The authors would like to acknowledge the work of Annemarie Baars and Jos&#233; Lozeman-van &#8216;t Klooster in performing, assisting and teaching experiments using the mHB set-up.

NOTE: The experiments have been approved by the Animal Experiments Committee of the University Medical Center Utrecht and Utrecht University, The Netherlands. Furthermore, the animal experiments followed the Principles of laboratory animal care and refer to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioural Research.25
1. Experimental Set-up
NOTE: The standard mHB apparatus consists of a grey PVC experimental box (100 x 50 x 50 cm) separated from an additional compartment (50 x 50 x 50 cm) in which the group mates of the experimental animal can be placed during the testing period by a transparent, perforated partition1. If the presence of group mates is unwanted or if individually housed animals are tested, replace the transparent partition by a partition made of grey PVC (Figure 1; see also Ohl et al. (2001)1). The mHB (with different measurements) used for cognitive testing is described in section 5 of the protocol.
<ol>
	<li>Place the board (60 x 20 x 0.5 cm; made of grey PVC) in the middle of the box. 
	NOTE: The board can contain 20 cylinders (&#7443; 1.5 cm)14 staggered in two lines or 23 cylinders (&#7443; 3 cm)8 staggered in three lines.</li>
	<li>Divide the area around the board by black lines into 10 rectangles (20 x 15 cm) and 2 squares (20 x 20 cm).</li>
	<li>Position a stage light above the board to create a larger contrast in light intensity between the board (representing an unprotected area comparable with the center of an open field or the light compartment of a light-dark transition test) and the box (protected area) in order to increase the aversive character of the board4,8.</li>
	<li>House the animals under a 12 hr reversed day-night cycle (e.g., lights off at 7:00 AM and lights on at 7:00 PM). 
	NOTE: However, the mHB can also be used under the conventional light schedule (see discussion on potential shortcomings)1,6.</li>
	<li>Perform the behavioral testing in the most active phase of the animals (e.g., between 10:00 AM and 2:00 PM)4,15.</li>
	<li>Maintain a habituation period of 2 weeks after arrival of the animals in the facilities. During this period have the same person performing the behavioral experiment handle the animals four times a week and include all handling procedures to be performed during actual testing. Handle the animals exclusively during the time of the day when the animals will be exposed to testing later on.</li>
	<li>Record the experiments for data storage on video and in order to optimize the results and to reduce the inter observer variability, practice the behavioral scoring using a video recording of previous experiments2. Make sure to standardize all actions and procedures executed by the observer.</li>
</ol>
2. Behavioral Testing &#8211; Without the Presence of Group Mates
<ol>
	<li>Perform behavioral testing in the room the animals are regularly housed in (to avoid possible effects of transportation to a testing location) and install all testing equipment before arrival of the animals in the facilities (to habituate the animals to the presence of the equipment).</li>
	<li>Pick up the animal by the base of the tail from its home cage and directly place it in the mHB.</li>
	<li>Place each animal in the apparatus in the same corner facing the wall (as indicated in Figure 1).</li>
	<li>Allow the animal to freely explore the mHB for a period of time (often 5 min1,6,16-18).</li>
	<li>Have an experienced observer live score the behavioral parameters using behavioral scoring software. Use the parameters listed in Table 1. 
	NOTE: Some behavioral parameters (e.g., motor- and exploratory behavior) might be scored automatically as e.g., discussed by Henry et al. (2010)19 after necessary adjustment of the mHB.</li>
	<li>Clean the apparatus with tap water and a paper towel after every trial in order to avoid a bias based on olfactory cues. 
	NOTE: Possible testing-order effects when testing socially housed animals should be kept in mind17,20.</li>
</ol>
3. Behavioral Testing &#8211; in the Presence of Group Mates
<ol>
	<li>In the case of group housing, measure the interaction with the experimental animal and its cage mates during testing.
	<ol>
		<li>Place the group mates in the group compartment before testing of the experimental animal to allow for habituation (mainly 10-30 min1,12). 
		NOTE: Testing under social-stress conditions is possible by placing a dominant cage-mate in the group compartment when testing a socially defeated individual13.</li>
		<li>Place the experimental animal in the test compartment and allow it to freely explore the mHB as described in section 2. 
		NOTE: Possible testing-order effects when testing socially housed animals should be kept in mind17,20.</li>
		<li>Have an experienced observer live score the behavioral parameters using behavioral scoring software. Use the parameters listed in Table 1.</li>
		<li>Clean the apparatus with tap water and a paper towel after every trial in order to avoid a bias based on olfactory cues.</li>
	</ol>
	</li>
</ol>
4. Novel Object Recognition and Food Intake Inhibition
<ol>
	<li>Familiarize the animals with an object (for instance a dice or a food pellet) in their home cage 2 days before the experiment.</li>
	<li>Place the familiarized object in the apparatus 2 cm apart from a novel object (for instance a bolt or unfamiliar food) in the corner across from the starting point.</li>
	<li>Measure the time the animal takes to approach the novel and familiar object/food. Use the parameters in Table 1.</li>
	<li>Clean the apparatus with tap water and a paper towel after every trial in order to avoid a bias based on olfactory cues.</li>
</ol>
5. Cognitive Testing
<ol>
	<li>Place a smaller board (35 x 22&#160;x 1 cm) with 10 cylinders in the middle of the box (Figure 1) for testing rats3,22. Reduce the box in size to 50 x 50 cm for testing mice21 by&#160;inserting a partition made of grey PVC.</li>
	<li>Scent all cylinders with a flavor animals are attracted to (e.g., vanilla) and bait all with a reward (e.g., a piece of almond, a highly palatable reward for mice and rats) beneath a grid so the animals cannot remove it.</li>
	<li>Cue cylinders (often three) with a colored ring (contrasting with the grey PVC) and bait them with a removable reward (e.g., 0.05 g piece of almond).</li>
	<li>Familiarize the animals with the reward daily in the 2 days before the experiment in their home cage by offering it with tweezers and making sure that the animals eat it.</li>
	<li>Have an experienced observer live score the behavioral parameters using behavioral scoring software. Measure the parameters listed in Table 2 in addition to the behavioral parameters mentioned in section 2 (Table 1) with the exception of the parameters related to object recognition or food intake inhibition.</li>
	<li>Stage 1: With each animal, perform four trials daily with a constant inter-trial interval (e.g., 30-60 min) until a constant time to finish a trial is reached (i.e., when all three almond pieces have been collected).</li>
	<li>Stage 2: Cue and bait three different cylinders and place the animals in the setup for four trials to test the reversal learning ability.</li>
	<li>Clean the apparatus with tap water and a paper towel after every trial in order to avoid a bias based on olfactory cues.</li>
</ol>

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The authors have nothing to disclose.

The mHB paradigm can be used to measure multiple dimensions of unconditioned behavior. The protocol can be slightly altered depending on the purpose of the experiment. In this protocol we discuss the settings, times and measurements usually used in our lab. However, slight deviations to the measurements of the apparatus have been used in the past and also the amount of cylinders on the board may vary3. Often studies employ a testing time of 5 min per trial, but other testing times can also be appropriate, i.e.,&#160;ending the test as soon as a cognitive trial has been completed successfully or in extending testing time if animals are extremely anxious or physically impaired. The time of day of testing was selected to be under red light condition since rodents are nocturnal animals and are most active in the early dark phase. Roedel et al. (2006) shows the effects of light or dark phase testing on behavioral and cognitive performance in DBA mice in the mHB16. Other studies have performed mHB experiments under white light conditions1,6, however, it should be noted that testing under white light conditions can induce behavioral inhibition and cognitive disruption (as shown in DBA mice)16.
Tables 1 and 2 contain a large amount of behavioral parameters to be measured. During data analysis this can lead to some parameters indicating a significant increase of for instance &#8216;latency to first entry board&#8217;, but not in other parameters of the same motivational system (in this case &#8216;Avoidance&#8217;). In some cases this may lead to inconclusive results. Guilloux et al. (2011) introduced integrated behavioral z-scores to the behavioral phenotyping in mice24. With the use of integrated behavioral z-scores the multiple parameters can be combined to a single z-score describing a particular motivational system. The subsequent z-scores can in their turn be more easily compared across behavioral tests and experiments.
Besides the described features of this paradigm, a more profound use became apparent in the study of Salomons et al. (2012). The habituation to novelty of two mouse strains (BALC/cJ and 129P3/J) in the mHB was compared, exhibiting a difference in behavioral flexibility indicating a non-adaptive behavioral profile of the 129P3/J mice4, mirroring impaired adaptive capacities and probably even pathological anxiety.
Concluding, the mHB allows measurement of multiple behavioral dimensions in a single experiment. By combining features from a traditional hole board and open field test, unconditioned behavior, social interaction, cognition and adaptive capacities, i.e., welfare can be investigated. This test can for example be used to evaluate behavioral changes due to pharmacological- and/or genetic manipulations, selective breeding and adaptive capacities. In comparison to classical test batteries, the number of animals needed is clearly reduced and stress experienced by the animals during testing is extremely low.

The modified hole board (mHB) is used to assess multiple dimensions of unconditioned behavior, mainly in mice and rats1. A number of widely used tests measure a single behavioral parameter which does not completely cover the entire phenotype of a behavioral dimension. The mHB was developed based on the concept that rodents can show their rich behavioral repertoire only in a rich testing environment2 and thus allows for complex ethological observations.
The set-up comprises the characteristics of the traditional hole board and the open field test, resulting in a single complex paradigm which overcomes the disadvantages of a test battery (i.e., in reducing the number of animals used1,3,4, circumventing the possible effects of test order5, and reducing time-effect and costs6).&#160; In contrast to most behavioral tests (e.g., H&#229;nell &#38; Marklund, 2014)7, an advantage of the mHB is that animals do not need to be food deprived in order to increase the motivation to solve the task. Additionally, social isolation can be circumvented during testing by placing group mates of the experimental animal in a (group-) compartment separated from the test compartment by a transparent perforated partition, allowing for visual, auditory and olfactory contact8,9.
The mHB has been (pharmacologically) validated for both mice and rats1,6. A wide range of behaviors can be measured, such as avoidance behavior, risk assessment, arousal, exploration, locomotor activity, habituation, social affinity and cognition2,8-10. Additionally, the mHB can be combined with a food intake inhibition test, as well as novel object recognition test10,11. Finally, the mHB can also be used to perform social stress experiments by testing socially defeated animals while placing a dominant individual in the group compartment12,13. This protocol for mice and rats will give an overview of the multiple applications of the mHB.

<table><tbody><tr><td>Cognitive mHB apparatus</td><td></td><td></td><td>The box for cognitive testing is 50x50 cm by inserting a partition made of grey PVC in the standard mHB. A smaller board (35x22x1 cm) with 10 cylinders is placed in the middle of the box. All cylinders are scented with a flavor animals are attracted to (e.g. vanilla) and all are baited with a reward (e.g. a piece of almond) beneath a grid so the animals cannot remove it. Cylinders (often three) are cued with a colored ring (contrasting with the grey PVC) and are baited with a removable reward (e.g. 0.05 g piece of almond).&amp;nbsp;</td></tr><tr><td>Vanilla flavor</td><td></td><td></td><td>Vanilla flavor dissolved in water (0.02%)</td></tr><tr><td>Camera</td><td></td><td></td><td>Camera that generates sufficient quality output to rescore the behavior from video.&amp;nbsp;</td></tr><tr><td>Stage light</td><td></td><td></td><td>A theather light that allows to adjust the light intensity and the dimensions of the surface that is lit.&amp;nbsp;</td></tr><tr><td>Behavioral scoring software</td><td></td><td></td><td>Behavioral scoring software.</td></tr></tbody></table>

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.

The large amount of parameters that can be measured in the mHB make this setup especially suitable to measure numerous behavioral dimensions. One example is the identification of behavioral adaptation to a novel environment by repeated exposure to the test. Salomons et al. (2010) studied the habituation of two inbred strains of mice (BALB/cJ and 129P3/J) to the mHB under two different light conditions (red light: contrast between box and board: 45 lux vs. white light: contrast between box and board: 115 lux (see also protocol section 1.3))4. BALC/cJ mice show a decreasing (habituating) latency until the first board entry (see Table 1) under red light conditions as shown in Figure 2A. Contrastingly, 129P3/J mice show no sign of habituation over trials. Figure 2B shows the experiment under white light conditions. BALB/cJ mice show again a decreasing latency to the first board entry over trials albeit that the animals show a slower habituation pattern compared to the red light condition. 129P3/J mice not only show again impaired habituation, but also a trend towards sensitization under the white light condition. Similarly, in a study by Salomons (2012) 129P2/OlaHsd mice showed an impaired behavioral flexibility in response to novelty compared to BALB/cOlaHsd mice23. The difference in habituation ability thus becomes apparent between two inbred strains of mice when tested in the mHB4.
The cognitive version of the mHB can for instance be used to measure cognitive impairments in mice. Van der Kooij et al. (2010) used this set-up to measure the cognitive functioning of C57BL/6J mice with mild cerebral hypoxia-ischemia (45 min of hypoxia; HI-45), severe HI (75 min of hypoxia; HI-75) and sham-control mice9. The ability to complete the trials (i.e., find the baited holes within 5 min) is shown in Figure 3A. The number of short-term memory mistakes (revisits to a baited hole), long-term memory mistakes (visits to a non-baited hole) and omission errors (no visit to a baited hole) are shown in Figure 3B-D respectively.
In order to confirm that the HI-45 group had no cognitive impairment, this group was tested against the sham-controls in a reversal task. The three baited holes were appointed to three different cylinders and the animals were tested for 4 trials. The reversal effect becomes apparent when comparing the last trial of the first stage with the first trial of the reversal stage. The duration to complete the four reversal trials gives an indication on the overall performance. Figure 4 shows the latency to complete the trial for both groups and a clear overall treatment effect is evident. This means that in the reversal task there is indeed an impairment in cognitive flexibility (re-learning) in the HI-45 group which became detectable using the mHB9.
<img alt="Figure 1" src="/files/ftp_upload/52529/52529fig1.jpg" /> 
Figure 1. Schematic overview of the modified hole board. (A) The set-up consists of a test compartment (box) with in the middle the (unprotected) board indicated with zigzag lines and the group compartment. <img alt="Figure 5" src="/files/ftp_upload/52529/52529fig5.jpg" />= novel object, <img alt="Figure 6" src="/files/ftp_upload/52529/52529fig6.jpg" />= familiar object, <img alt="Figure 7" src="/files/ftp_upload/52529/52529fig7.jpg" />= starting point. (B) Schematic overview of the cognitive version of the modified hole board for mice. (C) Side view of a cylinder as used in the cognitive version of the modified hole board. A piece of almond is placed beneath the grid of all cylinders. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52529/52529fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52529/52529fig2.jpg" /> 
Figure 2. Red light or white light conditions. (A) Latency until first board entry (mean + SEM) of BALB/cJ and 123P3/J mice measured under red light conditions (box 0-5 lux and board 45 lux). A repeated measures ANOVA with Huyn-Feldt adjustment revealed a strain (P = 0.025), trial (P &#60;0.001) and trial x strain interaction (P &#60;0.001) effect. Post hoc analyses: between strains: * = P &#60;0.0026, between two consecutive trials: $ = P &#60;0.0026 (BALB/cJ). (B) The latency of the first board entry (mean + SEM) under white light conditions (box 0-5 lux and board 120 lux). A repeated measures ANOVA with Huyn-Feldt adjustment revealed a strain (P = 0.031), trial (P &#60;0.001) and trial x strain interaction (P &#60;0.001) effect. Post hoc analyses: between strains: * = P &#60;0.0026, between two consecutive trials: $ = P &#60;0.0026 (BALB/cJ) and T = P &#60;0.0026 (123P3/J). This figure has been modified from Salomons et al. 20104. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52529/52529fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52529/52529fig3.jpg" /> 
Figure 3. Cognitive testing in the mHB (stage 1). All figures show mean + SEM. (A) The latency to complete the trial (sec) of the sham-control mice, HI-45 and HI-75. (B) Number of omission errors, (C) Non-baited hole visits and (D) Revisits for baited holes. * = P &#60;0.05, ** = P &#60;0.01, *** = P &#60;0.001 sham vs. HI-75, # = P &#60;0.05, ## = P &#60;0.01, ### = P &#60;0.001 HI-45 vs. HI-75. This figure has been modified from Van der Kooij et al. 20109. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52529/52529fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52529/52529fig4.jpg" /> 
Figure 4. Reversal learning task (stage 2). Latency to complete a trial (sec) in the reversal learning task (mean + SEM). Trial effects: # = P &#60;0.05, ## = P &#60;0.01 (first trial reversal task vs. previous trial). Treatment effect: * = P &#60;0.05, ** = P &#60;0.01 (HI-45 vs. sham). This figure has been modified from Van der Kooij et al. 20109.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>System</td>
			<td>Activity</td>
			<td>Measured parameter</td>
		</tr>
		<tr>
			<td>Avoidance</td>
			<td>Board entry</td>
			<td>Frequency, latency (s), duration (%) and average duration (s) on the board</td>
		</tr>
		<tr>
			<td>Risk assessment</td>
			<td>Stretched attends</td>
			<td>Frequency and latency (s) of stretched body postures (including hind limbs)</td>
		</tr>
		<tr>
			<td>Arousal</td>
			<td>Grooming</td>
			<td>Frequency, latency (s), duration (%) and average duration (s) self-grooming</td>
		</tr>
		<tr>
			<td></td>
			<td>Defecation</td>
			<td>Frequency and latency (s) of boli produced</td>
		</tr>
		<tr>
			<td></td>
			<td>Urination</td>
			<td>Frequency and latency (s) of urinations</td>
		</tr>
		<tr>
			<td>Directed exploration</td>
			<td>Hole visits</td>
			<td>Frequency and latency (s) of cylinder visits</td>
		</tr>
		<tr>
			<td></td>
			<td>Novel object exploration</td>
			<td>Frequency, latency (s), duration (%) and average duration (s) exploring the novel object</td>
		</tr>
		<tr>
			<td>Undirected exploration</td>
			<td>Rearing box</td>
			<td>Frequency and latency (s) of rearings in the box (front paws not touching the wall)</td>
		</tr>
		<tr>
			<td></td>
			<td>Rearing board</td>
			<td>Frequency and latency (s) of rearings on the board</td>
		</tr>
		<tr>
			<td></td>
			<td>Hole exploration</td>
			<td>Frequency and latency (s) of cylinder explorations</td>
		</tr>
		<tr>
			<td>Memory</td>
			<td>Familiar object exploration</td>
			<td>Frequency, latency (s), duration (%) and average duration (s) exploring the familiar object</td>
		</tr>
		<tr>
			<td>Social affinity</td>
			<td>Group interaction</td>
			<td>Frequency, latency (s), duration (%) and average duration (s) interacting with the group compartment</td>
		</tr>
		<tr>
			<td>Locomotor activity</td>
			<td>Line crossing</td>
			<td>Frequency and latency (s) of line crossings</td>
		</tr>
	</tbody>
</table>
Table 1: List of behavioral parameters
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Memory system</td>
			<td>Parameter</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>Long-term memory</td>
			<td>Wrong choice</td>
			<td>Visit to non-baited cylinder; nose below the rim</td>
		</tr>
		<tr>
			<td>Omission error</td>
			<td>Omission of a baited cylinder</td>
			<td>No visit to a baited cylinder</td>
		</tr>
		<tr>
			<td>Short-term memory</td>
			<td>Repeated choice</td>
			<td>Revisit to baited cylinder; nose below the rim</td>
		</tr>
		<tr>
			<td>Overall performance</td>
			<td>Total trial time</td>
			<td>Time until all baited cylinders have been visited</td>
		</tr>
	</tbody>
</table>
Table 2: List of cognitive parameters

Watch this Scientific Journal Video about The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats at JoVE.com

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

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 ...

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Universidad San Sebastian

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JoVE Journal

Behavior

17.7K Views.  Utrecht University. 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. 

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

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 ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

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 ...

Neuroscience

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 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 ...

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

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 Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

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 ...