Name: quantifying social motivation in mice using operant conditioning
Uploaded: 2015-08-08T14:00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about Quantifying Social Motivation in Mice Using Operant Conditioning at JoVE.com

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>

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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Modular Shuttle Box</td>
			<td>Med Associates Inc.</td>
			<td>ENV-010MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto-Guillotine Door&#160;</td>
			<td>Med Associates Inc.</td>
			<td>ENV-010B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Levers&#160;</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&#160;</td>
			<td>Med Associates Inc.</td>
			<td>ENV-253C</td>
			<td></td>
		</tr>
		<tr>
			<td>Melamine Sound-Attenuating Cubicle&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Lifecam Camera&#160;</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.&#160;</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. We always purchase the same brand for better consistency.&#160;</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.&#160;</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 ...

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Research

JoVE Journal

Behavior

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

An Operant Intra-/Extra-dimensional Set-shift Task for Mice

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric diseases. The most widely used neuropsychological tests for the evaluation of attentional set-shifting in human subjects are the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-dimensional set shift task (ID/ED). These tasks have proven clinical relevance and have been modified and successfully adapted for research in animal models. However, currently available tasks for rodents present several limitations, mainly due to their manual-based testing procedures, which are hampering translational advances in psychiatric medicine. To overcome these limitations and to better mimic the original version in primates, we present the development of a novel operant-based two-chamber ID/ED "Operon" task for rodents. We demonstrated the effectiveness of this novel task to measure different facets of cognitive flexibility in mice including attentional set formation and shifting, and reversal learning. Moreover, we show the high flexibility of this task in which three different perceptual dimensions can be manipulated with a high number of stimuli cues for each dimension. This novel ID/ED Operon task can be an effective preclinical tool for drug testing and/or large genetic screening relevant to the study of executive dysfunction and cognitive symptoms found in psychiatric disorders.

Attentional set-shifting cognitive abnormalities are a core feature of many psychiatric diseases. Here, we present the development of a novel automatic system for the effective study of attentional set-shifting abilities, executive functions and other cognitive abilities in mice with high translational validity to human studies.

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...