Sylvie Cloutier, Megan LaFollette, Brianna Gaskill, and Ruth Newberry wish to dedicate this article to the memory of Jaak Panksepp, our beloved co-author, who passed away during the final stages of completing this manuscript. We remember Jaak fondly as the &#8220;father of rat tickling&#8221; - his insights into animal emotions form the foundational inspiration for this work. This material is partially based upon work supported by a Washington State University Veterinary Summer Research Fellowship, an Animal Welfare Enhancement Award from the Animal Welfare Institute, and a &#8220;Grant for Laboratory Animal Science&#8221; from the American Association for Laboratory Animal Science. Publication of this article was sponsored by Petco Animal Supplies, Inc. We would like to thank Carol Dowell and Purdue University&#8217;s Laboratory Animal Program for allowing us to tickle their rats for the shoot.

All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals31,32 at facilities accredited by AAALAC International and were approved by the Washington State University Institutional Animal Care and Use Committee.&#160;The following protocol can&#160; expose personnel to laboratory animal allergens. Personal protective equipment should be worn as recommended by the institutional occupational health and safety program and health care providers. The protocol depicts procedures under typical laboratory housing conditions but could be adapted for use in higher-biosecurity facilities (e.g. by using a biosafety cabinet, etc.).&#160;
1. Preparation of Equipment and Setup
<ol>
	<li>Handling box
	<ol>
		<li>Prepare an animal&#39;s home cage or other enclosure for tickling, hereafter called the handling box.</li>
		<li>Make sure the handling box is large enough to provide space for the tickler&#39;s hand and for the rats to move around while preventing them from jumping out.</li>
		<li>Make sure the handling box is free of obstacles. Thus, if conducting tickling in the home cage, remove any objects such as huts, gnawing sticks, and crinkled paper.</li>
		<li>Line the handling box floor with bedding33. Other types of soft floor cover can be used but are not as effective at eliciting 50-kHz vocalizations13.</li>
		<li>Place the handling box on a table or countertop at a height that allows the handler&#39;s hand to comfortably reach the entire floor area of the handling box.</li>
		<li>Minimize the effect of novelty by habituating the rats to the handling box prior to tickling, if needed.</li>
	</ol>
	</li>
	<li>Timer: To indicate the start and end of 15 s bouts, place a timing device near the handling box where it can be easily read. Alternatively, an auditory time signal may be transmitted to the tickler through earphones.</li>
	<li>Vocalization recording
	<ol>
		<li>If vocalizations are being recorded to quantify different vocalization types for research purposes, use a microphone capable of capturing 10-120-kHz broadband sound, connected to a device capable of recording high frequency sounds.</li>
		<li>Suspend or attach the microphone at the top or side of the box, low enough to clearly detect the calls and avoid confusion with calls from rats in nearby cages, but out of physical reach of the rats.</li>
		<li>If vocalizations only need to be monitored to confirm proper tickling technique, then use a bat detector and set it up on the side of the box. If using a simple, heterodyne detector, set the device at 50-kHz.</li>
	</ol>
	</li>
	<li>Laboratory gloves: Use a bare or thinly gloved hand when tickling. The tickling technique was developed using a bare hand.&#160; However, a thinly gloved hand is also effective at inducing playful behavior and 50-kHz USV production while allowing the handler to sense the amount of pressure being applied to the rat&#39;s body and feel the rat&#39;s responses through the glove.</li>
</ol>
2. Tickling Procedure
<ol>
	<li>Put on the laboratory gloves, if required. Bring the home cage to a table or workbench. Gently transfer the rat into the handling box if tickling is not conducted in the home cage. Start the timer, and the audio recording if needed.</li>
	<li>For consistent application, alternate between 15 s of hand contact and 15 s release from contact for a total of 2 min. This duration of interaction has been found sufficient for increasing attraction of rats to humans11.
	<ol>
		<li>To begin the tickling session with a 15 s release, place the hand motionless inside one side of the handling box. The presence of the hand allows rats to solicit interaction by directing licks and light nips (nibbles) towards the hand, indicating the rat&#39;s interest to interact22. When this occurs, move the hand away from the rat, which can solicit playful chasing of the hand by the rat. Alternatively, leave the hand out of the box during the 15 s release.</li>
		<li>Follow the release with 15 s of tickling, initiate the interaction with a dorsal contact (2-4 s). Using one hand, vigorously touch the rat&#39;s nape (dorsal surface of the neck) with the fingertips using rapid finger movements as commonly performed in human tickling. Avoid contact with the rump as this area of the body is targeted during aggressive encounters34.</li>
		<li>Follow the dorsal contact with a&#160;pin. Place&#160;the thumb and middle finger under the rat&#39;s forelegs; lift the rat, just high enough to allow quickly turning the rat on its back while ensuring the tail does not get kinked. 
		NOTE: It may help to lightly push on the back of the rat with the little finger when turning the rat on its back.</li>
		<li>Hold the rat loosely, but firmly; be assertive but not forceful. Once the rat is on its back on the floor, hold the body down loosely while moving the fingers quickly and vigorously but gently on the belly, as is commonly used in human tickling.</li>
		<li>Even though the stimulation is rapid and assertive, keep the pinning brisk (2-4 s/pin) to minimize negative responses. Allow the rat to right itself and rapidly follow with a dorsal contact.</li>
		<li>Continue to alternate between dorsal contacts and pins. On average, there should be 4 to 5 pins/15 s35.</li>
		<li>Do not pick up the rat with both hands. During the 15 s of tickling, always keep the fingers moving and in contact with the rat&#39;s body, even in transitions between pins and dorsal contacts.</li>
		<li>Perform three additional 15-s tickles and 15-s releases for a total of 2 min.</li>
	</ol>
	</li>
	<li>If using a handling box, return the rat to its home cage. If using the home cage, place enrichment objects back in the cage. Clean the handling box with paper towels to remove feces and urine not absorbed by the bedding.</li>
	<li>Repeat with the next cage of rats, as needed.</li>
</ol>
3. Tickling Multiple Rats Simultaneously
<ol>
	<li>Rats can be tickled as a group36. If the handling duration is not relevant, alternate tickling each rat for 15 s. The duration of the tickling session can be extended beyond 2 min as needed so that each rat receives four bouts of tickling hand contact interspersed by release.</li>
	<li>If the handling duration is relevant for research results, then use the following procedures:
	<ol>
		<li>For pairs, tickle one rat for 15 s followed immediately by tickling the second rat for the next 15 s (i.e., during the release of the first rat), and so on. The tickler does not have a rest period.</li>
		<li>For triplets, use the procedure described in 3.2.1 to tickle two of the rats and tickle the third one at the same time as one of the others using the other hand.</li>
		<li>For groups of 4 or more rats, tickle each rat or pair of rats for 15 s. The duration of the tickling session can be extended beyond 2 min as needed.</li>
	</ol>
	</li>
</ol>

The authors declare that they have nothing to disclose.

This protocol describes a technique for rat tickling, a playful handling technique mimicking rat rough-and-tumble play behavior that is effective in generating vocalizations indicative of positive affect and increasing approach behavior when rats are handled for common husbandry or medical and research procedures such as IP injection.
Social play, a typically positive form of social contact, has been proposed to assist in the calibration of emotional reactions to frightening situations42,43, and generate resilience to anxiety and depression44. By mimicking rat social play, tickling appears to have similarly beneficial effects7. Thus, for the application of tickling to be successful at inducing positive affective states, it is important to keep in mind that the hand is intended to act as &#34;the play partner&#34; during the tickling. The hand is attractive because it simulates the behaviors that rats expect and enjoy from another rat during rough-and-tumble play. Hence, lifting and holding the rat with both hands is not mimicking rat play, and may not have the same effect. In fact, laboratory rats will learn to avoid non-playful restraints in which a handler holds them with one hand and strokes them with the other hand45. During rat tickling, it is also important to move the hand confidently and assertively, while being lively and playful. Keeping the pinning brisk (2-4 s/pin) should minimize negative responses because in rough-and-tumble play between rats, repeated pinning leads to uttering of 22-kHz USVs18.
Even though juvenile rats are more playful and show stronger responses to tickling than adult rats18, tickling in adulthood also has benefits10. Tickling rats when young has also been found to have benefits later in life36, suggesting that rats experience a prolonged positive effect on their emotional reaction to human presence. Sex differences in response to tickling are inconsistent, with reports of no sex differences in juvenile rats46,47, juvenile males producing more 50-kHz vocalizations and fewer 22-kHz vocalizations than juvenile females29,48,39, and older females producing more 50-kHz vocalizations than same-aged males29, similar to findings for social play49.
The effectiveness of the tickling technique can be assessed using rat movement patterns and vocalization. Rats will nibble and lick the handler&#39;s fingers during the 15-s periods of release following tickling11. Nibbles are light, non-injurious bites that have been described as play-solicitation behavior in young rats22. When the handler moves the hand around the handling box, the rat will follow and chase it. Rats, especially young ones, will also run, scamper around the box between tickling bouts, and show a distinct increase in activity after a tickling bout, similar to the running activity that characterizes rough-and-tumble play. If the rat remains immobile during releases, adjustment of the tickling technique, such as reducing pin duration or giving fewer pins, is probably needed. It is best to give pins of short duration for the first tickling session. Duration and number of pins can be increased as the rats get used to tickling. Rats utter 50-kHz USVs, including trills, during tickling and between tickling bouts. A gradual increase in the number of trills produced during the 15-s releases can be seen over the first two weeks of daily tickling before stabilizing47. Individual differences allow tickled rats to be divided into high and low callers that respond differentially to stress, anxiety, and anhedonia46,47,50.
Rat tickling has been used successfully to induce positive affect as assessed based on production of 50-kHz USVs during tickling13. It has recently been implemented as a technique to reduce fear of humans in the rats and mitigate their aversiveness of medical and research procedures such as injection. Evidence suggests that, when provided for only 2 min daily per animal, tickling is an effective and practical method for reducing avoidance of humans11. Considering that juvenile rats will play together for about 60 min daily51,52,53, tickling for 2 min daily is practical but presumably too brief to replicate the full effects of social play among rats. Pet rat owners, who generally have more time for handling their rats than laboratory caretakers, may provide rats with additional tickling as long as the rats continue to respond positively. Based on Burgdorf et al.44,45, the first 15 min of rough-and-tumble play seems to be the most positive for rats. In turn, it may be more effective to tickle rats for periods of up to 15 min, especially if for a justified reason they cannot be kept in a social group. However, based on play as &#34;training for the unexpected&#34;38, a more varied technique than the method described here might be needed to maintain the playfulness of longer tickling sessions. Additional tickling sessions each day may also be beneficial as long as other biologically important activities such as sleep and feeding are not impaired. As well as contributing to improving the well-being of laboratory and pet rats, the described standardized tickling technique can be used in continued research aimed at understanding the effects of social play and its components on behavioral, social, and brain development.
When tickling was performed surrounding injection, particularly when combined with previous tickling experience (ending 40-50 days earlier), it increased 50-kHz USV rates before and after injection, and reduced audible call rates during injection10. Thus tickling seems to have mitigated the adverse effects of IP injections, especially in rats accustomed to tickling as juveniles10. Even though tickling did not prevent discomfort associated with the injection as assessed based on production of audible calls, it increased 50-kHz USVs compared to the control condition, before, during and after the restraint procedure12. According to 50-kHz USV data, the most effective time point for applying tickling was just before rather than after the restraint procedure12. Tickling rats before the injection had a carry-over effect that elevated positive affect during the procedure12. Tickling can therefore minimize potential negative effects of routine medical procedures on rat welfare and behavior. More research is necessary to determine how the technique could be paired effectively with other injection techniques and other medical/experimental procedures.
Overall, we have demonstrated that rat tickling results in positive outcomes such as increased production of 50-kHz USV and approach behavior, as well as decreased avoidance of humans and resistance to handling. We recommend rat tickling as a viable method for improving rat welfare and modeling positive affective states.

Handling of small animals, such as domestic rats (Rattus norvegicus) kept in laboratories and as pets, can cause significant increases in behavioral and physiological indicators of stress1, as well as aversive responses suggestive of emotional distress2. Handling rats can trigger fear-based responses towards humans3,4, which may make further handling more difficult, increase the risk of injury to both animal and handler, and limit the validity of experimental data5.
To avoid negative effects of handling on experimental results and human-animal relationships, it is common practice to allow some time for newly acquired young animals to habituate to human interaction prior to assigning them to experimental treatments. During this acclimation period, caretakers may expose rats to a series of events that can be highly variable in both form and duration. These may include passive exposure to people, touching, lifting, petting, holding, talking, and offering food treats. Although combinations of such procedures have been reported to reduce fear of humans6, it is unclear which specific practices are most effective for use by busy animal caretakers. There are reports that rats can have either adverse responses, or at least no favorable response, to some of these individual techniques2,7,8,9.
In contrast, abundant evidence supports the conclusion that tickling, a handling technique that mimics aspects of the playful rough-and-tumble behavior of rats, is a robustly beneficial form of social contact, especially for juvenile rats10,11,12,13. Rats are highly social mammals, which is clear from observing the time they spend playing together as juveniles14 and their strong motivation to re-establish and maintain social contact with conspecifics15,16,17. Rat tickling draws upon the playful social nature of rats to convert handling into a positive experience. This concept was originally developed as a model for investigating the neurobiological basis of positive affective states evoked by social play behavior7. Since then, rat tickling has been used in over 56 different experiments, 22 of which compared tickling to other handling techniques13.
The rat&#39;s rough-and-tumble play is characterized by two key behaviors: &#34;dorsal contact&#34; and &#34;pin&#34; (Figure 1)18,19. During rough-and-tumble play, a rat initiates play by making dorsal contact with the partner&#39;s nape. The recipient responds by rolling supine and is pinned down by the initiator standing above19. The play continues with the rats taking turns being in each position, on top and below18. Human handlers are able to mimic the two key components of rough-and-tumble play by alternating between contact with a rat&#39;s nape (dorsal contact) and ventral surface (pin) using vigorous, quick movements of the fingers similar to those used when tickling a child. Of course, tickling is not an exact copy of rat rough-and-tumble play because rats cannot pin the human hand during tickling. Indeed, the level of engagement of individuals in rough-and-tumble play and tickling can differ20,21. Despite this, it appears that tickling invokes the same reward mechanisms as rough-and-tumble play among conspecifics. Tickling is actively solicited by the animals and can serve as a reward for operant learning, contributing evidence that being tickled is substantially rewarding for young rats7,11,12,13,22. Even though rats have also been reported to learn an operant task in exchange for gentle stroking as the reward, they were faster to approach the hand of a person offering tickling compared to stroking7.
The pins used in tickling may seem similar to common restraint procedures but there is evidence that rats perceive these types of handling differently. When rats are repeatedly restrained, such as for an injection procedure, they secrete excessive porphyrins resulting in chromodacryorrhea (&#34;red tears&#34;)8. Chromodacryorrhea occurs in response to acute stressors and causes the skin and fur around the eyes and nose to become stained a reddish color23. When repeatedly held down and restrained on the back as in a pin, rats showed less attraction to humans than when tickled12. These outcomes imply that the repeated brief restraints and releases during tickling are perceived differently than non-playful restraint. Thus, the precise technique used in tickling has important implications for generating a positive affective state as opposed to contributing to handling stress.
Monitoring the effect of tickling on rat affective state can be easily accomplished by attending to the rats&#39; vocalizations. Rats typically produce two categories of ultrasonic vocalizations (USVs), at around 22-kHz and 50-kHz, in addition to audible vocalizations. The 22-kHz USVs typically occur when anticipating aversive events and are accompanied by elevated brain cholinergic activity24. Therefore, 22-kHz USVs are deemed to indicate a negative affective state of anxiety. The 50-kHz USVs, especially of the frequency-modulated &#34;trill&#34; type (Figure 2), are generally associated with rewarding situations and stimuli and are accompanied by increased brain dopaminergic activity24,25. Accordingly, expression of abundant 50-kHz USVs is considered to reflect a positive affective state24,26,27,28. Finally, audible vocalizations are usually associated with, and therefore used as markers of, physical pain and discomfort. The differential production of these three forms of vocalizations thus offers a reliable record of changes in the affective state of rats26. Rats produce USVs at around 22-kHz during exposure to predators, exposure to painful stimuli such as foot shock, and inter-male fighting28. Stroking has also been reported to elicit multiple 22-kHz USVs in na&#239;ve rats, inferred as indicating negative affect2. In contrast, when tickled by humans, as when engaged in rough-and-tumble play with other rats, rats produce numerous 50-kHz USVs. It follows that the rate of 50-kHz USVs provides a graded measure of the positive affective response generated by tickling29,30.
Existing descriptions of &#34;how to tickle a rat&#34; are rather brief and can lead to variable results. Further, novice &#34;rat ticklers&#34; may be overly tentative in their attempts to mimic rough-and-tumble play. The words &#34;tickle vigorously&#34; are easier to understand when demonstrated visually. Therefore, the goal of this article is to provide a detailed description and demonstration of how to mimic rats&#39; positive playful interactions in an effective and consistent manner that reliably elicits positive responses from most rats when handled. This protocol describes a standardized tickling technique, and explains how tickling can be combined with procedures such as restraint and injection to mitigate potentially unpleasant affective experiences.

<table><tbody><tr><td>Table or workbench</td><td></td><td></td><td>No specific brand is recommended.</td></tr><tr><td>Handling box (rat home cage or another enclosure)</td><td></td><td></td><td>No specific brand is recommended.</td></tr><tr><td>Bedding or or other type of soft cover</td><td></td><td></td><td>No specific brand is recommended.</td></tr><tr><td>Gloves (thin laboratory gloves)</td><td></td><td></td><td>Optional.</td></tr><tr><td>Timing device</td><td>Deltaworks</td><td>Interval Timer:Timing for HIIT Training, Workouts&lt;br/&gt; https://itunes.apple.com/us/app/interval-timer-timing-for-hiit-training-workouts/id406473568?mt=8</td><td>Optional. Any timer will do, although it is convenient to use an interval timer if you are tickling multiple rats.</td></tr><tr><td>Ultrasonic microphone such as a bat detector or USB microphone</td><td>Magenta Electronics LTD</td><td>Bat4 Bat Detector&lt;br/&gt; https://www.magenta2000.co.uk/acatalog/Bat_Detector_Bat4.html</td><td>Optional. The Magenta bat detector is relatively inexpensive and easy to use, but can only detect vocalizations at one range at a time.</td></tr><tr><td></td><td>Dodotronic</td><td>Ultramic 200K&lt;br/&gt; http://www.dodotronic.com/ultramics/?v=7516fd43adaa</td><td>Optional. This microphone has worked well in our experiments. There are many other options for ultrasonic microphones.</td></tr><tr><td>Bioacoustic software</td><td>CIBRA</td><td>SeaWave&lt;br/&gt; http://www-3.unipv.it/cibra/seawave.html</td><td>Optional. Free software with limited recording capabilities.</td></tr><tr><td></td><td>CIBRA</td><td>SeaPro&lt;br/&gt; http://www-3.unipv.it/cibra/seapro.html</td><td>Optional. This software has extensive recording capabilities, and has worked well in our studies. There are many other software packages that work with bioacoustic microphones.</td></tr></tbody></table>

tickling, a technique for inducing positive affect when handling rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

Factors affecting, or potentially affecting, responses to tickling are summarized in Table 1. Positive outcomes from rat tickling have been indicated by multiple measures of rat welfare and positive affect. Common findings include increased production of 50-kHz USVs, increased approach behavior, and increased ease of handling. Reported effects of rat tickling at the genetic and cellular level include altered gene expression in the hypothalamus37, and increased neurogenesis in the dentate gyrus of the hippocampus38,39. Outcomes indicative of reduced generalized anxiety and fear of humans are also reported such as reduced latency to approach in 9 different experiments13, less freezing after fear conditioning40,41, and increased entries into the center of an open field arena by individually housed rats36. For further information see LaFollette et al., who present a systematic review of the tickling literature that identifies 22 reports investigating the outcomes of rat tickling in comparison to other techniques13.
Next, specific examples of results that can be obtained when the tickling technique is paired with restraint and intra-peritoneal (IP) injection procedures are outlined.
Sprague-Dawley rats (males; 85-94 days of age; n = 48) tickled for 2 min immediately before and after IP saline injection daily for 10 days, especially when combined with the juvenile tickling experience (2 min/day from 25-45 days of age), produced more 50-kHz USV just before (GLM ANOVA, F3, 44 = 9.59, p &#60; 0.0001; Figure 3A) and just after (F3, 44 = 8.74, p = 0.0001; Figure 3B) injection (Tukey-Kramer, p &#60; 0.05) compared to rats exposed to a passive hand10. During the actual brief delivery of the injection, production of 50-kHz USVs was curbed (mean &#177; SE, 15 &#177; 1.4 USVs/min) and handling treatment had no effect on their rate10. It was concluded that tickling, when applied both before and after an injection procedure, induced a positive affective state that moderated the aversiveness of the procedure, especially for those that had been exposed to tickling as juveniles.
To find out if tickling would be effective if reduced to before or after an injection rather than both and, if so, which time would be better, rat responses to tickling before, after, and before-after injection were compared. Rates of 50-kHz USVs and audible calls were measured as indicators of positive and negative affect, respectively12. Sprague-Dawley rats (males; 32-41 days of age; n = 96) were either restrained and given a daily IP saline injection (INJ) or restrained without receiving an injection (control, CON), and exposed to one of the following treatments: no tickling, the rats were not touched before and after the restraint procedure (TN); tickled for 2 min right before (TB), after (TA), or before and after (TBA) restraint12. In the 2 min before restraint, rats from treatments that received tickling before the restraint (TB, TBA) produced the most 50-kHz USVs (GLMM ANOVA, F3,88 = 49.19, p &#60; 0.0001; Figure 3C). TA and TN rats did not differ in the rate of USVs produced before the restraint procedure. During the procedure, any rat that received tickling (TA, TB, TBA) produced more 50-kHz USV than those that were not tickled (TN; F3,88 = 50.67, p &#60; 0.0001; Figure 3D). Among tickled rats, those receiving tickling prior to the procedure (TB, TBA) produced more 50-kHz USVs during the restraint compared to the ones tickled afterwards (TA; p &#60; 0.05). In the 2-min period after the procedure, rats in all tickled groups (TA, TB, TBA) produced more 50-kHz USVs than those that were not tickled (TN; F3,88 = 53.64, p &#60; 0.0001; Figure 3E). Furthermore, during the procedure, rats tickled before restraint (TB, TBA) produced fewer audible calls compared to rats that did not received tickling (TN), whereas rats tickled afterwards (TA) did not differ from those in the other groups (F3,88 = 3.67, p= 0.02; Figure 3F). The results demonstrate that tickling rats before restraining them for an IP injection produced signs of greater positive affect across the periods before, during, and after the procedure than tickling only after restraint12.
Caretakers applied one of the following handling treatments to rats (Sprague-Dawley males; n = 32) from 57 to 74 days of age: (1) Minimal handling once a week at cage change; (2) Exposure to a passive hand for 2 min daily; (3) 2 min of tickling each day, to mimic social play; (4) 2 min daily of restraint on the rat&#39;s back, simulating unreciprocated pinning by a dominant11. In the minute following an IP injection, rats from the Tickled and Passive treatments spent more time rearing in the front half of the cage, close to where the researcher was standing, compared to rats from the Minimal and Restraint treatments (GLM ANOVA, F3, 27 = 5.70, p = 0.004; Figure 3G). Additionally, Tickled rats uttered more 50-kHz USVs than rats in the Passive treatment when exposed to a familiar or unfamiliar hand for 3 min in a Human approach test (Log-transformed mean &#177; SE calls/min, Minimal: 0.7 &#177; 0.26, Passive: 0.6 &#177; 0.18, Tickled: 1.8 &#177; 0.30, Restraint: 1.4 &#177; 0.28; F3,28 = 4.04, p = 0.02). Thus, the simple presence of a passive hand aided habituation of rats to humans relative to minimal handling, but tickling was the most effective method for producing a positive response towards humans11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57190/57190fig1.jpg" /> 
Figure 1: Rat rough-and-tumble play behavior. (A) Dorsal contact; (B) Pin18. <a href="https://cloudflare.jove.com/files/ftp_upload/57190/57190fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57190/57190fig2.jpg" /> 
Figure 2: 50-kHz ultrasonic vocalizations of the frequency modulated &#34;trill&#34; type. Five trill vocalizations were produced in 1 s by a rat during tickling. The horizontal lines at 17-, 25-, and 70-kHz were produced by electronic equipment in the room (e.g., computer, microphone, camera).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57190/57190fig3.jpg" /> 
Figure 3: Responses of rats exposed to tickling prior to, or in combination with, experimental procedures such as intra-peritoneal (IP) injections. Rate (mean &#177; SE calls/min) of 50-kHz USVs produced (A) before and (B) after an injection procedure by adult male rats exposed to minimal handling (M) or tickling (T) as juveniles and a passive hand (P) or tickling (T) immediately before and after injection (yielding four treatment combination: M-P, T-P, M-T, T-T); from Cloutier et al.10. Rate of 50-kHz USVs (mean &#177; SE calls/min) produced (C) before, (D) during, and (E) after restraint (with or without an IP injection) once daily for 10 consecutive days by juvenile male rats that received no tickling (TN) or tickling before (TB), after (TA), or both before and after (TBA) the procedure; and (F) the rate of audible calls produced during restraint; from Cloutier et al.12. (G) Duration (mean &#177; SE s) of rearing nearby the researcher during the 1 min following an IP injection by male rats exposed to minimal, passive, tickle, or restraint handling by caretakers; from Cloutier et al.11. Bars with different letters (a, b, c) indicate differences in pairwise comparisons (Tukey-Kramer, p &#60; 0.05). <a href="https://cloudflare.jove.com/files/ftp_upload/57190/57190fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Factor</td>
			<td colspan="4">Effect</td>
			<td>References</td>
		</tr>
		<tr>
			<td>Intrinsic</td>
			<td colspan="4"></td>
			<td></td>
		</tr>
		<tr>
			<td>Age</td>
			<td colspan="4">No difference in 50-kHz ultrasonic vocalizations (USVs) between rats ages 24-44 and 89-92 days old</td>
			<td>18, 29, 42</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Decrease in 50-kHz USVs for rats from 71 days old to 148 days old</td>
			<td>29</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">17 day old males made many more 50-kHz USVs than 7-9 month old males</td>
			<td>43</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Rats tickled as both juveniles and adults showed the most positive results compared to rats only tickled as juveniles or adults.</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td colspan="4">Male rats produced more 50-kHz USVs at 44 and 71 days old, but females produced more 50-kHz USVs at 148 days old</td>
			<td>29</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Male rats produced more 50-kHz USVs than females at 44 and 58 days.</td>
			<td>43</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">No sex differences in 50-kHz USVs in 96 juvenile Long-Evans rats or 52 juvenile Wistar rats</td>
			<td>42, 44</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Female juvenile Wistar rats produced more 22-kHz USVs than males</td>
			<td>44</td>
		</tr>
		<tr>
			<td>Inter-individual differences</td>
			<td colspan="4">Rats that uttered more 50-kHz USVs showed decreased approach time to self-administered tickling, increased positive judgement bias after tickling, and increased hippocampal cell proliferation after tickling</td>
			<td>27, 37, 45</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">More playful rats produced more 50-kHz USVs</td>
			<td>43</td>
		</tr>
		<tr>
			<td>Strain</td>
			<td colspan="4">Not reported, but anecdotal evidence suggests rats from different strains and breeders differ in their response to tickling. Rat lines diverged when selected upon high vs. low 50-KHz USVs rate in response to tickling.</td>
			<td>22, 26, 42, 46</td>
		</tr>
		<tr>
			<td>Previous experience of tickling</td>
			<td colspan="4">Increased production of 50-kHz ultrasonic USVs</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Extrinsic</td>
			<td colspan="4"></td>
			<td></td>
		</tr>
		<tr>
			<td>Bedding</td>
			<td colspan="4">Rats produced more 50-kHz USVs when tickled on familiar bedding vs no bedding</td>
			<td>33</td>
		</tr>
		<tr>
			<td>Stress</td>
			<td colspan="4">Tickling rats 1 hour, but not 23 hours, after restraint stress reduced 50-kHz USVs production</td>
			<td>47, 48</td>
		</tr>
		<tr>
			<td>Timing</td>
			<td colspan="4">Tickling rats before injection was more effective in increasing 50-KHz USVs than tickling after injection</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Sex of handler</td>
			<td colspan="4">No effect found</td>
			<td>Panksepp, J. 2014, Unpublished data</td>
		</tr>
		<tr>
			<td>Familiarity of rat with handler</td>
			<td colspan="4">Rats produced more 50-kHz USVs and spent more time near a familiar compared to an unfamiliar tickler</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Handler tickling experience / vigor of tickling by handler</td>
			<td colspan="4">Affected production of 50-kHz USVs but not preference and approach behavior</td>
			<td>Cloutier, S. 2011, Unpublished data</td>
		</tr>
		<tr>
			<td>Duration of pins by handler / frequency of alternation between dorsal contacts and pins</td>
			<td colspan="4">Lower frequency of nibbles (interpreted as play solicitation) was directed to a hand imposing 15-s pins (restraint) than a hand giving tickles with multiple dorsal contacts and pins per 15 s</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Odorants (e.g., perfume, shampoo, shaving cream) used by handler</td>
			<td colspan="4">Not reported</td>
			<td></td>
		</tr>
		<tr>
			<td>Social group size</td>
			<td colspan="4">Individual housing increased positive responses to tickling compared to group housing in most experiments. Tickling group-housed rats with cage mates in the home cage reduced this difference</td>
			<td>7, 22, 27, 36</td>
		</tr>
		<tr>
			<td>Light intensity</td>
			<td colspan="4">Tickling at 1000 lux reduced 50-kHz USVs compared to 25 lux.</td>
			<td>43</td>
		</tr>
		<tr>
			<td>Noise</td>
			<td colspan="4">Not reported</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage cleaning before tickling</td>
			<td colspan="4">Induced production of 22-kHz USVs in some rats</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Time of day</td>
			<td colspan="4">No effect on 50-kHz USVs production when performed at the beginning or end of the light period. Tickling rats at the beginning/end of the light period vs middle of light or dark period has not yet been compared</td>
			<td>Cloutier, S. 2011, Unpublished data</td>
		</tr>
	</tbody>
</table>
Table 1: List of factors affecting, or potentially affecting, the responses of rats to tickling, a playful handling technique intended to mimic rat rough-and-tumble play.

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Tickling, a Technique for Inducing Positive Affect When Handling Rats

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

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Research

JoVE Journal

Behavior

38.7K Views.  Washington State University. This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Video: Tickling, a Technique for Inducing Positive Affect When Handling Rats

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

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

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

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

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Neuroscience

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

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 ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Handling Techniques to Reduce Stress in Mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

Positive Reinforcement-Based Training for Routine Rabbit Husbandry Procedures

Training Laboratory Rabbits to Refine Routine Husbandry Procedures

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving both animal welfare and scientific quality. Clicker training was utilized to develop training protocols for rabbits to refine stressful routine husbandry procedures usually associated with lifting (i.e., being picked up from the floor)/restraining (i.e., being held in the arms of a human) them. Thirteen female New Zealand White rabbits were trained over three weeks. All rabbits learned the predefined goal behaviors: they followed the target stick, jumped onto the weighing scale, entered a transport box, and reared while placing their front paws onto the trainer&#39;s hand. In addition, ten animals jumped from the floor onto the sitting trainer&#39;s lap and allowed the trainer to lift their paws off the surface while sitting on the trainer&#39;s lap. For some individuals, the protocols had to be adapted by additional interim steps. At the end of the training, the rabbits reliably showed the expected goal behaviors, even after short and long training breaks. With few exceptions, a familiar person other than the trainer could elicit the goal behaviors from the rabbits (generalization), though further sessions were required for generalization. In the voluntary approach test, the rabbits preferred interacting with the trainer in the 1st trial but spent as much time with an unfamiliar person as with the trainer in the 2nd trial. The behavioral observations suggested that picking the rabbits up with the transport box, as described in the protocol, instead of restraining them with the scruff of their neck and lifting them on the arm, was less aversive. All in all, the training protocols were feasible and can serve as a refinement strategy in laboratory animal facilities. In the interest of animal welfare, the training protocols should be applied wherever possible.

Author Spotlight: Training of Laboratory Animals for Gentle and Stress-Free Handling

Training protocols involving positive reinforcement for routine husbandry procedures in rabbits were developed and proved to be successful, as the rabbits reliably showed the trained behaviors at the weekly general examination. Picking the rabbits up with the transport box was less aversive than the conventional handling technique.

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving ...

Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.