Name: measuring skeletal muscle thermogenesis in mice and rats
Uploaded: 2022-07-27T14:40:00
Description: Watch this Scientific Journal Video about Measuring Skeletal Muscle Thermogenesis in Mice and Rats at JoVE.com

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...

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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>
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			<td>1-1/4 in. Ratcheting PVC Cutter</td>
			<td>BrassCraft</td>
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			<td>1 mL Syringes</td>
			<td>Fisher Scientific</td>
			<td>BD 309659</td>
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			<td>Betadine Swabs</td>
			<td>Fisher Scientific</td>
			<td>19-898-945</td>
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			<td>Booster Coil</td>
			<td>BioMedic Data Systems</td>
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			<td>Transponder Accessory</td>
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			<td>Electric Clippers</td>
			<td>Andis</td>
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			<td>40 Ultraedge Clipper Blade</td>
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			<td>Flexible Mirror Sheets</td>
			<td>Amazon</td>
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			<td>Self Adhesive Non Glass Mirror Tiles</td>
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			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>89259-940</td>
			<td></td>
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		<tr>
			<td>Heating Pad</td>
			<td></td>
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		</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>
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			<td>Magnetic Strips</td>
			<td>Amazon</td>
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			<td></td>
		</tr>
		<tr>
			<td>Magnets</td>
			<td>Amazon</td>
			<td></td>
			<td>DIYMAG Magnetic Hooks 40lbs</td>
		</tr>
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			<td>Needles</td>
			<td>Med-Vet Intl.</td>
			<td>26400</td>
			<td></td>
		</tr>
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			<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>
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			<td>Predator (Ferret) Odor Towels</td>
			<td>Marshall BioResources</td>
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			<td>PVC pipe</td>
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			<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>
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			<td>Stainless Strainers Interval Seasonings Tea Infuser</td>
			<td>Amazon</td>
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		</tr>
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			<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>
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			<td>Surgical Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
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			<td>Surgical Sheets</td>
			<td></td>
			<td></td>
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		<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>
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			<td>Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>22-246-073&#160;</td>
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			<td>Wedge pieces for PVC pipe</td>
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	</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...

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.

measuring-skeletal-muscle-thermogenesis-in-mice-and-rats

Research

JoVE Journal

Behavior

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 ...