Name: novel object recognition test for the investigation of learning and memory in mice
Uploaded: 2017-08-30T13:00:00
Description: Watch this Scientific Journal Video about Novel Object Recognition Test for the Investigation of Learning and Memory in Mice at JoVE.com

Work cited and previously published by the author was supported by a grant from the National Institute of Mental Health (MH088480). The author would like to thank her former mentor, Dr. James O'Donnell for his support in that project. This publication is supported by a grant from the National Institute of Health (T32 DA007135).

All procedures performed here were submitted to and approved by the Animal Care and Use committee and were conducted following NIH guidelines.
1. Object Selection and Experimental Setup
<ol>
	<li>Select objects that are different enough to be easily discriminated by mice, but have a similar degree of complexity (texture, shape, color patterning and brightness, etc.) in order to minimize any potential induced object preference that may bias the results (see Ennaceur 2010 for ...

<ol>
	<li>Ennaceur, A., Meliani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+one-trial+test+for+neurobiological+studies+of+memory+in+rats+III.+Spatial+vs.+non-spatial+working+memory.">A new one-trial test for neurobiological studies of memory in rats III. Spatial vs. non-spatial working memory.</a> Behav. Brain Res. 51 (1), 83-92 (1988).</li><li>Akkerman, 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=Object+recognition+testing:+methodological+considerations+on+exploration+and+discrimination+measures.">Object recognition testing: methodological considerations on exploration and discrimination measures.</a> Behav. Brain Res. 232 (2), 335-347 (2012).</li><li>Antunes, M., Biala, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+object+recognition+memory:+neurobiology,+test+procedure,+and+its+modifications.">The novel object recognition memory: neurobiology, test procedure, and its modifications.</a> Cogn. Process. 13 (2), 93-110 (2012).</li><li>Leger, M., 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=Object+recognition+test+in+mice.">Object recognition test in mice.</a> Nat. Protoc. 8 (12), 2531-2537 (2013).</li><li>van Goethem, N. P., 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=Object+recognition+testing:+Rodent+species,+strains,+housing+conditions,+and+estrous+cycle.">Object recognition testing: Rodent species, strains, housing conditions, and estrous cycle.</a> Behav. Brain Res. 232 (2), 323-334 (2012).</li><li>Lueptow, L. M., Zhang, C. -. G., O'Donnell, J. 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Brain Res. 215 (2), 244-254 (2010).</li><li>Berlyne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novelty+and+curiosity+as+determinants+of+exploratory+behavior.">Novelty and curiosity as determinants of exploratory behavior.</a> Br. J. Psychol. 41 (1-2), 68-80 (1950).</li><li>Ennaceur, A., Delacour, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+one-trial+test+for+neurobiological+studies+of+memory+in+rats+I.+Behavioral-data.">A new one-trial test for neurobiological studies of memory in rats I. Behavioral-data.</a> Behav. 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Pharmacol. 436 (1-2), 83-87 (2002).</li><li>Bertaina-Anglade, V., Enjuanes, E., Morillon, D., Drieu la Rochelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+object+recognition+task+in+rats+and+mice:+A+simple+and+rapid+model+in+safety+pharmacology+to+detect+amnesic+properties+of+a+new+chemical+entity.">The object recognition task in rats and mice: A simple and rapid model in safety pharmacology to detect amnesic properties of a new chemical entity.</a> J. Pharmacol. Toxicol. 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Brain Res. 285, 140-157 (2015).</li><li>Balderas, I., Moreno-Castilla, P., Bermudez-Rattoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+D1+receptor+activity+modulates+object+recognition+memory+consolidation+in+the+perirhinal+cortex+but+not+in+the+hippocampus.">Dopamine D1 receptor activity modulates object recognition memory consolidation in the perirhinal cortex but not in the hippocampus.</a> Hippocampus. 23 (10), 873-878 (2013).</li><li>Akkerman, S., Blokland, A., Prickaerts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+the+gap:+delayed+manifestation+of+long-term+object+memory+improvement+by+phosphodiesterase+inhibitors.">Mind the gap: delayed manifestation of long-term object memory improvement by phosphodiesterase inhibitors.</a> Neurobiol. Learn. 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Brain Res. 236 (1), 16-22 (2013).</li><li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housing,+husbandry+and+handling+of+rodents+for+behavioral+experiments.">Housing, husbandry and handling of rodents for behavioral experiments.</a> Nat. Protoc. 1 (2), 936-946 (2006).</li><li>&#350;&#305;k, A., van Nieuwehuyzen, P., Prickaerts, J., Blokland, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+different+mouse+strains+in+an+object+recognition+task.">Performance of different mouse strains in an object recognition task.</a> Behav. Brain Res. 147 (1-2), 49-54 (2003).</li><li>Prut, L., Belzung, C., Rabelias, U. 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The ORT is an efficient and flexible method for studying learning and memory in mice. When setting up an experiment, it is important to consider a number of variables that may affect the outcome. As discussed in the representative results, the strain of mouse will affect both exploration time and retention interval. A decrease in exploration time may skew or mask results in an absolute discrimination analysis2,3,5,<su...

The object recognition test (ORT), also known as the novel object recognition test (NOR), is a relatively fast and efficient means for testing different phases of learning and memory in mice. It was originally described by Ennaceur and Delacour in 1988 and used primarily in rats1; however, since then, it has been successfully adapted for use in mice2,3,4,5,6,7. The test relies on as few as three sessions: one habituation session, one training session, and one test session. Training simply involves visual exploration of two identical objects, while the test session involves replacing one of the previously explored objects with a novel object. Because rodents have an innate preference for novelty, a rodent that remembers the familiar object will spend more time exploring the novel object7,8,9.
The main advantage of the ORT over other rodent memory tests is that it relies on rodents&#39; natural proclivity for exploring novelty8. Therefore, there is no need for numerous training sessions or any positive or negative reinforcement to motivate behavior. This means that the ORT is much less stressful, relative to other tests10,11,12,13,14,15, and requires significantly less time to run than other commonly used memory tests, such as the Morris water maze or Barnes maze, which both can take up to a week or longer. Consequently, the conditions of the ORT more closely resemble those used in studying human cognition, increasing the ecological validity of the test over many other rodent memory tests. Similarly, because ORT is a simple visual recall task, it has been successfully adapted for use in numerous species, including humans and non-human primates, to assess different inter-species aspects of declarative memory 2,16,17. Finally, the ORT can be easily modified to examine different phases of learning and memory (i.e., acquisition, consolidation, or recall), to assess different types of memory (e.g., spatial memory), or to assess different retention intervals (i.e., short-term vs long-term memory).
The versatility of the ORT provides a platform for innumerable research applications. Studies can make use of pharmacologic agents to either disrupt or enhance memory. Varying the time of drug administration before or after training, or prior to testing can hint at the underlying neural mechanisms that lead to disrupted or enhanced memory6,18,19,20. In a similar way, optogenetic technology can be used at these same various time points to look at the neural activation/inhibition that contributes to the different phases of learning and memory. The ORT is also appropriate for assessing differences in transgenic animals, in lesion studies, or in neurodegenerative models or in aging studies21,22,23,24,25,26,27,28. The time between training and testing, known as the retention interval, can be altered to assess any of these changes on short- and long- term memory26. Ultimately, the ORT can be used as a tool to study pharmacological, genetic, and neurological changes to learning and memory, or these tools can be used to study the basis of learning and memory in the ORT.

<table border="0" cellpadding="0" cellspacing="0" width="805">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Open Field Box</td>
			<td style="width:71px;">Panlab/Harvard Apparatus</td>
			<td style="width:119px;">LE800SC</td>
			<td style="width:399px;">Available in grey, white, or black</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ANY-maze</td>
			<td style="width:71px;">Stoelting Co.</td>
			<td style="width:119px;">60000</td>
			<td style="width:399px;">Behavior tracking system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EthoVisionXT 12</td>
			<td style="width:71px;">Noldus</td>
			<td style="width:119px;"></td>
			<td style="width:399px;">Behavior tracking system; requires 3 point tracking</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Video Camera</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td style="width:399px;">Video camera should be mounted directly overhead of the apparatus</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">70% Ethanol&#160;</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP2818-4</td>
			<td style="width:399px;">Prior to starting testing and in between trials, each object should be carefully cleaned. The floor and walls of the apparatus should also be cleaned.&#160;</td>
		</tr>
	</tbody>
</table>

novel object recognition test for the investigation of learning and memory in mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

A general experimental setup for the ORT is shown in Figure 2. On habituation day (T0) mice are placed in the empty arena for 5 min. Twenty-four hours later, mice are placed back in the chamber with 2 identical objects and allowed to freely explore for up to 10 min (T1). On testing day (T2), the mice are again placed in the arena, but with one familiar object and one novel object, and allowed to explore for up to 10 min. The retention interval, the time betwe...

Watch this Scientific Journal Video about Novel Object Recognition Test for the Investigation of Learning and Memory in Mice at JoVE.com

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

The overall goal of this behavioral assay is to test various aspects of learning and memory in mice. This method can help answer numerous questions in the area of rodent cognition, from the basics of developing a deeper understanding, for certain aspects of learning in memory and mice, as well as more complex issues, such as cognitive impairment, including disease models, such as Alzheimer's disease, to the exploration of potential cognitive enhancement. The main advantage of the ORT, is its simplicity, efficiency and flexibility and studying learning and memory in mice.Demonstrating this procedure will be Andrei Jeltyi, a lab technician from our lab. Begin by selecting mole sized objects that can be easily discriminated by the animal but have a similar degree of complexity in features, such as, texture, shape or brightness to minimize any potential induced object preference that may bias the results. Additionally, to further reduce induced preferences, ensure that mice are able to climb on both objects or neither object.The ability to climb on an object may increase interest in exploration. Then to minimize the stress of bright lighting, use defused low lighting with the center of the maze illuminated around 20 lucks. Acclimate mice to any new testing rooms for at least one hour prior to use each day and keep the temperature and humidity similar to regular housing conditions.Note, the mice should be handled one to two times a day for at least one minute, over the course of one to two weeks prior to testing. Lastly, for the main arena, use a square chamber made from white or black non-porous plastic contrasted to the color of the mouse. Begin by removing the mouse from its home cage and placing it in the middle of the open empty arena.Allow for free exploration of the arena for five minutes. Once the home cage is empty, save it for use as a holding cage the next day. At the end of five minutes, remove the mouse and place it in a holding cage.Do not return the mouse to its original home cage, or this may affect the behavior of the remaining mice to be tested. Then thoroughly clean the apparatus between mice, using 70%volume per volume ethanol. Next, begin the training phase or T1, by placing two identical objects in opposite quadrons of the arena.For example, one in the north west corner and one in the south east corner. Fasten any objects that are too light to the floor with removal mounting putty. Counter balance the use of each set of objects as well as the location of the novel object to each of the four corners of the arena.Be sure to know, which diagonal is used for which animal, so that the diagonal use on the training day is the shape as that used on the testing day for each mouse. 24 hours after habituation, remove the mouse from its home cage and place it in the center of the arena, equidistant from the two identical objects to begin training. Allow free exploration for a minimum of five minutes, if fusing a strain of mice that are known to have low locomotor or exploration activity, extend the trial to 10 minutes for all mice in the cohorts.At the end of training trial, remove the mouse and place it in the holding cage. Once the home cage is empty, save it for use as the holding cage on testing day. Be sure to clean the apparatus and objects between mice.Next, start the testing phase or T2, by placing the familiar object used during training and one novel object in opposite quadrons of the arena. Use the same locations as you use during training for each mouse. At the T1 to T2 interval of choosing, remove the mouse from its home cage and place it in the center arena, equidistant from the familiar object and the novel object.Allow free exploration for 10 minutes. Finally, at the end of the testing trial, remove the mouse and place it in the holding cage. For analysis, exploration is defined as the nose directed towards the object within two to three centimeters with active vibracy sweeping.However, the mouse sitting on the object, is not counted towards exploration time. For both T1 and T2, score the first five minutes and calculate the total exploration time for both objects for each session. If the mouse does not meet the minimum exploration time of 20 seconds for both objects, continue scoring past five minutes until total exploration exceeds 20 seconds.Extend T1 and T2 time to 10 minutes for strains of mice that have low exploration and do not meet this minimum criterion by five minutes as observed during pilot testing. Finally, if mice do not reach a 20 second minimum of exploration for both objects for either T1 or T2 at 10 minutes, exclude them from the analysis as it cannot be confirmed that the animal spent enough time exploring to learn and discriminate. The object recognition task is an efficient and flexible method for studying learning and memory in mice, in which phosphodiesterase 2 inhibitors have been shown to improve during this task.As compared to vehicle, administration of the PDE2 inhibitors bays 60-7550 or ND7001, significantly enhanced memory in a dose dependent manner, when given 30 minutes prior to training. When administered at different time points, relative to training and testing, the PDE2 inhibitor bay 60-7550 significantly enhanced his memory when given 30 minutes prior to training, as well as immediately after training and 30 minutes prior to recall. Once mastered, this technique can be completed in two to three days if it's performed properly.While attempting this procedure, it's important to remember to run pilot studies and carefully choose the T1 to T2 interval, as choosing a wrong interval may result in a misaffect. Following this procedure, other methods like the Barn's maze can be performed in order to answer additional questions like differences in learning, which cannot be fully addressed using the object recognition tests. After its development, this technique paved the way for researchers in the field of learning and memory to explore the basic biological mechanisms of memory, as well as explore potential mechanisms of various cognitive deficits in rodents.After watching this video, you should have a good understanding of how to set up and design an appropriate experiment using the object recognition test, including running pilot studies, choosing objects and selecting the appropriate T1 to T2 interval. Thanks for watching and good luck with your experiments.

Research

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