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>
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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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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&lt;br/&gt; 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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JoVE Journal

Behavior

4.9K Views.  Doshisha University. Dit protocol biedt een beter diermodel voor het onderzoeken van de effecten van de loutere aanwezigheid van een verbonden dier op de motorprestaties. Dit protocol stelt ons in staat om de effecten van verbonden dieren op zowel de snelheid en nauwkeurigheid van de motorische prestaties van ratten in een enkel experiment te evalueren. Monteer voor aanvang van de experimenten een zoemer van 400 hertz 75 decibel op de scheidingswand van het apparaat.En het opzetten van een pellet dispenser...

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

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Neuroscience

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

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, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.

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. ...

Acquisition of a High-precision Skilled Forelimb Reaching Task in Rats

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor learning and execution is of critical importance in order to understand the principles and processes that underlie motor function. Here we present a paradigm to study movement acquisition within a daily session of training (within-session) representing the fast learning component and primary acquisition as well as skilled motor learning over several training sessions (between-session) representing the slow learning component and consolidation of the learned task. This behavioral paradigm increases the degree of difficulty and complexity of the motor skill task due to two features: First, the animal realigns its body prior to each pellet retrieval forcing renewed orientation and preventing movement execution from the same angle. Second, pellets are grasped from a vertical post that matches the diameter of the pellet and is placed in front of the cage. This requires a precise grasp for successful pellet retrieval and thus prevents simple pulling of the pellet towards the animal. In combination with novel genetics, imaging and electrophysiological technologies, this behavioral method will aid to understand the morphological, anatomical and molecular underpinnings of motor learning and memory.

A paradigm is presented to analyze the acquisition of a high-precision skilled forelimb reaching task in rats.

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

An Innovative Running Wheel-based Mechanism for Improved Rat Training Performance

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity for reducing the severity of the effects of the stroke&#160;in rats. This system provides more effective animal exercise training than commercially available systems such as treadmills and motorized running wheels (MRWs). In contrast to an MRW that can only achieve speeds below 20 m/min, rats are permitted to run at a stable speed of 30 m/min on a more spacious and high-density rubber running track supported by a 15 cm wide acrylic wheel with a diameter of 55 cm in this work. Using a predefined adaptive acceleration curve, the system not only reduces the operator error but also trains the rats to run persistently until a specified intensity is reached. As a way to evaluate the exercise effectiveness, real-time position of a rat is detected by four pairs of infrared sensors deployed on the running wheel. Once an adaptive acceleration curve is initiated using a microcontroller, the data obtained by the infrared sensors are automatically recorded and analyzed in a computer. For comparison purposes, 3 week training is conducted on rats using a treadmill, an MRW and a PRW. After surgically inducing middle cerebral artery occlusion (MCAo), modified neurological severity scores (mNSS) and an inclined plane test were conducted to assess the neurological damages to the rats. PRW is experimentally validated as the most effective among such animal mobility systems. Furthermore, an exercise effectiveness measure, based on rat position analysis, showed that there is a high negative correlation between the effective exercise and the infarct volume, and can be employed to quantify a rat training in any type of brain damage reduction&#160;experiments.

This study presents an innovative running wheel-based animal mobility system to quantify an effective exercise activity in rats. A rat-friendly testbed is built, using a predefined adaptive acceleration curve, and a high correlation between the effective exercise rate and the infarct volume suggests the protocol&#39;s potential for stroke prevention experiments.

This study presents an animal mobility system, equipped with a positioning running wheel (PRW), as a way to quantify the efficacy of an exercise activity ...

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 ...

Investigating Motor Skill Learning Processes with a Robotic Manipulandum

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be time-intensive and ambiguous to quantify beyond simple success rates. Here, we describe the training procedure for reach-and-pull tasks with ETH Pattus, a robotic platform for automated forelimb reaching training that records pulling and hand rotation movements in rats. Kinematic quantification of the performed pulling attempts reveals the presence of distinct temporal profiles of movement parameters such as pulling velocity, spatial variability of the pulling trajectory, deviation from midline, as well as pulling success. We show how minor adjustments in the training paradigm result in alterations in these parameters, revealing their relation to task difficulty, general motor function or skilled task execution. Combined with electrophysiological, pharmacological and optogenetic techniques, this paradigm can be used to explore the mechanisms underlying motor learning and memory formation, as well as loss and recovery of function (e.g. after stroke).

A paradigm is presented for training and analysis of an automated skilled reaching task in rats. Analysis of pulling attempts reveals distinct subprocesses of motor learning.

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

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

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