We thank the Faculty Research Council at Azusa Pacific University for grant support. We also thank Ashley Schneider, Michael Gregg, and Hannah Sample for technical support.

This research was approved by the Azusa Pacific University Institutional Animal Care and Use Committee. All procedures involving animals were carried out following the recommendations of the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.
1. Modifying Apparatuses for Social Motivation and Valence Comparison Testing Paradigms
<ol>
	<li>In order to test mice in operant tasks of social behavior, modify center channel modular shuttle boxes. These shuttle boxes measure 44 cm x 17.3 cm and are made of Plexiglas and stainless steel.</li>
	<li>Modify a programmable guillotine door by mounting a wire grid to the face of the doorframe. The wire grid should allow limited social contact but not allow the mice to cross from one chamber to the other.</li>
	<li>Using the programmable guillotine door, divide each box into two chambers: each measuring approximately 22 cm x 17.3 cm. Designate one chamber as the test chamber and the other as the target chamber.</li>
	<li>Place mouse levers in the test chamber on the wall opposite of the guillotine door. Place a liquid dipper food receptacle between the two mouse levers.</li>
	<li>Program the mouse levers to either open the guillotine door or present a liquid food reward, depending upon the testing paradigm.</li>
	<li>Connect a handheld button to the apparatus and program it to open the guillotine door (for manual shaping of test mice).</li>
	<li>Fit each shuttle box with an eight-channel I/R controller to monitor activity across the metal grid floor of each chamber. Mount a USB digital video camera above the test chamber and connect it to a computer. Enclose the entire apparatus within a melamine sound-attenuating cubicle.</li>
	<li>Run the operant programs using customized programs written in the laboratory.</li>
</ol>
2. Shaping the Mice for the Social Motivation Paradigm
<ol>
	<li>Place a test mouse in the test chamber and an age and sex-matched stimulus mouse in the target chamber.</li>
	<li>Train the test mouse to press the levers to open the guillotine door using the method of shaping or reinforcement of successive approximations to the desired operant response. During this shaping procedure, deliver reinforcement manually by pressing the handheld button programmed to control the guillotine door. 
	Note: Reinforcement consists of opening the guillotine door for 15 sec, thereby allowing access to the stimulus mouse through the wire grid.</li>
	<li>Observe the mouse in the test chamber using the camera mounted above the test chamber.</li>
	<li>Train each mouse in a series of 30 min daily sessions until they demonstrate at least 10 operant responses (lever presses) in 3 out of 5 consecutive testing sessions. Rotate mice through shuttle boxes using random assignment.</li>
	<li>If mice do not reach this criterion after 30 daily training sessions, remove the mice from the experiment and designate as non-learners. 
	Note: The number of daily training sessions may be extended beyond 30 depending on researcher preferences.</li>
	<li>Alternate the stimulus mice used during shaping so that a different mouse is used every other day.</li>
	<li>Clean shuttle boxes between each training session using 70% ethyl alcohol. Additionally, following the completion of testing each day, clean all equipment using a disinfecting detergent.</li>
</ol>
3. Testing the Mice in the Social Motivation Paradigm
<ol>
	<li>Place a trained test mouse in the test chamber and an age and sex-matched stimulus mouse in the target chamber. During testing, use a pool of at least 10 stimulus mice to assign to each test mouse so that stimulus mice are only repeated after 10 daily test sessions. Do not use stimulus mice used during shaping for the testing.</li>
	<li>Program the guillotine door to open on a progressive ratio schedule of reinforcement so that the number of operant responses (lever presses) necessary to obtain the social reward of 15 sec access to the target mouse arithmetically increases by a fixed rate of 3 each trial.</li>
	<li>Program the test sessions to end when the test mouse stops lever pressing for five consecutive minutes.</li>
	<li>Record the last completed (reinforced) ratio as the breakpoint. Use this dependent measure as an index of social motivation. Test each mouse for 20 consecutive daily sessions or for at least 10 test sessions after asymptotic performance levels are observed.</li>
	<li>Wipe down shuttle boxes between each testing session using 70% ethyl alcohol and following the completion of testing each day using a disinfecting detergent.</li>
</ol>
4. Valence Comparison of Social Versus Food Reward
<ol>
	<li>After all mice complete the social motivation paradigm, test these same mice in a task designed to compare the valence of the social reward with that of a food reward.</li>
	<li>Program one lever to open the guillotine door and present the social reward for 15 sec and program the opposite lever to present a liquid food reward for 15 sec. 
	Note: The liquid food reward used in this protocol consisted of evaporated milk sweetened with 0.2% sucrose. Prepare sufficient amounts (typically 500 ml) for repeatedly presenting rewards of 0.02 ml to each mouse.</li>
	<li>Assign test mice alternating lever/reward contingencies to control for potential lever biases among mice. Thus, assign half of the mice in each testing group to a left lever/food reward and right lever/social reward contingency and the other half to a left lever/social reward and right lever/food reward contingency.</li>
	<li>Train the mice for valence comparison of social versus food reward.
	<ol>
		<li>Place a test mouse in the test chamber and place an age and sex-matched stimulus mouse in the target chamber.</li>
		<li>Make only one lever/reward contingency active during each training session. Training consists of six 1 hr sessions that alternate between contingencies each day. Alternate stimulus mice assigned to each test mouse every other social reward session.</li>
		<li>Maintain the mice on their ad libitum chow diet during training and testing with the food reward. Food deprivation is unnecessary for operant conditioning in mice with the use of the evaporated milk and sucrose solution18.</li>
	</ol>
	</li>
	<li>Testing the mice for the valence comparison of social versus food reward.
	<ol>
		<li>Program the schedule of reinforcement at a fixed ratio of 3:1, so that every third lever press is reinforced, but only by its respective associated reward.</li>
		<li>Program test sessions to run for 60 min.</li>
		<li>Place a test mouse in the test chamber and place an age and sex-matched stimulus mouse in the target chamber. Note: The same pool of 10 stimulus mice used in the social motivation task can be used again but assigned to the test mice using a 10-day rotation.</li>
		<li>Test the mice in 20 daily sessions following the 6 days of training or for at least 10 daily sessions following the observation of asymptotic performance levels. The primary dependent measures for this paradigm is the total number of lever presses for each reward type.</li>
		<li>Once again, wipe down shuttle boxes between testing sessions using 70% ethyl alcohol and following each test day using a disinfecting detergent.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Silverman, J. L., Yang, M., Lord, C., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+phenotyping+assays+for+mouse+models+of+autism.">Behavioural phenotyping assays for mouse models of autism.</a> Nat Rev Neurosci. 11, 490-502 (2010).</li><li>File, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+social+interaction+as+a+method+for+detecting+anxiolytic+activity+of+chlordiazepoxide-like+drugs.">The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs.</a> J Neurosci Methods. 2, 219-238 (1980).</li><li>File, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+predicting+clinical+efficacy+of+anxiolytic+drugs:+social+behaviour.">Animal models for predicting clinical efficacy of anxiolytic drugs: social behaviour.</a> Neuropsychobiology. 13, 55-62 (1985).</li><li>File, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiolytic+action+of+a+neurokinin1+receptor+antagonist+in+the+social+interaction+test.">Anxiolytic action of a neurokinin1 receptor antagonist in the social interaction test.</a> Pharmacol Biochem Behav. 58, 747-752 (1997).</li><li>File, S. E., Hyde, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+social+interaction+be+used+to+measure+anxiety.">Can social interaction be used to measure anxiety.</a> Br J Pharmacol. 62, 19-24 (1978).</li><li>Winslow, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+social+recognition+and+preference.">Mouse social recognition and preference.</a> Curr Protoc Neurosci. 8, Unit 8 16 (2003).</li><li>Angelis, L., File, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+effects+of+three+benzodiazepines+in+the+social+interaction+anxiety+test+in+mice.">Acute and chronic effects of three benzodiazepines in the social interaction anxiety test in mice.</a> Psychopharmacology (Berl). 64, 127-129 (1979).</li><li>Gheusi, G., Bluthe, R. M., Goodall, G., Dantzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethological+study+of+the+effects+of+tetrahydroaminoacridine+(THA)+on+social+recognition+in+rats.">Ethological study of the effects of tetrahydroaminoacridine (THA) on social recognition in rats.</a> Psychopharmacology (Berl). 114, 644-650 (1994).</li><li>Carter, C. S., DeVries, A. C., Getz, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+substrates+of+mammalian+monogamy:+the+prairie+vole+model).">Physiological substrates of mammalian monogamy: the prairie vole model).</a> Neurosci Biobehav Rev. 19, 303-314 (1995).</li><li>Moy, S. S., 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=Sociability+and+preference+for+social+novelty+in+five+inbred+strains:+an+approach+to+assess+autistic-like+behavior+in+mice.">Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice.</a> Genes Brain Behav. 3, 287-302 (2004).</li><li>Pearson, B. L., 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=Absence+of+social+conditioned+place+preference+in+BTBR+T+tf/J+mice:+relevance+for+social+motivation+testing+in+rodent+models+of+autism.">Absence of social conditioned place preference in BTBR T+tf/J mice: relevance for social motivation testing in rodent models of autism.</a> Behav Brain Res. 233, 99-104 (2012).</li><li>Matthews, T. J., Abdelbaky, P., Pfaff, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+and+sexual+motivation+in+the+mouse.">Social and sexual motivation in the mouse.</a> Behav Neurosci. 119, 1628-1639 (2005).</li><li>Lee, A., Clancy, S., Fleming, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mother+rats+bar-press+for+pups:+effects+of+lesions+of+the+mpoa+and+limbic+sites+on+maternal+behavior+and+operant+responding+for+pup-reinforcement.">Mother rats bar-press for pups: effects of lesions of the mpoa and limbic sites on maternal behavior and operant responding for pup-reinforcement.</a> Behav Brain Res. 108, 215-231 (2000).</li><li>Kogan, J. H., Frankland, P. W., Silva, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+memory+underlying+hippocampus-dependent+social+recognition+in+mice.">Long-term memory underlying hippocampus-dependent social recognition in mice.</a> Hippocampus. 10, 47-56 (2000).</li><li>Sekiguchi, R., Wolterink, G., van Ree, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+duration+of+retroactive+facilitation+of+social+recognition+in+rats.">Short duration of retroactive facilitation of social recognition in rats.</a> Physiol Behav. 50, 1253-1256 (1991).</li><li>Bluthe, R. M., Gheusi, G., Dantzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadal+steroids+influence+the+involvement+of+arginine+vasopressin+in+social+recognition+in+mice.">Gonadal steroids influence the involvement of arginine vasopressin in social recognition in mice.</a> Psychoneuroendocrinology. 18, 323-335 (1993).</li><li>Thor, D. H., Holloway, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anosmia+and+play+fighting+behavior+in+prepubescent+male+and+female+rats.">Anosmia and play fighting behavior in prepubescent male and female rats.</a> Physiol Behav. 29, 281-285 (1982).</li><li>Martin, L., Sample, H., Gregg, M., Wood, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+operant+social+motivation+paradigms+using+BTBR+T+tf/J+and+C57BL/6J+inbred+mouse+strains.">Validation of operant social motivation paradigms using BTBR T+tf/J and C57BL/6J inbred mouse strains.</a> Brain Behav. 4, 754-764 (2014).</li></ol>

The authors have no competing financial interests.

The operant social motivation paradigms described in this article are powerful tools for the characterization of social behavior in mouse models of human disease. Rather than allow a test mouse to passively choose a social encounter, these paradigms require effort from the test mouse in order to obtain a social reward. The amount of effort that a test mouse is willing to produce to obtain the social reward provides a quantitative measure of social motivation. Furthermore, through the valence comparison paradigm, motivated behavior can be compared across different reward types. Both of these operant paradigms can be used to assess sexual and parental motivations as well by presenting the appropriate stimulus on the opposite side of the guillotine door.
The development of these social motivation paradigms involved the troubleshooting of several key variables. For example, through pilot studies the amount of time that the social reward is presented was lowered from 30 sec to 15 sec. While this time is sufficient for social interactions between test and stimulus mice, future research may optimize the amount of time for the social reward. Similarly, the progressively increasing ratio of lever presses to social rewards was lowered from a fixed rate of 5 to 3 based upon pilot research.
The number of days necessary to reach asymptotic lever pressing behavior was initially assessed through 30 days of daily test sessions. It was determined that most mice reach asymptote in 7 days so the number of test sessions was lowered to 20. Daily performance does fluctuate however, so averaging the data over the last 10 days ensures asymptotic performance while also providing a better index of true mean performance than fewer test days. Researchers should monitor their data to ensure that asymptotic performance is reached within 10 test days or modify the protocol as necessary.
Training sessions were also lowered from 1 hr to 30 min. The number of training sessions required does depend on the strain of mouse and some mice may be arbitrarily designated as nonlearners after a fixed number of training sessions18. The number of training days necessary to reach the criterion for advancement to the testing paradigm provides another index of social motivation and is thus worthy of tracking. Researchers may also want to experiment with different food reward options for the valence comparison paradigm. However, it is important that the test mice are initially trained using a purely social reward and then tested in the social motivation paradigm before beginning the valence comparison paradigm.
These operant social motivation paradigms can be modified and improved upon by measuring additional variables than those indicated in the above protocol. For example, the location of each mouse in his respective chamber can be tracked using currently available commercial software. In addition, specific behaviors including nose-to-nose contact or ultrasonic vocalizations can be scored during social interactions. Researchers may further experiment with adjustments to the ratios used in the social motivation paradigm or with trials involving the absence of a social partner or even comparisons involving other stimuli behind the guillotine door such as a video of a conspecific or even a puppet. For the valence comparison paradigm, analyses of choice dynamics across trials can provide further information regarding shifts in motivation due to satiation for a particular reward type18.
While these operant social motivation paradigms provide a much-needed tool for quantifying social motivation in mice, they do have some limitations. First, these paradigms require mice to have the cognitive ability to make learned associations between lever pressing and the social reward. Certain mouse models may have deficits in both cognitive and social abilities making it difficult to assess social motivation using these paradigms. Second, the nature of the social encounter is limited as the mice are only allowed to interact across a wire grid. However, mice are still able to successfully engage in many typical social behaviors, most notably, nose-to-nose investigations. One final limitation of these methods is that they are labor-intensive and require much time to be completed. The training and testing through both paradigms requires a minimum of two months of daily sessions. Some mice may take much longer to learn the lever-pressing association stretching the required number of sessions. While the paradigms certainly do not qualify as high-throughput techniques, the data that they generate is highly valuable and worth the required labor investment. However, to provide a fuller characterization of the social behavior of a particular mouse model, it is recommended that these paradigms be used in conjunction with other established methods of measuring social behavior in mice.

In recent years, the linkage of genetic and environmental factors to disorders of social behavior including autism spectrum disorder, has led to the development of mouse models designed to explore the role of these factors in social behavior. This has created a demand for new techniques for the characterization of social behavior in mice including a quantitative measure of social motivation1.
There are several established paradigms that can be used to demonstrate social motivation in mice, however; they all have severe limitations on their ability to quantify the strength of the motivated behavior. For example, some of the earliest behavioral assessments included simple observations of social interactions between two rodents that have never met. Several behaviors including approaching, following, sniffing, and grooming are often measured, and may provide an index of social motivation. These studies were initially conducted in rats2-5, and then later adapted for use in mice6,7. Habituation/dishabituation social recognition paradigms in which the test animal is exposed repeatedly to the same stimulus animal (habituation) and then exposed to a novel animal (dishabituation) also provide a measurement of social motivation8. As the labels suggest, typical animals will display a habituated behavioral response to the repeated presence of the same stimulus animals but will demonstrate dishabituation, or a return to baseline, when exposed to the novel animal. The habituation response likely demonstrates a decline in the motivation of the test mouse to interact with the stimulus mouse. Social choice paradigms allowing a test mouse to choose between an empty chamber and a stimulus mouse restricted to a different chamber have also been used to assess social motivation. A trial typically begins by placing the test mouse in a neutral, center chamber. Some versions of this task involve tethering the stimulus mouse to one chamber9. A modification of this task employed the use of a wire enclosure over the stimulus mice avoiding tethering of stimulus animals10. More recently, a social conditioned place preference task was developed in which mice were conditioned to associate visually distinct chambers with either the presence or absence of a social partner, and then allowed to explore the empty chambers following conditioning11. More time spent in the chamber where social pairings previously occurred is considered to be an index of social motivation.
All of the above paradigms provide valuable data related to the social phenotyping of mice. However, while one can observe whether a mouse demonstrates a &#8220;desire&#8221; to seek social interaction in the above paradigms, the strength of this &#8220;desire&#8221;, i.e., social motivation, is difficult to measure. The strength of motivated behavior has traditionally been measured in rodents by creating a scenario in which they have to work in order to gain access to the source of their motivation. This usually involves training rodents in an operant paradigm involving food as the motivating factor. However, this method has been employed previously to investigate social motivation in rats, but either involving access to a sexual partner for male rats or, for females, access to their own pups (reviewed in Matthews et al., 2005) 12,13.
Prior to the development of the social motivation paradigms described below, there was only one other study that investigated social motivation outside of sexual motivation using an operant paradigm with mice12. In this series of experiments, outbred Swiss Webster mice were trained to press a lever in order to gain access to a same-sex conspecific. In one experiment, a significantly reduced latency to begin responding for access to the target mouse was demonstrated in individually-housed mice compared to group-housed mice suggesting increased motivation to seek social access in these socially isolated mice. In another experiment of that same study, a comparison was made between saline and oxytocin antagonist (atosiban) infused mice. Interestingly, the drug-infused mice demonstrated a significantly increased latency to begin responding for access to the target mouse suggesting reduced motivation for a social reward in these mice.
While these experiments represent a noble attempt to quantify social motivation in mice using operant conditioning, they were also highly limited and are greatly improved upon using the methods described below. For example, an apparent fundamental flaw in the first experiment described above (experiment 2 of the actual study) was the researchers&#8217; use of only 2 target mice alternated across testing days. Previous research has shown that social recognition in group-housed mice can last for at least 7 days leading to reduced social investigation with repeated exposure to the same target14. However, social recognition in individually-housed rats and mice dissipates within 1 - 2 hr so that social investigation durations remain at baseline levels if a repeated exposure to the same target is at least 2 hr after the initial exposure15-17. The reduced latency of individually-housed mice to begin responding for social access reported in the Matthews et al. experiment may have been a consequence of the lack of social recognition of the target mice across testing days12. This potential confound is controlled for in the below methods by using a novel target mouse on each test day. Also, in both experiments from the Matthews et al. study described above, there were no reported differences in the total number of lever presses made between groups. It is possible that a difference in total lever presses could have been detected if the researchers utilized a progressively increasing ratio of responses to reward as used in our research design outlined below. In addition, access to the target mouse was controlled manually by the experimenter, by lifting up a box that covered the target. The use of an automatically-controlled guillotine door for access to the target mouse eliminates the possibility of artifact due to the presence of the experimenter. In fact, in order to fully automate the social motivation operant paradigms, the research equipment was customized as detailed below.

<table><tbody><tr><td>Modular Shuttle Box</td><td>Med Associates Inc.</td><td>ENV-010MC</td><td></td></tr><tr><td>Auto-Guillotine Door&amp;nbsp;</td><td>Med Associates Inc.</td><td>ENV-010B</td><td></td></tr><tr><td>Mouse Levers&amp;nbsp;</td><td>Med Associates Inc.</td><td>ENV-3010M</td><td></td></tr><tr><td>Liquid Dipper with Food Receptacle</td><td>Med Associates Inc.</td><td>ENV-202M-S</td><td></td></tr><tr><td>Eight-Channel I/R Controller&amp;nbsp;</td><td>Med Associates Inc.</td><td>ENV-253C</td><td></td></tr><tr><td>Melamine Sound-Attenuating Cubicle&amp;nbsp;</td><td>Med Associates Inc.</td><td>ENV-016MD</td><td></td></tr><tr><td>MED-PC IV Software Program</td><td>Med Associates Inc.</td><td>MED-PC IV&amp;nbsp;</td><td></td></tr><tr><td>Microsoft Lifecam Camera&amp;nbsp;</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Wire Grid</td><td></td><td></td><td>Wire grids can be purchases from a hardward store. We cut wire grids from stainless steel mouse cage tops.&amp;nbsp;</td></tr><tr><td>70% Ethyl Alcohol</td><td></td><td></td><td>Used to clean shuttle boxes between mice. Specific brand is unnecessary.</td></tr><tr><td>Evaportated Milk</td><td>Carnation</td><td></td><td>0.02 ml combined with 0.2% sucrose for each reward per mouse. We always purchase the same brand for better consistency.&amp;nbsp;</td></tr><tr><td>Sucrose</td><td></td><td></td><td>Any brand of pure granular sugar will due as it is dissolved in the evaporated milk.&amp;nbsp;</td></tr></tbody></table>

quantifying social motivation in mice using operant conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This protocol has been used to compare the prosocial C57BL/6J inbred mouse strain with the BTBR inbred mouse strain which has been previously reported to show decreases in social behavior10,18. It has also been used to characterize social motivation in different knockout mouse strains hypothesized to have either increased or decreased social motivation. Previously published results comparing the C57BL/6J and BTBR inbred mouse strains are shown in Figure 1. These results demonstrated decreased social motivation for the BTBR mice compared to the C57BL/6J mice. The Valence Comparison paradigm also demonstrated decreased food motivation for the BTBR mice suggesting an overall decrease in motivated behavior for this strain (Figure 1B).
Figure 2A presents some unpublished preliminary data comparing wild-type siblings on the C57BL/6 background strain with heterozygous and homozygous knockout siblings with reduced or absent protein product from a gene (gene X) hypothesized to promote social motivation. Figure 2B presents some unpublished preliminary data comparing wild-type siblings on the C57BL/6 background strain to heterozygous knockout siblings with reduced protein product from a gene (gene Y) hypothesized to reduce social motivation. The preliminary data from each of these studies support each respective hypothesis and further demonstrate the value of the described operant social motivation paradigms.
<img alt="Figure 1" src="/files/ftp_upload/53009/53009fig1.jpg" /> 
Figure 1. Previously published results from the social motivation and valence comparison paradigms. (A) The mean breakpoint of individually-housed (IH) B6 mice, group-housed (GH) B6 mice, and group-housed BTBR mice across the last 10 days of testing in the social motivation task. There were a total of 9 mice in each group. Individually and group-housed B6 mice performed similarly but there was a significant difference between group-housed B6 and BTBR mice (t = 2.741, df = 16, p = 0.015). (B) The mean number of food rewards and social rewards for the group-housed B6 and BTBR mice across the last 10 sessions of the valence comparison paradigm. Both mouse groups obtained significantly more food rewards than social rewards (BTBR: t = 3.551, df = 8, p = 0.007; B6: t = 9.478, df = 8, p &#60; 0.001). However, the BTBR mice acquired significantly fewer food rewards than the B6 mice and also the fewest number of social rewards (t = 3.321, df = 16, p = 0.004). Figure modified from Reference 18.
<img alt="Figure 2" src="/files/ftp_upload/53009/53009fig2.jpg" /> 
Figure 2. Representative results from unpublished research utilizing the social motivation paradigm.&#160; (A) Mean breakpoint (last reinforced ratio) achieved before the mice stopped lever pressing for 5 min. For gene X, a total of 12 WT, 7 HET, and 6 KO mice were tested in the social motivation paradigm. ANOVA revealed a significant difference between genotypes (F = 7.18, df = 2, 22, p = 0.004). Post hoc Tukey HSD tests showed that KO mice performed significantly below WT controls. (B) For gene Y, a total of 8 HET and 6 WT mice were tested. An independent samples t-test revealed a significant difference between genotypes (t = 2.26, df = 8.97, p = 0.05). * indicates significant difference from WT mice at the .05 alpha level.

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Quantifying Social Motivation in Mice Using Operant Conditioning

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

quantifying-social-motivation-in-mice-using-operant-conditioning

Universidad San Sebastian

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JoVE Journal

Behavior

18.0K Views.  Azusa Pacific University. This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior. 

Video: Quantifying Social Motivation in Mice Using Operant Conditioning

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Neuroscience

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

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

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

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

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Behavioral Tasks for Examining Identity Recognition In Mice

Social animals, like rodents, are able to recognize and differentiate between the identity of familiar individuals. Recognizing the identity of familiar individuals is important for developing social structures such as hierarchy, kinship, and family. However, mechanisms underlying the recognition of social identity remain unclear. Most rodent studies of social recognition are based on the propensity of rodents to interact with a novel social target, a phenomenon known as social novelty. However, behavioral tasks for examining social novelty cannot reveal the recognition of familiar conspecifics based on their identities. Presented here are behavioral tasks allowing for the examination of identity recognition in C57BL/6 mice by associating two familiar mice with or without a valenced experience. Subjects had interactions with two mice either without (neutral) or with a valenced experience (negative or positive) and became familiar with these mice. The negatively valenced mouse was associated with shocks, while the positively valenced mouse was associated with a food reward. Following training, the recognition of the identity of these familiar mice can be revealed in a social discrimination test, which is represented as the preference for the positively valenced mouse and avoidance of the negatively valenced mouse compared to the neutral mouse. Behavioral tasks for identity recognition could be useful in probing social memory mechanisms and the pathophysiology of disorders with impaired social cognition, such as autism spectrum disorder or schizophrenia.

By pairing negatively or positively valenced experiences with individual social targets, we developed behavior tasks for examining the identity recognition of C57BL/6 mice. These tasks allow for the study of mechanisms of social memory of individual social targets in healthy and disease-related mouse models with impaired social cognition.

Social animals, like rodents, are able to recognize and differentiate between the identity of familiar individuals. Recognizing the identity of familiar ...