Name: mimicking a space mission to mars using hindlimb unloading and partial weight bearing in rats
Uploaded: 2019-04-04T14:00:00
Description: Watch this Scientific Journal Video about Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats at JoVE.com

This work was supported by the National Aeronautics and Space Administration (NASA: NNX16AL36G). Authors would like to thank Carson Semple for providing the drawings included in this manuscript.

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center under protocol number 067-2016.
NOTE: Male Wistar rats aged of 14 weeks at baseline (day 0) are used. Rats are housed individually in custom cages 24 h prior baseline to allow for acclimation.
1. Hindlimb unloading
NOTE: The pelvic harness can be put on either anesthetized or awake animals. Here, the de...

<ol>
	<li>Desplanches, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Functional+Adaptations+of+Skeletal+Muscle+to+Weightlessness.">Structural and Functional Adaptations of Skeletal Muscle to Weightlessness.</a> International Journal of Sports Medicine. 18 (S4), (1997).</li><li>Fitts, R. H., Riley, D. R., Wildrick, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+microgravity+environment&#8239;:+Invited+review&#8239;:+microgravity+and+skeletal+muscle.">Physiology of a microgravity environment&#8239;: Invited review&#8239;: microgravity and skeletal muscle.</a> Journal of Applied Physiology. 89, 823-839 (2000).</li><li>Fitts, R. H., Riley, D. R., Widrick, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+adaptations+of+skeletal+muscle+to+microgravity.">Functional and structural adaptations of skeletal muscle to microgravity.</a> The Journal of Experimental Biology. 204 (Pt 18), 3201-3208 (2001).</li><li>Narici, M. V., De Boer, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disuse+of+the+musculo-skeletal+system+in+space+and+on+earth.">Disuse of the musculo-skeletal system in space and on earth.</a> European Journal of Applied Physiology. 111 (3), 403-420 (2011).</li><li>di Prampero, P. E., Narici, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+microgravity:+from+fibres+to+human+motion.">Muscles in microgravity: from fibres to human motion.</a> Journal of Biomechanics. 36 (3), 403-412 (2003).</li><li>Wagner, E. B., Granzella, N. P., Saito, H., Newman, D. J., Young, L. R., Bouxsein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+weight+suspension:+a+novel+murine+model+for+investigating+adaptation+to+reduced+musculoskeletal+loading.">Partial weight suspension: a novel murine model for investigating adaptation to reduced musculoskeletal loading.</a> Journal of Applied Physiology (Bethesda, Md.&#8239;: 1985). 109 (2), 350-357 (2010).</li><li>Sung, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+and+hind+limb+unloading+induce+similar+changes+in+electrical+impedance+characteristics+of+mouse+gastrocnemius+muscle.">Spaceflight and hind limb unloading induce similar changes in electrical impedance characteristics of mouse gastrocnemius muscle.</a> Journal of Musculoskeletal and Neuronal Interactions. 13 (4), 405-411 (2013).</li><li>Mcdonald, K. S., Blaser, C. A., Fitts, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-velocity+and+power+characteristics+of+rat+soleus+muscle+fibers+after+hindlimb+suspension.">Force-velocity and power characteristics of rat soleus muscle fibers after hindlimb suspension.</a> Journal of Applied Physiology. 77 (4), 1609-1616 (1994).</li><li>Chowdhury, P., Long, A., Harris, G., Soulsby, M. E., Dobretsov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+of+simulated+microgravity:+a+comparative+study+of+hindlimb+unloading+via+tail+versus+pelvic+suspension.">Animal model of simulated microgravity: a comparative study of hindlimb unloading via tail versus pelvic suspension.</a> Physiological Reports. 1 (1), e00012 (2013).</li><li>Morey, E. R., Sabelman, E. E., Turner, R. T., Baylink, D. 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+rat+model+simulating+some+aspects+of+space+flight.">A new rat model simulating some aspects of space flight.</a> The Physiologist. 22 (6), (1979).</li><li>. National Space Exploration Campaign Report Available from: <a target="_blank" href="https://www.nasa.gov/sites/default/files/atoms/files/nationalspaceexplorationcampaign.pdf">https://www.nasa.gov/sites/default/files/atoms/files/nationalspaceexplorationcampaign.pdf</a> (2018)</li><li>Mortreux, M., Nagy, J. A., Ko, F. C., Bouxsein, M. L., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+partial+gravity+ground-based+analogue+for+rats+via+quadrupedal+unloading.">A novel partial gravity ground-based analogue for rats via quadrupedal unloading.</a> Journal of Applied Physiology. 125, 175-182 (2018).</li><li>Ellman, 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=Combined+effects+of+botulinum+toxin+injection+and+hind+limb+unloading+on+bone+and+muscle.">Combined effects of botulinum toxin injection and hind limb unloading on bone and muscle.</a> Calcified Tissue International. 94 (3), (2014).</li><li>Swift, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+Weight+Bearing+Does+Not+Prevent+Musculoskeletal+Losses+Associated+with+Disuse.">Partial Weight Bearing Does Not Prevent Musculoskeletal Losses Associated with Disuse.</a> Medicine &#38; Science in Sports &#38; Exercise. 45 (11), 2052-2060 (2013).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+technical+aspects.">Hindlimb unloading rodent model: technical aspects.</a> Journal of Applied Physiology. 92 (4), 1367-1377 (2002).</li><li>Andreev-Andrievskiy, A. A., Popova, A. S., Lagereva, E. A., Vinogradova, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shift+versus+body+size:+changes+of+hematological+parameters+and+body+fluid+volume+in+hindlimb-unloaded+mice,+rats+and+rabbits.">Fluid shift versus body size: changes of hematological parameters and body fluid volume in hindlimb-unloaded mice, rats and rabbits.</a> Journal of Experimental Biology. 221 (Pt 17), (2018).</li></ol>

This model presents the first ground-based analogue developed to investigate successive mechanical unloading levels and aims to mimic a trip to and stay on Mars.
Many steps of this protocol are critical to ensure its success and need to be closely examined. First, it is crucial to monitor the wellbeing of the animals and ensure that they are maintaining a normal behavior (i.e., performing tasks such as eating, resting, and exploring), particularly during the PWB state where they maintain a rel...

Extraterrestrial targets, including the Moon and Mars, represent the future of human space exploration, but both have considerably weaker gravity than Earth. While the consequences of weightlessness on the musculoskeletal system have been extensively studied in astronauts1,2,3,4,5 and in rodents6,7,8,9, the latter thanks to the well-established hindlimb unloading (HLU) model10, very little is known about the effects of partial gravity. Martian gravity is 38% of Earth&#8217;s and this planet has become the focus of long-term exploration11; hence, it is crucial to understand the muscular alterations that may occur in this setting. To do so, we developed a partial weight bearing (PWB) system in rats12, based on previous work done in mice6,13, which was validated using both muscle and bone outcomes. However, the exploration of Mars will be preceded by a prolonged period of microgravity, which was not addressed in our previously described model12. Therefore, in this study, we altered our model to mimic a trip to Mars, comprised of a first phase of total hindlimb unloading and immediately followed by a second phase of partial weight bearing at 40% of normal loading.
Unlike most HLU models, we chose to use a pelvic harness (based on the one described by Chowdhury et al.9) rather than a tail suspension to improve animals&#8217; comfort and to be able to move seamlessly and effortlessly from HLU to PWB in a matter of minutes. In conjunction, we used the cages and suspension devices that we previously developed and described extensively12. In addition to providing reliable/consistent data, we also previously demonstrated that the fixed attachment point of the suspension system at the center of the rod did not prevent the animals from moving, grooming, feeding, or drinking. In this article, we will describe how to unload the animals&#8217; hind limbs (both totally and partially), verify their achieved gravity levels, as well as how to functionally assess the resulting muscular alterations using grip force and wet muscle mass. This model would be extremely useful for researchers seeking to investigate the consequences of partial gravity (either artificial or extra-terrestrial) on an already compromised musculoskeletal system, thus allowing them to investigate how organisms adapt to partial reloading, and for the development of countermeasures that could be developed to maintain health during and after human spaceflight.

<table border="0" cellpadding="0" cellspacing="0" style="width:1668px;" width="1667">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">10G Insulated Solid Copper Wire</td>
			<td style="width:320px;">Grainger</td>
			<td style="width:133px;">4WYY8</td>
			<td style="width:987px;">100 ft solid building wire with THHN wire type and 10 AWG wire size, black</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 Custom design plexiglass walls</td>
			<td>P&#38;K Custom Acrylics Inc.</td>
			<td>N/A</td>
			<td>2 clear plexiglass custom wall 3/16&#34; tick, width 12 3/16&#34;, height 18 13/16&#34;, 1 rounded slot 0.25 in of diameter located at the center top of the wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Transpore Surgical Tape</td>
			<td>Fisher Scientific</td>
			<td>18-999-380</td>
			<td>Transpore Surgical Tape&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Accessory Grasping Bar Rat</td>
			<td style="width:320px;">Harvard Apparatus</td>
			<td style="width:133px;">76-0479</td>
			<td>Accessory grasping bar rat, front or hind paws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analytical Scale</td>
			<td>Fisher Scientific</td>
			<td>01-920-251</td>
			<td>OHAUS Adventurer Analytic Balance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animal Scale</td>
			<td>ZIEIS by Amazon</td>
			<td>N/A</td>
			<td>70 lb capacity digital scale big top 11.5&#34; x 9.3&#34; dura platform z-seal 110V adapter 0.5 ounce accuracy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Bra Extenders</td>
			<td>Luzen by Amazon</td>
			<td>N/A</td>
			<td>17 pcs 2 hook 3 rows assorted random color women spacing bra clip extender strap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Force Gage</td>
			<td>Wagner Instruments</td>
			<td>DFE2-010</td>
			<td>50 N Capacity Digital Grip Force Meter Chatillon DFE II</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gauze</td>
			<td>Fisher Scientific</td>
			<td>13-761-52</td>
			<td>Non-sterile Cotton Gauze Sponges&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Key rings and swivel claps</td>
			<td>Paxcoo Direct by Amazon</td>
			<td>N/A</td>
			<td>PaxCoo 100 pcs metal swivel lanyard snap hook with key rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lobster Claps</td>
			<td>Panda Jewelry International Limited by Amazon</td>
			<td>N/A</td>
			<td>Pandahall 100 pcs grade A stainless steel lobster claw clasps 13x8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tether Jacket - Large</td>
			<td>Braintree Scientific</td>
			<td>RJ L</td>
			<td>Rodent Jacket</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Tether Jacket - Medium</td>
			<td>Braintree Scientific</td>
			<td>RJ M</td>
			<td>Rodent Jacket</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone tubing</td>
			<td>Versilon St Gobain Ceramics and Plastics</td>
			<td>ABX00011</td>
			<td>SPX-50 Silicone Tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Chains</td>
			<td>Super Lover by Amazon</td>
			<td>N/A</td>
			<td>4.5m 15FT stainless steel cable chain link in bulk 6x8mm</td>
		</tr>
	</tbody>
</table>

mimicking a space mission to mars using hindlimb unloading and partial weight bearing in rats

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been routinely employed since 1979 and the development of hind limb unloading (HLU). However, the next steps in space exploration now include to travel to Mars where the gravity is 38% of Earth&#8217;s gravity. Since no human being has experienced this level of partial gravity, a sustainable ground-based model is necessary to investigate how the body, already impaired by the time spent in microgravity, would react to this partial load. Here, we used our innovative partial weight-bearing (PWB) model to mimic a short mission and stay on Mars to assess the physiological impairments in the hind limb muscles induced by two different levels of reduced gravity applied in sequential fashion. This could provide a safe, ground-based model to study the musculoskeletal adaptations to gravitational change and to establish effective countermeasures to preserve astronauts&#8217; health and function.

Taking advantage of the new cages that we previously designed and described in detail12, we used a stainless steel chain-based suspension device that is suitable for both hindlimb unloading (HLU, Figure 1) and partial weight-bearing (PWB, Figure 2). The critical advantage of our design is the ability to go from one type of unloading to the other in a matter of minutes while maintaining an identical environ...

Watch this Scientific Journal Video about Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats at JoVE.com

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

By using an innovative ground-based analogue model, we are able to simulate a space mission including a trip to (0 g) and a stay on Mars (0.38 g) in rats. This model allows for a longitudinal assessment of the physiological changes occurring during the two hypo-gravitational stages of the mission.

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been ...

Our protocol offers a significant advantage by creating the possibility to expose animals to successive unloading levels. In the past, ground-based models have used either hindlimb unloading or partial weight-bearing separately. The main advantage of this technique is the use of a single apparatus and environment to provide numerous possibilities such as mimicking a trip to, and short stay on Mars.This model will help understand the physiological state upon landing on Mars and the physiological challenges for astronauts exposed to several unloading levels during their mission. This model could help researchers explore a wide range of physiological changes including but not limited to behavior, memory, performance or stress. Demonstrating the procedure will be Daniela Riveros, a Research Fellow in our laboratory.To perform hindlimb unloading, place the fully anesthetized rat on the bench with anesthetic gas coming from a nose cone with 2%isoflurane and an oxygen flow of 1.5 liters per minute. Place the rat in a prone position and put the pelvic harness on in a rostrocaudal movement. Gently bend the pelvic harness to provide a snug fit while being careful not to squeeze the hind limbs to prevent abrasions and discomfort.Attach the stainless steel chain with the swivel clasp to the top of the pelvic harness, where a hook is attached at the base of the tail. Remove the rat from anesthesia and place the animal in a custom cage with the chain extended at its maximum. Once the rat is fully awake and mobile, shorten the chain using the top swivel clasp until the hind limbs can no longer reach the floor.Convert the hindlimb unloading suspension device into a partial weight-bearing suspension device by adding the triangle-shaped part, composed of stainless steel chains and a back rod. Place a tether jacket of the appropriate size on the fore limbs of the rat, and close it using the back bra extender. Attach one clasp of the triangle-shaped part of the hook located on the back bra extender, and the opposite clasp on the hook located on the pelvic harness at the base of the tail.Place the rat on top of the scale to record the loaded body weight including the weight of the entire apparatus without shortening the chain. Shorten the chain until the scale displays 40%of the loaded body weight and record the achieved gravity level. With the rod, remove the entire apparatus from the scale and place the rat back in its cage.To assess the hindlimb grip force, hold the rat with the traditional restraint by placing one hand underneath the fore limbs. Gently hold the tail with the second hand. Approach the grip bar with the rear paws, and make sure that both paws are fully resting on the bar.Finally, gently pull the rat straight back until it releases its grip. Repeat three times and record the maximal force displayed on the transducer. Calculate the average of the three measurements to account for fatigue.One week of HLU followed by one week of PWB40 resulted in a significant body weight loss similar to what was observed in rats exposed at only PWB40 for the same duration. However, these rats continued losing weight over time while subsequently being exposed to PWB40. One week of total unloading led to a decrease in hindlimb grip force compared to age-matched controls.However, after one subsequent week of PWB40, no further change regarding grip force was observed. Additionally, rats that underwent total unloading followed by partial weight-bearing displayed a significantly greater grip force loss compared to the PWB40 group. PWB40 in HLU-PWB40 groups had significantly lower wet mass of soleus, gastrocnemius, and tibialis anterior muscles than age-matched controls.However, no significant difference between the wet mass of the PWB40 and HLU-PWB40 groups were observed. The success of this method relies on the accuracy of the partial weight-bearing level. Frequent monitoring is crucial to assess animal's wellness and unloading daily.This method is non-invasive and therefore doesn't prevent the utilization of additional testing procedures regarding all systems. Longitudinal measures are also easy to implement if the loading time remains controlled. This method was entirely developed in the laboratory and the utilization of custom equipment can be challenging for new teams.This technique opens the way for extensive studies in which partial weight-bearing is at play. This could include clinical situations such as use of assistive equipment, for example crutches, canes or casts, as well as aqua-therapy.

mimicking-space-mission-to-mars-using-hindlimb-unloading-partial

Research

JoVE Journal

Behavior

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 ...

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 ...

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 ...

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and &#945;7 nicotinic acetylcholine (&#945;7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

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

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

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

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

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

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

A Laboratory Method to Measure Contagious Yawning in Rats

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. Communication is also the way sexes interact during courtship and how rivals settle disputes without fighting. However, there are some behavioral patterns for which it is difficult to test the existence of a communicatory function, because several types of sensory modalities are likely involved. For example, contagious yawning is a communicatory act in mammals that potentially occurs through sight, hearing, smell, or a combination of these senses depending on whether the animals are familiar to one another. Therefore, to test hypotheses about the possible communicatory role of such behaviors, a suitable method is necessary to identify the participating sensory modalities.
The method proposed here aims to obtain yawn contagion curves for familiar and unfamiliar rats and evaluate the relative participation of visual and olfactory sensory modalities. The method uses inexpensive materials, and with some minor changes, it can also be used with other rodent species such as mice. Overall, the method involves the substitution of clear dividers (with or without holes) with opaque dividers (with or without holes) that either allow or prevent communication between rats placed in adjacent cages with holes in adjoining sides. Accordingly, four conditions can be tested: olfactory communication, visual communication, both visual and olfactory communication, and neither visual nor olfactory communication. As social interaction occurs between the rats, these test conditions simulate what may occur in a natural environment. In this respect, the method proposed here is more effective than traditional methods that rely on video presentations whose biological validity can raise concerns. Nonetheless, it does not discriminate between the potential role of hearing and roles of smell and vision in yawn contagion.

The method described here aims to obtain yawn contagion curves in pairs of familiar or unfamiliar male rats. Cages with holes separated by either clear or opaque partitions with (or without) holes are used to detect whether visual, olfactory, or both types of sensory cues can stimulate yawn contagion.

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. ...

Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut

Operant conditioning chambers are used to perform a wide range of behavioral tests in the field of neuroscience. The recorded data is typically based on the triggering of lever and nose-poke sensors present inside the chambers. While this provides a detailed view of when and how animals perform certain responses, it cannot be used to evaluate behaviors that do not trigger any sensors. As such, assessing how animals position themselves and move inside the chamber is rarely possible. To obtain this information, researchers generally have to record and analyze videos. Manufacturers of operant conditioning chambers can typically supply their customers with high-quality camera setups. However, these can be very costly and do not necessarily fit chambers from other manufacturers or other behavioral test setups. The current protocol describes how to build an inexpensive and versatile video camera using hobby electronics components. It further describes how to use the image analysis software package DeepLabCut to track the status of a strong light signal, as well as the position of a rat, in videos gathered from an operant conditioning chamber. The former is a great aid when selecting short segments of interest in videos that cover entire test sessions, and the latter enables analysis of parameters that cannot be obtained from the data logs produced by the operant chambers.

This protocol describes how to build a small and versatile video camera, and how to use videos obtained from it to train a neural network to track the position of an animal inside operant conditioning chambers. This is a valuable complement to standard analyses of data logs obtained from operant conditioning tests.

Operant conditioning chambers are used to perform a wide range of behavioral tests in the field of neuroscience. The recorded data is typically based on ...