belt and shocker.</li>
		<li>Start the stopwatch. Take measurements of the test subjects in the same order in which the mice were set up in the treadmills. Record the temperatures and clock time of the measurements following the desired time points. 
		NOTE: Temperature can reliably be measured from outside the treadmill while a mouse is inside an enclosed treadmill during walking activity. For rats, the treadmill size and transponder-reader&#160;distance limitations may require an experimenter to keep the back of the treadmill open to insert the reader inside the treadmill, closer to the subject.</li>
		<li>When the test is complete, turn off the shockers and treadmills; return the mice to their home cages. Transfer the mice to the housing location.</li>
		<li>Clean the treadmills using liquid detergent and water, paying specific attention to remove any residual PO.</li>
		<li>When experiments are complete, euthanize the animals (e.g., using CO2 inhalation), and visually confirm the transponder location.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64264/64264fig02.jpg" /> 
Figure 2: Activity-controlled temperature testing. (A) Workflow of activity-controlled temperature testing with a pharmacological agent using treadmill walking. (B) Facility images of treadmills. Left, an image of full equipment setup. Right, a closer image of individual treadmills and shockers. (A) was created with Biorender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64264/64264fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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K., Bartness, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+of+the+sympathetic+innervation+of+adipose+tissues+by+melanocortin+receptor+stimulation.">Differential activation of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation.</a> Endocrinology. 148 (11), 5339-5347 (2007).</li><li>Vaughan, C. H., Shrestha, Y. B., Bartness, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+melanocortin+receptor-containing+node+in+the+SNS+outflow+circuitry+to+brown+adipose+tissue+involved+in+thermogenesis.">Characterization of a novel melanocortin receptor-containing node in the SNS outflow circuitry to brown adipose tissue involved in thermogenesis.</a> Brain Research. 1411, 17-27 (2011).</li><li>Kort, W. J., Hekking-Weijma, J. M., TenKate, M. T., Sorm, V., VanStrik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchip+implant+system+as+a+method+to+determine+body+temperature+of+terminally+ill+rats+and+mice.">A microchip implant system as a method to determine body temperature of terminally ill rats and mice.</a> Laboratory Animals. 32 (3), 260-269 (1998).</li><li>Mei, 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=Body+temperature+measurement+in+mice+during+acute+illness:+Implantable+temperature+transponder+versus+surface+infrared+thermometry.">Body temperature measurement in mice during acute illness: Implantable temperature transponder versus surface infrared thermometry.</a> Scientific Reports. 8, 3526 (2018).</li><li>Warn, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+body+temperature+measurement+of+mice+as+an+early+predictor+of+death+in+experimental+fungal+infections.">Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections.</a> Laboratory Animals. 37 (2), 126-131 (2003).</li><li>Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+and+sensitive+method+for+measuring+thermal+nociception+in+cutaneous+hyperalgesia.">A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.</a> Pain. 32 (1), 77-88 (1988).</li><li>Fiebig, K., Jourdan, T., Kock, M. H., Merle, R., Thone-Reineke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+infrared+thermography+for+temperature+measurement+in+adult+male+NMRI+nude+mice.">Evaluation of infrared thermography for temperature measurement in adult male NMRI nude mice.</a> Journal of the American Association for Laboratory Animal Science. 57 (6), 715-724 (2018).</li><li>Franco, N. H., Geros, A., Oliveira, L., Olsson, I. A. S., Aguiar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThermoLabAnimal+-+A+high-throughput+analysis+software+for+non-invasive+thermal+assessment+of+laboratory+mice.">ThermoLabAnimal - A high-throughput analysis software for non-invasive thermal assessment of laboratory mice.</a> Physiology &amp; Behavior. 207, 113-121 (2019).</li><li>Koganti, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+KATP+channel+expression+in+skeletal+muscle+by+targeted+oligonucleotide+delivery+promotes+activity-linked+thermogenesis.">Disruption of KATP channel expression in skeletal muscle by targeted oligonucleotide delivery promotes activity-linked thermogenesis.</a> Molecular Therapy. 23 (4), 707-716 (2015).</li><li>Bal, N. C., Periasamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+of+sarcoendoplasmic+reticulum+calcium+ATPase+pump+activity+by+sarcolipin+as+the+basis+for+muscle+non-shivering+thermogenesis.">Uncoupling of sarcoendoplasmic reticulum calcium ATPase pump activity by sarcolipin as the basis for muscle non-shivering thermogenesis.</a> Philosophical Transactions of the Royal Society B. 375 (1793), 20190135 (2020).</li><li>Hicks, 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=Body+temperature+and+cardiac+changes+induced+by+peripherally+administered+oxytocin,+vasopressin+and+the+non-peptide+oxytocin+receptor+agonist+WAY+267,464:+a+biotelemetry+study+in+rats.">Body temperature and cardiac changes induced by peripherally administered oxytocin, vasopressin and the non-peptide oxytocin receptor agonist WAY 267,464: a biotelemetry study in rats.</a> British Journal of Pharmacology. 171 (11), 2868-2887 (2014).</li><li>Kasahara, 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=Oxytocin+receptor+in+the+hypothalamus+is+sufficient+to+rescue+normal+thermoregulatory+function+in+male+oxytocin+receptor+knockout+mice.">Oxytocin receptor in the hypothalamus is sufficient to rescue normal thermoregulatory function in male oxytocin receptor knockout mice.</a> Endocrinology. 154 (11), 4305-4315 (2013).</li><li>Kasahara, 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=Role+of+the+oxytocin+receptor+expressed+in+the+rostral+medullary+raphe+in+thermoregulation+during+cold+conditions.">Role of the oxytocin receptor expressed in the rostral medullary raphe in thermoregulation during cold conditions.</a> Frontiers in Endocrinology. 6, 180 (2015).</li><li>Yuan, J., Zhang, R., Wu, R., Gu, Y., Lu, 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+oxytocin+to+rectify+metabolic+dysfunction+in+obese+mice+are+associated+with+increased+thermogenesis.">The effects of oxytocin to rectify metabolic dysfunction in obese mice are associated with increased thermogenesis.</a> Molecular and Cellular Endocrinology. 514, 110903 (2020).</li><li>Scholl, J. L., Afzal, A., Fox, L. C., Watt, M. J., Forster, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+anxiety-like+behaviors+in+rats.">Sex differences in anxiety-like behaviors in rats.</a> Physiology &amp; Behavior. 211, 112670 (2019).</li></ol>

The authors declare that they have no conflicts of interest.

This temperature testing protocol provides the field with an avenue to measure skeletal muscle thermogenesis directly. This is critical as research delves into identifying the mechanisms underlying muscle thermogenesis33. The method provides two cost-effective protocols for measuring skeletal muscle thermogenesis under contextual and pharmacological conditions. This protocol emphasizes the importance of both habituation and acclimation within these procedures. Habituation is used to repeatedly int...

Within metabolic research, the examination of skeletal muscle thermogenesis is a promising new avenue for probing body weight homeostasis. The published literature supports the idea that the thermogenic responses of one of the body&#39;s largest organ systems&#8212;the skeletal muscle&#8212;provide an avenue for increasing energy expenditure and other metabolic effects, thereby effectively rebalancing systems within diseases such as obesity1,2,3. If the muscle can be considered a thermogenic organ, studies must utilize a practical methodology to study thermogenic changes within this organ. The desire to understand the endothermic impact of skeletal muscles and the utility of this methodology for studying non-shivering muscle thermogenesis are not specific to metabolic studies. Disciplines including evolution4, comparative physiology5, and ecophysiology6,7 have shown a vested interest in understanding the ways in which muscle thermogenesis may contribute to endothermy and how this mechanism adapts to the environment. The presented protocol provides the critical methods necessary to address these questions.
The provided method can be utilized in the assessment of both contextual and pharmacological stimuli modulation of muscle temperature, including the unique technique of providing predator odor (PO) to shift the context to replicate predator threat. Prior reports have demonstrated the ability of PO to rapidly induce a sizable increase in muscle thermogenesis8. Moreover, pharmacological stimuli can also alter muscle temperature. This has been demonstrated in the context of PO-induced muscle thermogenesis, where pharmacological blockade of peripheral &#946;-adrenergic receptors, using nadolol, inhibited the ability of PO to induce muscle thermogenesis without significantly affecting contractile thermogenesis during treadmill walking8. Central administration of melanocortin receptor agonists in rats has also been used to discern brain mechanisms altering thermogenesis9,10.
Provided here is a preliminary investigation of the ability of the neurohormone oxytocin (Oxt) to alter muscle thermogenesis in mice. Similar to predator threat, social encounters with a same-sex conspecific increase body temperature, a phenomenon referred to as social hyperthermia11. Given the relevance of Oxt to social behavior12, it has been speculated that Oxt is a mediator of social hyperthermia in mice. Indeed, an oxytocin receptor antagonist decreases social hyperthermia in mice11, and mouse pups lacking Oxt show deficits in behavioral and physiological aspects of thermoregulation, including thermogenesis13. Given that Harshaw et al. (2021) did not find evidence supporting &#946;3 adrenergic receptor-dependent brown adipose tissue (BAT) thermogenesis with social hyperthermia11, it has been posited that social hyperthermia may be driven by Oxt&#39;s induction of muscle thermogenesis.
To measure skeletal muscle thermogenesis, the following protocol uses the implantation of preprogrammed IPTT-300 transponders adjacent to the muscle of interest within a mouse or rat8,10,14,15. These transponders are glass-encapsulated microchips that are read using corresponding transponder readers. Little to no research has utilized this technology in this capacity, though studies have suggested a need for the specificity provided by this method16,17. Previous investigations have shown the reliability of this method and a variety of ways in which temperature transponders can be used in comparison with other temperature-testing methods18 or in conjunction with surgical methods (e.g., cannulation19). However, studies of this nature rely on different strategic placements to measure overall body temperature20,21,22 or specified tissues such as BAT23,24,25.
Rather than measuring temperature from these locations or while using ear or rectal thermometers26, the method described here provides specificity for the muscle of interest. The ability to target a site by directly implanting transponders adjacent to the muscles of interest is more effective for probing muscle thermogenesis specifically. It provides a new avenue in addition to those provided by surface infrared thermometry27,28 or cutaneous temperature measurements via thermocouple29. Furthermore, the data provided through this method offer a range of avenues of research, avoiding the need for large, expensive, high-tech equipment and software such as infrared thermography30,31,32.
This method has been successfully used to measure temperature in the quadriceps and gastrocnemius, either unilaterally or bilaterally. This method has also been effective in conjunction with stereotaxic surgery14,15. Within ~7-10 cm&#160;of the transponder limb, portable transponder readers (DAS-8027/DAS-7007R) are used to scan, measure, and display the temperature. This distance has been critical and valuable to prior investigations8,9,10 because it minimizes potential stressors and temperature-altering variables such as animal handling during the testing procedures. Using timers, measurements can then be recorded and collected over a period of time without direct interaction with the animals.
To further minimize the disturbance of mice during testing, this method describes the assembly and use of risers made of PVC piping to give the experimenter access to the bottom of the home cages during testing. Using the risers in tandem with the digital reader, temperature measurements of the transponder limb can be made without any animal interaction after the stimulus is placed. At a minimal cost, this method can be used in conjunction with pharmacological and contextual stimuli, making it quite accessible for researchers. Additionally, this method can be employed with a substantial number of subjects (~16 mice or ~12 rats) at a time, saving time in increasing the overall throughput for any research project.
Introduced in this method is a&#160;crafted mechanism for presenting odors to mice using stainless steel mesh tea infuser balls, from now on referred to as &#34;tea balls&#34;. Though these tea balls are ideal for containing any odor material, in these studies, towels that served as in-cage bedding over 2-3 weeks for ferrets, a natural predator of mice and rats, are placed within each treatment tea ball. Each towel is cut into 5 cm x 5 cm squares. This aliquoting is also repeated with otherwise identical odorless control towels. Presenting these odors without a barrier (i.e., tea ball) led to mice shredding the fibers within their cages, increasing physical activity. This behavior was not as salient in rats. Tea balls provide a ventilated casing to the towel, giving full access to the odor while staying protected for the entirety of the experimental trial. These tea balls can be sanitized in accordance with animal use protocols, prepared, and introduced directly after surgery to begin habituating the animals to the structure along with the control stimulus. Mice can then live with the additional enrichment, decreasing the salience of the acute stimulus presentation.
Habituation to the presence of the tea ball is only one aspect of habituation that is critical to this method. The described habituation protocol also consists of repeated exposure to the testing procedure to normalize the testing environment (i.e., personnel, transportation and movement to the testing location, exposure to stimulus). This extended habituation minimizes nuanced responses from the animals and focuses measurements on the desired dependent variables (e.g., pharmacological or contextual stimuli). Previous assessment of this protocol has identified four trials as the minimum number of habituations necessary before temperature testing within home cages in rats8. If testing is separated by long periods (more than 2-3 weeks), the animals must be habituated again. For repeated habituation, a minimum of one to two trials are sufficient. However, if temperature tests are separated by more prolonged bouts of time, repeating more trials may be necessary.
In the continued effort to accustom mice and rats to the testing procedure, an acclimation period before stimulus presentation should be included in every experimental trial. This acclimation time is critical to rebalance temperature and activity after being shifted to the testing location. Rodents tend to have sharp temperature increases due to translocation. Acclimation should consist of a minimum of 1 h without interaction from the experimenter on the day of testing before any addition of a pharmacological agent or contextual stimuli. This is necessary each day of testing.
In the outlined home-cage temperature tests, mice have the free range of their home cage to roam in response to the tested stimulus. This can cause variable shifts in activity, impacting the accuracy of temperature readings and, therefore, the analysis of the thermogenic effects of the independent variable (e.g., pharmacological or contextual stimulus). In recognition of the potential changes in temperature due to activity level, a protocol is included below describing the use of temperature during treadmill walking. The published literature describes the successful use of this procedure in rats, and it is currently being employed with mice8,10,14,15. Treadmill walking maintains a constant speed of activity for the testing subject. For this study, treadmills are strictly used to control activity level and, therefore, are set to the lowest available speed on the treadmill to promote walking for mice and a similarly low setting for rats.
The following procedure is outlined for the temperature measurement of unilateral gastrocnemius in mice and predator odor presentation. The design can be used in conjunction with pharmacological agents and is transferrable to rats and other skeletal muscle groups (i.e., quadriceps) in mice. For rats, transponders can be placed in the gastrocnemius bilaterally and in brown adipose tissue. Due to size and distance limitations, only one transponder can be used per mouse. Minor modifications (e.g., the removal of contextual stimuli) can be made to assess thermogenic responses to pharmacological agents.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1012M-2 Modular Enclosed Metabolic Treadmill for Mice, 2 Lanes w/ Shock</td>
			<td>Columbus Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1012R-2 Modular Enclosed Metabolic Treadmill for Rats, 2 Lanes w/ Shock</td>
			<td>Columbus Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-1/4 in. Ratcheting PVC Cutter</td>
			<td>BrassCraft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringes</td>
			<td>Fisher Scientific</td>
			<td>BD 309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Swabs</td>
			<td>Fisher Scientific</td>
			<td>19-898-945</td>
			<td></td>
		</tr>
		<tr>
			<td>Booster Coil</td>
			<td>BioMedic Data Systems</td>
			<td></td>
			<td>Transponder Accessory</td>
		</tr>
		<tr>
			<td>Electric Clippers</td>
			<td>Andis</td>
			<td></td>
			<td>40 Ultraedge Clipper Blade</td>
		</tr>
		<tr>
			<td>Flexible Mirror Sheets</td>
			<td>Amazon</td>
			<td></td>
			<td>Self Adhesive Non Glass Mirror Tiles</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>89259-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Induction Chamber (isoflurane)</td>
			<td>Kent Scientific</td>
			<td>VetFlo-0730</td>
			<td>3.0 L Low Cost Chambers for Traditional Vaporizers</td>
		</tr>
		<tr>
			<td>Ketoprophen</td>
			<td>Med-Vet Intl.</td>
			<td>RXKETO-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Strips</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnets</td>
			<td>Amazon</td>
			<td></td>
			<td>DIYMAG Magnetic Hooks 40lbs</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Med-Vet Intl.</td>
			<td>26400</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin/Polymixin/Bacitracin with Hydrocortisone Ophthalmic Ointment, 3.5 g</td>
			<td>Med-Vet Intl.</td>
			<td>RXNPB-HC</td>
			<td></td>
		</tr>
		<tr>
			<td>Oasis Absorbable Suture</td>
			<td>Med-Vet Intl.</td>
			<td>MV-H821-V</td>
			<td></td>
		</tr>
		<tr>
			<td>Predator (Ferret) Odor Towels</td>
			<td>Marshall BioResources</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVC pipe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex Wound Clip Remover</td>
			<td>CellPoint Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex Wound Clip, 7 mm (mouse)</td>
			<td>CellPoint Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex Wound Clip, 9 mm (rat)</td>
			<td>CellPoint Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Srerile Autoclip, 7 mm (mouse)</td>
			<td>CellPoint Scientific</td>
			<td></td>
			<td>Wound Clip Applier (mouse)</td>
		</tr>
		<tr>
			<td>Stainless Strainers Interval Seasonings Tea Infuser</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Autoclip, 9 mm (rat)</td>
			<td>CellPoint Scientific</td>
			<td></td>
			<td>Wound Clip Applier (rat)</td>
		</tr>
		<tr>
			<td>Sterile Saline</td>
			<td>Med-Vet Intl.</td>
			<td>RX0.9NACL-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Towels (Control/Habituation)</td>
			<td>Amazon</td>
			<td></td>
			<td>100% Cotton Towels, white</td>
		</tr>
		<tr>
			<td>Transponders</td>
			<td>BioMedic Data Systems</td>
			<td></td>
			<td>Model: IPTT-300</td>
		</tr>
		<tr>
			<td>Transponders Reader</td>
			<td>BioMedic Data Systems</td>
			<td></td>
			<td>Model: DAS-8027-IUS/ DAS-7007R</td>
		</tr>
		<tr>
			<td>Versaclean</td>
			<td>Fisher Scientific</td>
			<td>18-200-700</td>
			<td>liquid detergent</td>
		</tr>
		<tr>
			<td>Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>22-246-073&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wedge pieces for PVC pipe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring skeletal muscle thermogenesis in mice and rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Transponders were unilaterally implanted into the right gastrocnemius of ten 4-6-month-old, wild-type (WT) mice bred from the SF1-Cre strain (Tg(Nr5a1-cre)7Lowl/J, Strain #012462, C57BL/6J and FVB backgrounds; female N = 5; male N = 5). After recovery, the mice were habituated to a home cage temperature-testing procedure that did not include a contextual stimulus (e.g., PO). Temperature measurements using a transponder wand were recorded within their housing room and after transfer to the testing location. Mice were give...

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Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

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Behavior

2.8K Views.  Kent State University. Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed). 

Video: Measuring Skeletal Muscle Thermogenesis in Mice and Rats

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Measuring Attentional Biases for Threat in Children and Adults

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this domain has focused almost exclusively on adult participants, completely ignoring the topic of threat detection over the course of development. One of the biggest reasons for the lack of developmental work in this area is likely the absence of a reliable paradigm that can measure perceptual biases for threat in children. To address this issue, we recently designed a modified visual search paradigm similar to the standard adult paradigm that is appropriate for studying threat detection in preschool-aged participants. Here we describe this new procedure. In the general paradigm, we present participants with matrices of color photographs, and ask them to find and touch a target on the screen. Latency to touch the target is recorded. Using a touch-screen monitor makes the procedure simple and easy, allowing us to collect data in participants ranging from 3 years of age to adults. Thus far, the paradigm has consistently shown that both adults and children detect threatening stimuli (e.g., snakes, spiders, angry/fearful faces) more quickly than neutral stimuli (e.g., flowers, mushrooms, happy/neutral faces). Altogether, this procedure provides an important new tool for researchers interested in studying the development of attentional biases for threat.

Here we describe a touch-screen visual search paradigm that can be used to study threat detection across the lifespan. The paradigm has already been used in various studies demonstrating that both children and adults detect threatening stimuli like snakes, spiders, and angry faces faster than non-threatening stimuli.

Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...