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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Immun. 24 (5), 850-856 (2010).</li><li>Boleij, H., Salomons, A. R., van Sprundel, M., Arndt, S. S., Ohl, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+all+mice+are+equal:+Welfare+implications+of+behavioural+habituation+profiles+in+four+129+mouse+substrains.">Not all mice are equal: Welfare implications of behavioural habituation profiles in four 129 mouse substrains.</a> PLoS One. 7 (8), e42544 (2012).</li><li>Enard, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+humanized+version+of+Foxp2+affects+cortico-basal+ganglia+circuits+in+mice.">A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.</a> Cell. 137 (5), 961-971 (2009).</li><li>Bartolomucci, A., Fuchs, E., Koolhaas, J. M., Ohl, F., Gould, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+social+defeat:+Stress+protocols+and+behavioral+testing.">Acute and chronic social defeat: Stress protocols and behavioral testing.</a> Mood and anxiety related phenotypes in mice. 42, 261-275 (2010).</li><li>Erhardt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+chronic+social+stress+on+behaviour+and+vasopressin+gene+expression+in+the+PVN+of+DBA/2OlaHsd+mice-influence+of+treatment+with+the+CRHR1-antagonist+R121919/NBI+30775.">Consequences of chronic social stress on behaviour and vasopressin gene expression in the PVN of DBA/2OlaHsd mice-influence of treatment with the CRHR1-antagonist R121919/NBI 30775.</a> J. Psychopharmacol. 23 (1), 31-39 (2009).</li><li>Salomons, A. R., Kortleve, T., Reinders, N. 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Dis. 38 (2), 266-272 (2010).</li><li>Roedel, A., Storch, C., Holsboer, F., Ohl, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+or+dark+phase+testing+on+behavioural+and+cognitive+performance+in+DBA+mice.">Effects of light or dark phase testing on behavioural and cognitive performance in DBA mice.</a> Lab. Anim. 40 (4), 371-381 (2006).</li><li>Arndt, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+housing+of+mice--impact+on+behaviour+and+stress+responses.">Individual housing of mice--impact on behaviour and stress responses.</a> Physiol. Behav. 97 (3-4), 385-393 (2009).</li><li>Salomons, A. R., Arndt, S. 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Rev. 26 (8), 907-923 (2002).</li><li>Ohl, F., Roedel, A., Binder, E., Holsboer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+high+and+low+anxiety+on+cognitive+performance+in+a+modified+hole+board+test+in+C57BL/6+and+DBA/2+mice.">Impact of high and low anxiety on cognitive performance in a modified hole board test in C57BL/6 and DBA/2 mice.</a> Eur. J. Neurosci. 17 (1), 128-136 (2003).</li><li>Gordan, M. L., Jungwirth, B., Ohl, F., Kellermann, K., Kochs, E. 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Neurosci. 12 (4), 794-803 (2012).</li><li>Guilloux, J., Seney, M., Edgar, N., Sibille, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+behavioral+z-scoring+increases+the+sensitivity+and+reliability+of+behavioral+phenotyping+in+mice:+Relevance+to+emotionality+and+sex.">Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: Relevance to emotionality and sex.</a> J. Neurosci. Methods. 197 (1), 21-31 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2010).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Name of Reagent/ Equipment</td>
			<td style="width:133px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:380px;">Comments/Description</td>
		</tr>
		<tr height="254">
			<td height="254" style="height:255px;width:211px;">Standard mHB apparatus</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:380px;">The standard mHB apparatus consists of a grey PVC experimental box (100x50x50 cm) separated from a group compartment (50x50x50 cm) by a transparent, perforated partition. If the presence of group mates is unwanted or if individually housed animals are tested, the transparent partition is replaced by a partition made of grey PVC. The board (60x20x0.5 cm; made of grey PVC) is placed in the middle of the box and can contain 20 cylinders (&#7443; 1.5 cm) staggered in two lines or 23 cylinders (&#7443; 3 cm) staggered in three lines positioned on the board. The area around the board is divided by black lines into 10 rectangles (20x15 cm) and 2 squares (20x20 cm).</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:211px;">Cognitive mHB apparatus</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</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).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Vanilla flavor</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Vanilla flavor dissolved in water (0.02%)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Camera</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Camera that generates sufficient quality output to rescore the behavior from video.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Stage light</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>A theather light that allows to adjust the light intensity and the dimensions of the surface that is lit.&#160;</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:211px;">Behavioral scoring software</td>
			<td style="width:133px;">N/A</td>
			<td style="width:119px;">N/A</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/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/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/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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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

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys maintained in semi-free ranging conditions. Data accumulated during the last five years have consistently demonstrated the efficiency of this protocol to investigate individual/physical cognition in monkeys, and have further shown that this procedure reduces stress level during animal testing3. This paper demonstrates that networks of ALDM can also be used to investigate different facets of social cognition and in-group expressed behaviors in monkeys, and describes three illustrative protocols developed for that purpose. The first study demonstrates how ethological assessments of social behavior and computerized assessments of cognitive performance could be integrated to investigate the effects of socially exhibited moods on the cognitive performance of individuals. The second study shows that batteries of ALDM running in parallel can provide unique information on the influence of the presence of others on task performance. Finally, the last study shows that networks of ALDM test units can also be used to study issues related to social transmission and cultural evolution. Combined together, these three studies demonstrate clearly that ALDM testing is a highly promising experimental tool for bridging the gap in the animal literature between research on individual cognition and research on social cognition.

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device ALDM Test Systems

Fagot &#38; Paleressompoulle1 have published an automated learning device (ALDM) aimed at testing individual cognitive abilities in semi-free ranging monkeys. The main goal of our protocol is to use a network of ALDM test units to study social cognition in non-human primates.

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys ...

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 ...

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

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

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

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

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 ...

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

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

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

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

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

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and &#945;7 nicotinic acetylcholine (&#945;7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...