This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) to Y.S. (grant number: JP18J10733) from the Japan Society for the Promotion of Science.

This experimental protocol was approved by the Doshisha Committee of Animal Experiment.
NOTE: Conduct all experimental sessions during the light period.
1. Animals
<ol>
	<li>Use 15 experimentally na&#239;ve male albino Wistar rats that weigh&#160;300&#8722;350 g. House them in individual cages of a controlled breeding room with the appropriate temperature and humidity (23 &#177; 2 &#176;C, 70%) and ad libitum access to the water. Maintain the light/dark cycle at 12 h/12 h (the light period begins at 8:00 AM).</li>
	<li>Assign 10 rats as subjects, and the remaining as confederate rats using randomization.</li>
	<li>Maintain rats&#8217; weights at 85&#8722;90% of their free-feeding weights throughout the experiment by food deprivation. 
	NOTE: The assignment of confederate is to reduce the number of animals&#160;required and time for one study.&#160;</li>
</ol>
2. Apparatus
NOTE: The overview of the apparatus is shown in Figure 1. The apparatus was developed and modified in reference to previous studies16,17.
<ol>
	<li>Construct a clear acrylic box (19 cm x 110 cm x 20 cm) with a central partition (5 cm wide) and insert two guillotine doors. Place the guillotine doors 15 cm away from each end of the box. Mount a buzzer (400 Hz, 75 dB) on the partition, and set up a pellet dispenser for serving a reward pellet (45 mg) into a food receptacle on the subject&#8217;s side of the box.</li>
	<li>Set up a metal lever (grip bar: &#966;3 mm, 5 cm high) on a shelf inside the central partition, which has a slit (1.5 cm wide) on each wall facing the runway (Figure 2), in order to allow the subject rat to access the lever by using its forelimbs. On the confederate&#8217;s side, however, insert a transparent wall in front of the partition&#8217;s wall that prohibits the confederate from accessing the lever.</li>
	<li>Place a switch for activating the dispenser under the lever that is pressed by the lever-pull movement of rats. Tie the proper length of gut to the lever, which is needed for the experimenter to pull up the lever.</li>
	<li>Use Arduino Mega 2560 REV3 to control the guillotine doors, buzzer, and pellet dispenser, and to obtain the value from the infrared sensors and switch of the dispenser. Prepare an air compressor (25 L) to operate the air cylinder and open the guillotine door.</li>
	<li>Place a video camera near the partition of the outside of the apparatus, in order to record lever-pull performance of the rats from a lateral view (60 frames per second [fps]). Use an adequate video playback software to conduct frame-by-frame analysis of the video recording.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/60176/60176fig1.jpg" /> 
Figure 1: A schematic of the apparatus used in this protocol. A central partition divides the box into two fields. There is a guillotine door on each side of the box, and the door divides the field into the start area and runway. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig01largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/60176/60176fig2.jpg" /> 
Figure 2: The central partition of the apparatus. Rats can grasp a bar and pull the lever through a slit of the partition. A switch of the pellet dispenser is set under the lever, and one lever-pull action results in one pellet delivery. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig02largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Procedure
<ol>
	<li>Handling and habituation for the reward pellets
	<ol>
		<li>Before the training procedures, handle all rats for 10 min/day for 3 days by the experimenter.</li>
		<li>As a habituation for the reward pellets, give each rat 3 g of reward pellets for 3 days after handling.</li>
	</ol>
	</li>
	<li>Habituation to the apparatus
	<ol>
		<li>Subject rats
		<ol>
			<li>On the third day of habituation for the reward pellets, before the experimenter gives daily pellets, place each rat in the runway of the subject&#8217;s side of the apparatus for 15 min for habituation, with the guillotine door closed.</li>
		</ol>
		</li>
		<li>Confederate rats 
		NOTE: There are two habituation sessions.
		<ol>
			<li>At the first habituation, place each rat on the runway of the confederate&#8217;s side of the apparatus for 15 min during the same period as step 3.2.1.1.</li>
			<li>At the second habituation, follow the method in step 3.2.2.1 on the day after subject rats completed training for the run-and-pull sequence (as mentioned in section 3.6). 
			NOTE: Conduct habituation sessions for confederate rats and subject rats separately, i.e., one rat at a time. Do not let a rat meet another rat in the apparatus until the test phase starts.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Magazine training 
	NOTE: For the subject rats, conduct a magazine training session on the next day of the habituation to the apparatus as follows. If rats do not eat all the food pellets, then conduct the magazine training session again on the next day.
	<ol>
		<li>Put one reward pellet on the food receptacle on the confederate&#8217;s side to exclude the effect of the olfactory property of the pellet, which the confederate eats in the test phase (refer to section 3.7).</li>
		<li>Place a subject rat on the runway of the subject&#8217;s side, with the guillotine door closed.</li>
		<li>Deliver a reward pellet into the food receptacle 60 times using the pellet dispenser on a variable time 30-s schedule. Ensure that the subject rat eats all the delivered pellets.</li>
	</ol>
	</li>
	<li>Shaping for the lever-pull action 
	NOTE: Figure 3 is a flowchart of the experiment.
	<ol>
		<li>Put one reward pellet on the food receptacle on each subject&#8217;s side and confederate&#8217;s side. Do not place the confederate rat in shaping sessions.</li>
		<li>Place the subject rat on the runway of the subject&#8217;s side with the guillotine door closed. Then train the rats to pull down the lever. Shape the lever-pull behavior gradually with following five reinforcement criteria (A-E): (A) Approach the slit. (B) Touch the grip bar to the rat&#8217;s muzzle&#160;or forelimbs, with the lever beforehand pulled down to the subject&#8217;s side. (C) Touch the grip bar, with the lever inclined to the rat&#8217;s side (at an angle of 60&#176;&#8722;30&#176;). (D) Grasp and pull down the bar to rat&#8217;s side using the same condition as in criterion C. (E) Grasp and pull down the bar to rat&#8217;s side with the lever standing upright.</li>
		<li>Finish daily sessions when 60 rewards are given, or 30 min has elapsed. When a rat completes criterion E 40 times in a session, the lever-pull shaping phase is finished on that day. 
		NOTE: Almost all rats finish the daily shaping session within 20 min. Wistar rats need about 3 days to reach criterion E.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/60176/60176fig3.jpg" /> 
Figure 3: A flowchart of the experimental procedure. Subject rats go through the training phases and test phases in this order. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig03largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Training for the lever-pull action
	<ol>
		<li>Place the subject rat on the start area. Do not place the confederate rat in the training sessions. Present the buzzer tone for 5 s, and then open the door. When the subject rat pulls down the lever, deliver a food pellet and pull the lever up using a gut tied to the lever.</li>
		<li>After rats pull down the lever for 10 times (first 3 days of the phase) or 6 times (last 3 days of the phase) and consume all reward pellets, close the door and move them to the start area by the experimenter&#8217;s hand.</li>
		<li>After an inter-trial interval (ITI) of 20 s, repeat steps 3.5.1 and 3.5.2. Finish daily sessions when each rat has earned 60 pellets. 
		NOTE: This training also aims to habituate rats to the buzzer tone and door opening because Wistar rats ordinarily show freezing behavior at first in response to door movement. A daily session for each rat requires about 15 min. Six days are needed for this training phase.</li>
	</ol>
	</li>
	<li>Training phase for the run-and-pull sequence
	<ol>
		<li>Conduct the same procedure in step 3.5.1.</li>
		<li>When rats pull down the lever one time and consume a reward pellet, close the door and move them to the start area by the experimenter&#8217;s hand.</li>
		<li>After an ITI of 20 s, start the next trial (steps 3.6.1 and 3.6.2). Finish the daily session when each rat has earned 30 pellets. 
		NOTE: A daily session for each rat requires about 20 min. About 10 days are needed for rats&#8217; scores to reach asymptote.</li>
	</ol>
	</li>
	<li>Test phases</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/60176/60176fig4.jpg" /> 
Figure 4: Description of each test condition. In single phases, the subject rat performed the task solitarily. In the pair phases, place the confederate rat in the runway on the opposite side to the subject rat. A transparent wall in front of the partition precludes the confederate rat from accessing the lever. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig04largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: Test phases include sessions in two conditions, single or pair (Figure 4). In the single condition, rats perform the task solitarily; that is, trials are identical to the session of the training for the run-and-pull sequence (section 3.6). In the pair condition, the confederate rat is present at the opposite side of the box. The confederate rat cannot access the lever because of the clear acrylic wall in front of the partition.
<ol start="1">
	<li>Conduct a single session that is identical to the sessions in the training phase for the run-and-pull sequence (section 3.6). Finish the daily session when each rat has earned 30 pellets. Put one reward pellet on the food receptacle on the confederate&#8217;s side, especially during sessions in the single condition.</li>
	<li>In pair sessions, give the confederate rat a reward pellet during the ITI of the subject rat in order to keep the confederate rat near the partition.</li>
</ol>
4. Data Analysis
<ol>
	<li>Index of performance accuracy
	<ol>
		<li>Record videos of rats&#8217; lever-pull movement using the video camera near the partition of the outside of the apparatus. After all sessions are completed, confirm the evaluation by frame-by-frame analysis of the video recordings using an adequate video playback software.</li>
		<li>Evaluate whether the rat&#8217;s pulling movement of a trial was the first-hit or not by visual observation of the experimenter during the experiment. 
		NOTE: The first-hit trial is defined as the trial in which rats could grasp and pull down the lever during their first attempt to the lever.</li>
		<li>Calculate first-hit rates for each subject as the proportion of &#64257;rst-hit trials to all trials in each session (for the run-and-pull training phase) or in each phase (for test phases) to use in the analysis.</li>
	</ol>
	</li>
	<li>Indices of performance speed
	<ol>
		<li>Calculate the time needed to complete a trial from the value of the switch of the pellet dispenser: the time needed to complete a trial = (the time when the switch is pressed) - (the time when the door is opened). Then divide the time needed for completion of a trial into three sections using values from the infrared sensors (Figure 5). 
		NOTE: Start latency (Figure 5a) is defined as the time from the door opening to the arrival of the rat at the &#64257;rst sensor. Similarly, running time (Figure 5b) is the time from the arrival at the &#64257;rst sensor to the arrival at the second sensor. Lever-pull latency (Figure 5c) is the time from the arrival at the second sensor to the time point when the switch of the dispenser is pressed.</li>
		<li>Use only the durations from the &#64257;rst-hit trials for analysis. Calculate median values of each subject for each session (of the training sessions) and each phase (of the test phases) for analysis.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/60176/60176fig5.jpg" /> 
Figure 5: Measurement of the indices of performance speed. (a) Start latency: the duration from the door opening to the arrival of the rat at the &#64257;rst sensor. (b) Running time: the duration from the arrival of the rat at the &#64257;rst sensor to its arrival at the second sensor. (c) Lever-pull latency: the duration from the arrival of the rat at the second sensor to the completion of a lever-pull response. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig05largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Statistical analysis
	<ol>
		<li>For the indices in the training phase of the run-and-pull sequence, conduct one-way repeated-measure analysis of variance (ANOVA) with the number of sessions as the within-subject factor for each index.</li>
		<li>For the indices in the test phase, conduct two-way repeated-measure ANOVA with the number of the phase (phase 1 or 2) and condition (pair or single) as the within-subject factors for each index in the test phases. Statistical signi&#64257;cance was set at &#945; = 0.05.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Allport, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Social Psychology. , (1924).</li><li>Guerin, 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> Social Facilitation. , (1993).</li><li>Zajonc, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+facilitation.">Social facilitation.</a> Science. 149 (3681), 269-274 (1965).</li><li>Markus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+mere+presence+on+social+facilitation:+an+unobtrusive+test.">The effect of mere presence on social facilitation: an unobtrusive test.</a> The Journal of Social Psychology. 14 (4), 389-397 (1978).</li><li>Schmitt, B. H., Gilovich, T., Goore, N., Joseph, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mere+presence+and+social+facilitation:+one+more+time.">Mere presence and social facilitation: one more time.</a> The Journal of Experimental Social Psychology. 22 (3), 242-248 (1986).</li><li>Pinto, A., Oates, J., Grutter, A., Bshary, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaner+wrasses+Labroides+dimidiatus+are+more+cooperative+in+the+presence+of+an+audience.">Cleaner wrasses Labroides dimidiatus are more cooperative in the presence of an audience.</a> Current Biology. 21 (13), 1140-1144 (2011).</li><li>Gipson, C. D., 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=Social+facilitation+of+d-amphetamine+self-administration+in+rats.">Social facilitation of d-amphetamine self-administration in rats.</a> Experimental and Clinical Psychopharmacology. 19 (6), 409-419 (2011).</li><li>Levine, J. M., Zentall, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+conspecific's+presence+on+deprived+rats'+performance:+social+facilitation+vs+distraction/imitation.">Effect of a conspecific's presence on deprived rats' performance: social facilitation vs distraction/imitation.</a> Animal Learning &amp; Behavior. 2 (2), 119-122 (1974).</li><li>Reynaud, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+facilitation:+a+meta-analysis+of+241+studies.">Social facilitation: a meta-analysis of 241 studies.</a> Psychological Bulletin. 94 (2), 265-292 (1983).</li><li>Strauss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+facilitation+in+motor+tasks:+a+review+of+research+and+theory.">Social facilitation in motor tasks: a review of research and theory.</a> Psychology of Sport and Exercise. 3 (3), 237-256 (2002).</li><li>Sekiguchi, Y., Hata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mere+presence+of+conspecifics+on+the+motor+performance+of+rats:+higher+speed+and+lower+accuracy.">Effects of the mere presence of conspecifics on the motor performance of rats: higher speed and lower accuracy.</a> Behavioural Processes. 159, 1-8 (2019).</li><li>Ogura, Y., Matsushima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+facilitation+revisited:+increase+in+foraging+efforts+and+synchronization+of+running+in+domestic+chicks.">Social facilitation revisited: increase in foraging efforts and synchronization of running in domestic chicks.</a> Frontiers in Neuroscience. 5, 91 (2011).</li><li>Dorfman, A., Nielbo, K. L., Eilam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traveling+companions+add+complexity+and+hinder+performance+in+the+spatial+behavior+of+rats.">Traveling companions add complexity and hinder performance in the spatial behavior of rats.</a> PLoS One. 11, e0146137 (2016).</li><li>Takano, Y., Ukezono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+task+to+examine+the+mirror+system+in+rats.">An experimental task to examine the mirror system in rats.</a> Scientific Reports. 4, 6652 (2014).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+an+action+pattern:+stability+in+rat+(Rattus+norvegicus)+grasping+movements+as+a+function+of+changing+food+pellet+size.">Skilled reaching an action pattern: stability in rat (Rattus norvegicus) grasping movements as a function of changing food pellet size.</a> Behavioural Brain Research. 116 (2), 111-122 (2000).</li><li>Cohen, 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> Statistical Power Analysis for the Behavioral Sciences. , (1988).</li></ol>

The authors have nothing to disclose.

This task enables us to evaluate the effect of the mere presence of others on speed and accuracy of motor performance. The effect sizes reported herein would be large enough. We recalculated &#951;2 (there was not much difference between &#951;2 and &#951;2G in this experiment), and these effect sizes are regarded as medium (&#951;2 &#62; 0.06) or large (&#951;2 &#62; 0.14) according to the criterion presented by Cohen18. For this reason, we considered that the differences seen in this study are meaningful and reliable. Results in the experiment were almost in line with those from studies in humans4,10, and the result of Ogura and Matsushima&#8217;s study14, which investigated the effect of co-action on the speed and accuracy of running and pecking behavior in chicks. Although studies about social facilitation in humans investigated the effect of social conditions on both speed and accuracy of behavior, most previous studies in animals have not investigated the mere presence effect on performance accuracy. The protocol presented herein provides a better animal model for investigating the effect of mere presence on motor performance.
As a limitation, the difference between the first-hit rate in pair and single conditions could be interpreted as an effect of practice. Despite presumably sufficient training, there could be room for improvement in the performance of rats. There were no more changes in the indices of performance accuracy and performance speed in the last four sessions of the training phase for the run-and-pull sequence. However, in the test phases, the first-hit rate increased continuously. This increase might be interpreted as the effect of practice. Additionally, this experimental design (A-B-A-B design) cannot exclude the effect of practice from the effect of conditions. Future experiments should (1) use the A-B-B-A design or another appropriate experimental design to exclude the effect of practice, and (2) consider extending the training phase for the run-and-pull sequence.
This protocol can be used in a study with a between-subject design, although the answer to the questions &#8220;Which design is appropriate? Within subject or between subject?&#8221; would be on a case-by-case basis. Generally, a study using the between-subject design can exclude the practice effect that might be observed in this study. However, the between-subject design requires more animals and more time for one study (i.e., if using the protocol in this article, 7&#8722;8 h per day would be needed to test all the rats in 1 day using one apparatus). A study that uses the within-subject design can reduce the number of animals required and time for one study, but the experimenter must control the effect of practice. Time and cost should be carefully weighed before the experimenter choses the experimental design.
With minor modification, this task can be applied to investigate the effect of co-action or social facilitation by mirror10 and other social situations that have been studied in humans on the performance speed and performance accuracy of rats. To investigate the effect of co-action, separate the lever in the central partition into two levers, and arrange the levers so that each lever can be pulled by one side of the box. To investigate the effect of using a mirror, change the acrylic clear wall on the confederate&#8217;s side of the box to the mirror. In the same manner, the effect of an invisible confederate behind the opaque wall could be investigated. Future studies using these modifications will contribute to comprehensively understanding social facilitation by multifaceted comparison of motor performance between species.

The effect of social conditions on one&#8217;s performance has been investigated in humans and animals for a long time, since Allport1 referred to &#8220;social facilitation&#8221; as &#8220;an increase in response merely from the sight or sound of others making the same movement&#8221;2. Although Allport1 did not distinguish the social situation (co-action or the mere presence of another), it has been shown that the mere presence of other(s) affects one&#8217;s performance speed or frequency3,4,5,6. Additionally, in animals, the mere presence of other conspecific results in a higher response rate or higher response speed during a lever-press task in rats7,8 and higher response rate in rhesus monkeys during a simple cognitive task9.
In humans, it has been shown that social situations affect not only response frequency or speed but also accuracy of performance10. Based on a meta-analysis by Bond and Titus11, Strauss12 argued that situations used in studies on social facilitation would have a different effect depending on features of the task used. Especially, decrement in the performance would be expected when the study used a task that placed high demands on one&#8217;s ability to control his/her body precisely and to perform with some extent of speed, which tends to be scored by its qualitative aspects (e.g., accuracy of the performance)13.
Except for a couple studies14,15, however, most studies on social facilitation in animals have not focused on the accuracy of performance. For instance, Takano and Ukezono16 investigated the effect of mere presence in rats using a skilled reaching task17. They required rats to turn around and then grasp a reward pellet on a shelf by using their forelimb. The authors reported only the performance speed, despite the fact that the task could provide an index of performance accuracy. Conversely, Ogura and Matsushima14 examined the effect of co-action on the pecking accuracy as well as running speed in chicks. The result showed that the pecking accuracy was lower and running speed was higher in the co-action situation than in the solitary situation.
Although Ogura and Matsushima14 focused on the qualitative aspect of an action for the first time, their study was about the effect of co-action. Most social conditions, including co-action, inevitably imply the presence of another. To examine the effect unique to co-action, it is indispensable to dissociate the effect of mere presence from that of co-action on an individual&#8217;s performance. However, the study did not investigate the effect of mere presence. To our knowledge, no study has examined the effect of mere presence on the accuracy of performance in animals.
We modified the task used in Takano and Ukezono&#8217;s study16 to evaluate the effects of mere presence on both performance speed and accuracy. This method enables us to examine the effect of social conditions, especially the mere presence of a conspecific, on performance accuracy and performance speed in rats in one experiment.

<table>
	<tbody>
		<tr>
			<td>45 mg Dustless Precision Pellets 
			Rodent, Purified</td>
			<td>Bio-Serv.</td>
			<td>F0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Mega 2560 REV3</td>
			<td>Arduino S.r.l.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellets Dispenser with Feeder (Rats)</td>
			<td>Harvard Apparatus</td>
			<td>76-0353</td>
			<td></td>
		</tr>
		<tr>
			<td>Power DVD 14</td>
			<td>CyberLink</td>
			<td>None</td>
			<td>Use an adequate video playback program which enables frame-by-frame playback.</td>
		</tr>
		<tr>
			<td>Run-and-pull task apparatus</td>
			<td>Bio Medica Corp.</td>
			<td>Custom-made item</td>
			<td>The set of apparatus (box), an air compressor, and a control device for air cylinders which receives inputs from Arduino.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>JVC</td>
			<td>GZ-R300</td>
			<td></td>
		</tr>
	</tbody>
</table>

a task for assessing the impact of a partner on the speed and accuracy of motor performance in rats

To our knowledge, no study has examined the effect of mere presence on accuracy of performance in animals. Therefore, we developed an experimental task to measure rats&#8217; motor performance (speed and accuracy) in a social condition. Rats were trained to run on a runway and pull down a lever at the end of the runway. In testing, rats performed the task solitarily (single) or in the presence of a confederate rat beyond the lever (pair or a social condition). As indices of the performance speed, we measured the time needed to start running, run through the runway, and pull down the lever. As the index of performance accuracy, we counted the number of trials in which rats could pull down the lever during their first attempt. One-way and two-way repeated-measure analyses of variance were used to analyze the data. This run-and-pull task enabled us to examine the effect of the presence of another conspecific on both speed and accuracy of motor performance in one experiment. The results showed that rats performed the task faster but less accurately in pair sessions than in single sessions. This protocol would be a valid animal model to examine the effect of mere presence on speed and accuracy of motor performance in rats.

Training phase for the run-and-pull sequence
Figure 6 shows the mean &#177; standard error of the mean (SEM) scores from the training phase for the run-and-pull sequence. The mean &#64257;rst-hit rate (Figure 6A) increased gradually during the first half of the training phase and then stopped at about 85%. The ANOVA results showed that the main effect of the number of sessions was signi&#64257;cant (F(7,63) = 3.74, p = 0.002, &#951;2G = 0.211). Multiple comparisons revealed that there were no signi&#64257;cant differences among the latter four sessions (all p values &#62; 0.60).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60176/60176fig6.jpg" /> 
Figure 6: Mean &#177; SEM scores from sessions in the training phase for the run-and-pull sequence. (A) Index of performance accuracy. (B) Indices of performance speed. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig06largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similarly, indices of performance speed (Figure 6B; start latency, running time, and lever-pull latency) decreased continuously during the &#64257;rst four sessions, and all values stabilized at about 600 ms among the latter four sessions. For all indices, ANOVA showed that main effects of the number of sessions were significant (start latency: (F(7,63) = 6.21, p &#60; 0.001, &#951;2G = 0.279; running time: (F(7,63) = 3.98, p = 0.001, &#951;2G = 0.170; lever-pull latency: (F(7,63) = 11.85, p &#60; 0.001, &#951;2G = 0.350). Multiple comparisons by sessions resulted in no signi&#64257;cant difference among the latter four sessions for all measures (all p values &#62; 0.12).
Figure 7 shows the mean &#177; SEM scores from sessions in the test phase. Regarding the index of performance accuracy, the &#64257;rst-hit rate (Figure 7A) in pair phases was lower than in the single phases. Additionally, the &#64257;rst-hit rate in the second phase was higher than in the &#64257;rst phase in the two conditions. The results of ANOVA showed significant main effects of the condition (F(1,9) = 6.25, p = 0.034, &#951;2G = 0.114) and phases (F(1,9) = 14.1, p = 0.005, &#951;2G = 0.147), but the interaction was not significant (F(1,9) = 0.15, p = 0.703, &#951;2G = 0.002).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60176/60176fig7.jpg" /> 
Figure 7: Mean &#177; SEM scores from sessions in the test phase. Index of performance accuracy (A: first-hit rate) and indices of performance speed (B: start latency, C: running time, and D: lever-pull latency). *** p &#60; 0.001, ** p &#60; 0.01, * p &#60; 0.05. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig07largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Regarding indices of performance speed, the start latency in pair phases (Figure 7B) was shorter than in single phases. The ANOVA results showed that for the start latency, only the main effect of the condition was signi&#64257;cant (F(1,9) = 23.1, p = 0.001, &#951;2G = 0.065), while the main effect of phases and the interaction was not signi&#64257;cant (phases: F(1,9) = 0.03, p = 0.878, &#951;2G &#60; 0.001; interaction: F(1,9) = 0.002, p = 0.970, &#951;2G &#60; 0.001). Similarly, a difference was observed between conditions for the lever-pull latency (Figure 7D). As with the start latency, for the lever-pull latency, ANOVA showed a significant main effect of the condition (F(1,9) = 23.3, p = 0.001, &#951;2G = 0.183). There was no significant main effect of phases (F(1,9) = 2.72, p = 0.133, &#951;2G = 0.028) and the interaction (F(1,9) = 1.07, p = 0.327, &#951;2G = 0.002). For the running time, there was no signi&#64257;cant effect (Figure 7C, condition: F(1,9) = 3.03, p = 0.116, &#951;2G = 0.004; phases: F(1,9) = 4.46, p = 0.063, &#951;2G = 0.010; interaction: F(1,9) = 0.29, p = 0.602, &#951;2G &#60; 0.001).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60176/60176fig1.jpg" /> 
Figure 1: A schematic of the apparatus used in this protocol. A central partition divides the box into two fields. There is a guillotine door on each side of the box, and the door divides the field into the start area and runway. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig01largev3.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/60176/60176fig2.jpg" /> 
Figure 2: The central partition of the apparatus. Rats can grasp a bar and pull the lever through a slit of the partition. A switch of the pellet dispenser is set under the lever, and one lever-pull action results in one pellet delivery. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig02largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60176/60176fig3.jpg" /> 
Figure 3: A flowchart of the experimental procedure. Subject rats go through the training phases and test phases in this order. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig03largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60176/60176fig4.jpg" /> 
Figure 4: Description of each test condition. In single phases, the subject rat performed the task solitarily. In the pair phases, place the confederate rat in the runway on the opposite side to the subject rat. A transparent wall in front of the partition precludes the confederate rat from accessing the lever. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig04largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60176/60176fig5.jpg" /> 
Figure 5: Measurement of the indices of performance speed. (a) Start latency: the duration from the door opening to the arrival of the rat at the &#64257;rst sensor. (b) Running time: the duration from the arrival of the rat at the &#64257;rst sensor to its arrival at the second sensor. (c) Lever-pull latency: the duration from the arrival of the rat at the second sensor to the completion of a lever-pull response. This figure has been modified from Sekiguchi and Hata12. <a href="https://www.jove.com/files/ftp_upload/60176/60176fig05largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Task for Assessing the Impact of a Partner on the Speed and Accuracy of Motor Performance in Rats

A procedure to measure the speed and accuracy of rats&rsquo; motor performance in a social condition is described. The protocol enables us to investigate the effect of the mere presence of others on speed and accuracy of motor performance in one experiment.

To our knowledge, no study has examined the effect of mere presence on accuracy of performance in animals. Therefore, we developed an experimental task to ...

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4.8K Views.  Doshisha University. A procedure to measure the speed and accuracy of rats’ motor performance in a social condition is described. The protocol enables us to investigate the effect of the mere presence of others on speed and accuracy of motor performance in one experiment.

Video: A Task for Assessing the Impact of a Partner on the Speed and Accuracy of Motor Performance in Rats

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 ...

The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents

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This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, which are modifications of the standard task, increase flexibility of the task and can be combined with other manipulations to more fully characterize behavior.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content

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The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The ...

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

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

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

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

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

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

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This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that involves finger-pressing movements while sitting, the modified SRT task requires participants to step with both feet while maintaining a standing posture. This stepping task necessitates whole body actions that impose postural challenges. The foot-stepping task complements the classic SRT task in several ways. The foot-stepping SRT task is a better proxy for the daily activities that require ongoing postural control, and thus may help us better understand sequence learning in real-life situations. In addition, response time serves as an indicator of sequence learning in the classic SRT task, but it is unclear whether response time, reaction time (RT) representing mental process, or movement time (MT) reflecting the movement itself, is a key player in motor sequence learning. The foot-stepping SRT task allows researchers to disentangle response time into RT and MT, which may clarify how motor planning and movement execution are involved in sequence learning. Lastly, postural control and cognition are interactively related, but little is known about how postural control interacts with learning motor sequences. With a motion capture system, the movement of the whole body (e.g., the center of mass (COM)) can be recorded. Such measures allow us to reveal the dynamic processes underlying discrete responses measured by RT and MT, and may aid in elucidating the relationship between postural control and the explicit and implicit processes involved in sequence learning. Details of the experimental set-up, procedure, and data processing are described. The representative data are adopted from one of our previous studies. Results are related to response time, RT, and MT, as well as the relationship between the anticipatory postural response and the explicit processes involved in implicit motor sequence learning.

The &quot;Motor&quot; in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task

We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning.

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that ...

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 ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...

Generating Acute and Chronic Experimental Models of Motor Tic Expression in Rats

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic generation is associated with abnormal inhibition of the basal ganglia, particularly its primary input structure, the striatum. In animal models of both rodents and non-human primates, local application of GABAA antagonists, such as bicuculline and picrotoxin, into the motor parts of the striatum induces local disinhibition resulting in the expression of motor tics.
Here, we present acute and chronic models of motor tics in rats. In the acute model, bicuculline microinjections through a cannula implanted in the dorsal striatum elicit the expression of tics lasting for short time periods of up to an hour. The chronic model is an alternative enabling the extension of tic expression to periods of several days or even weeks, utilizing continuous infusion of bicuculline via a sub-cutaneous mini-osmotic pump.
The models enable the study of the behavioral and neural mechanisms of tic generation throughout the cortico-basal ganglia pathway. The models support the implantation of additional recording and stimulation devices in addition to the injection cannulas, thus allowing for a wide variety of usages such as electrical and optical stimulation and electrophysiological recordings. Each method has different advantages and shortcomings: the acute model enables the comparison of the kinematic properties of movement and the corresponding electrophysiological changes before, during and after tic expression and the effects of short-term modulators on tic expression. This acute model is simple to establish; however, it is limited to a short period of time. The chronic model, while more complex, makes feasible the study of tic dynamics and behavioral effects on tic expression over prolonged periods. Thus, the type of empirical query drives the choice between these two complementary models of tic expression.

We present protocols for generating acute and chronic experimental models of tic expression in freely behaving rats. The models are based on striatal cannula implantation and subsequent GABAA antagonist application. The acute model uses transient injections whereas the chronic model utilizes prolonged infusions via a subcutaneous implanted mini-osmotic pump.

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic ...