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...

<ol>
	<li>Periasamy, M., Herrera, J. L., Reis, F. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+thermogenesis+and+its+role+in+whole+body+energy+metabolism.">Skeletal muscle thermogenesis and its role in whole body energy metabolism.</a> Diabetes Metabolism Journal. 41 (5), 327-336 (2017).</li><li>Rowland, L. A., 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=The+role+of+skeletal-muscle-based+thermogenic+mechanisms+in+vertebrate+endothermy.">The role of skeletal-muscle-based thermogenic mechanisms in vertebrate endothermy.</a> Biological Reviews of the Cambridge Philosophical Society. 90 (4), 1279-1297 (2015).</li><li>Maurya, S. 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=Sarcolipin+is+a+key+determinant+of+the+basal+metabolic+rate,+and+its+overexpression+enhances+energy+expenditure+and+resistance+against+diet-induced+obesity.">Sarcolipin is a key determinant of the basal metabolic rate, and its overexpression enhances energy expenditure and resistance against diet-induced obesity.</a> Journal of Biological Chemistry. 290 (17), 10840-10849 (2015).</li><li>Grigg, G., 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=Whole-body+endothermy:+Ancient,+homologous+and+widespread+among+the+ancestors+of+mammals,+birds+and+crocodylians.">Whole-body endothermy: Ancient, homologous and widespread among the ancestors of mammals, birds and crocodylians.</a> Biological Reviews of the Cambridge Philosophical Society. 97 (2), 766-801 (2022).</li><li>Franck, J. 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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.

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			<td>BioMedic Data Systems</td>
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			<td>BioMedic Data Systems</td>
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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...

Watch this Scientific Journal Video about Measuring Skeletal Muscle Thermogenesis in Mice and Rats at JoVE.com

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. ...

This protocol is significant, as it provides a direct and cost-effective method for measuring skeletal muscle temperature. Moreover, it can be used in various contexts and under the stimulation of pharmacological agents. The main advantage of this technique is the number of animals that can be tested simultaneously.The minimal animal disturbance described here, avoids the need for high-tech methods like infrared thermography. Individuals may have difficulty measuring the temperature of the animals within the home cage or on treadmills while in motion. This can be mitigated by practicing measuring techniques during habituation trials.Begin by warming the enclosed transponders between gloved hands and make sure to maintain sterility, while working with transponders. Measure the temperature using a temperature scanner and check whether the transponder reads the temperature changes. After anesthetizing the mouse as described in the manuscript, use surgical scissors to make a shallow cut through the skin on the right hind limb.Place the sharp edge of a pre-programmed and uncapped sterile transponder into the incision, parallel to the gastrocnemius. Make sure the green plunger faces up and is visible. Keep pushing the transponder applicator into the incision until the opening of the transponder applicator is no longer visible.Turn the applicator at 180 degrees, so that the green plunger faces down toward the mouse's limb and is no longer visible to the experimenter. Once the transponder applicator is placed adjacent to or partially enclosed in the gastrocnemius, push the green plunger, allowing the applicator's pressure to guide the investigator's hand away from the mouse. Using forceps, hold the open skin together.Place a wound clip with a sterile autoclip, bend if required. Use absorbable sutures before the sterile autoclip to close the fascia layer. Using the transponder reader, check the temperature of the mouse muscle.After removing the mouse from the anesthesia, place it in a clean home cage on top of a heating pad and ensure that the home cage includes a tea ball with an odorless towel to begin habituation. Assign a location to the riser in the testing room, and to avoid confounding variables, separate the risers set to receive different contextual stimuli by a minimum of two meters. Using magnetic strips, attach surgical sheets across the riser and create a visual barrier to minimize temperature fluctuations due to mouse activity, in response to the experimenter's movement.Prepare tea balls with control and predator odor towels. Transfer the animal to the prepared testing room. Place the animal in a preassigned location on the riser and avoid changing the riser's location between habituation and testing procedures.Remove the home cage ball from the mouse's home cage. Recover the cage with a cloth or surgical sheet and allow the mouse to acclimate to the testing space for one to two hours. After acclimation is complete, use the scanner to measure and record the baseline temperature of the subject.Uncover the cage and place the tea ball onto the floor of the home cage. Replace the cage lid and cloth covering. Start the stopwatch and measure the temperatures of the test subjects in the same order of tea ball placement.Record the temperatures and clock time of measurements, following the desired time points. Prepare the treadmills for testing, ensuring the shockers are functional. Ensure that the animals use the same treadmill for habituation and testing procedures.Move the mouse to the testing room, and allow it to acclimate for one to two hours in its home cage. Before moving the mouse to the treadmill, measure and record its baseline temperature. For easy placement and removal, adhere the control or predator odor towels to the ceiling or underneath the front of the treadmill.Guide the mouse into the assigned treadmill. Turn on the treadmill belt and shocker, start the stopwatch, and take measurements of the test subjects in the same order they were set up in the treadmills. Record the temperatures and clock time of the measurements, following the desired time points.Once the test is complete, turn off the shocker and treadmill and return the mouse to its home cage. Clean the treadmill using liquid detergent and water and remove any residual predator odor. Repeated habituation trials significantly decreased the muscle temperature of mice, showing mice habituated to the testing environment and protocol.The combined sex analysis of trial four showed no significant difference between before move and baseline temperature measurements, demonstrating the effectiveness of one hour acclimation to the testing context. Pharmacological stimulation using oxytocin in mice showed decreased muscle temperature relative to baseline with a maximal decrease after 30 minutes. Muscle temperatures were normalized 60 minutes after oxytocin injection.A sustained increase in muscle temperature was observed in Sprague Dawley rats, after removing predator odor as contextual stimuli. Sprague Dawley rats displayed a robust increase in temperature in response to ferret odor, compared to control odor. In the presence of other aversive odors, ferret odor produces and maintains a robust thermogenic change compared to all other conditions.While attempting this procedure, it is critical to habituate the animals to the testing protocol and to schedule one to two hours of acclimation each day of testing.

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JoVE Journal

Behavior

2.7K Görüntüleme.  Kent State University. Bu protokol, iskelet kası sıcaklığını ölçmek için doğrudan ve uygun maliyetli bir yöntem sağladığı için önemlidir. Ayrıca, çeşitli bağlamlarda ve farmakolojik ajanların uyarımı altında kullanılabilir. Bu tekniğin temel avantajı, aynı anda test edilebilecek hayvan sayısıdır.Burada tarif edilen minimum hayvan rahatsızlığı, kızılötesi termografi gibi yüksek teknolojili yöntemlere duyulan ihtiyacı ortadan kaldırır. Bireyler, hareket halindeyken e...