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

Summary

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

Abstract

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.

Introduction

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 mice^2,3,4,5,6,7. 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 object^7,8,9.

The main advantage of the ORT over other rodent memory tests is that it relies on rodents' 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 tests^{10,11,12,13,14,15}, 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 memory^6,18,19,20. 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 studies^{21,22,23,24,25,26,27,28}. 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.

Protocol

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

1. 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 a comprehensive description of object choice⁷).
   1. Test for innate preference and discrimination (see Steps 2 & 3).
   2. Use objects that are mouse-sized or only slightly larger to encourage exploration (Figure 1). To reduce induced preference, ensure that mice are able to climb on both objects or neither object, though ability to climb on an object may increase interest in exploration (time spent just sitting on an object is not counted towards exploration time.)
   3. Have at least 2 copies of each object. However, to minimize potential odor cues during testing, have at least 3 of each object, duplicates for training and one for testing.
   4. Use objects made of non-breakable material, as loss of an object due to damage during experimentation could interfere with the continuity of testing, and potentially cause harm or injury to the animal.
   5. Thoroughly clean the objects before and between use (70% vol/vol ethanol is appropriate).
2. To minimize the stress of bright lighting, use diffuse, low lighting, with the center of the maze illuminated around 20 lux. Use a temperature and humidity similar to regular housing conditions. If mice are being moved from a housing room to a different room for the experiment, acclimate mice to their new room for at least 1 h prior to use each day.
3. Handle mice well prior to training. Because the test relies on the natural tendency of rodents to explore novelty, reduce any stress or anxiety from handling that may interfere with their desire to explore the arena, and subsequently, the objects. Ideally, handle mice 1 - 2 times/day for at least 1 min, 1 to 2 weeks prior to testing^15,29.
4. For the main arena, use a square chamber (around 40 cm x 40 cm x 40 cm) made from white or black, non-porous plastic, contrasted to the color of the mouse. Alternatively, use a round arena ed. Specifically, when using anxious mice that may sit in the corners of a square arena, a round arena may be preferable to encourage exploratory behavior^18,30.
   1. On training day, place 2 identical objects on the diagonal (i.e. one in the NW corner and one in the SE corner). If objects are too light and can be moved by the mouse, fasten them to the floor. Removable mounting putty works well.
   2. For testing day, use the 3^rd copy of each object to place one familiar object and one novel object on the same diagonal as the training day (Figure 2).
   3. Counterbalance the use of each set of objects so that each object is used equally as a familiar object and as a novel object. Also, counterbalance the location of the novel object to each of the 4 corners of the arena. Be sure to note which diagonal is used for which animal so that the diagonal used on the training day is the same as that used on the testing day, for each mouse.
   4. Thoroughly clean the apparatus and objects to remove odor cues before and between use (70% vol/vol ethanol is appropriate).
5. Place the camera directly overhead of the apparatus for optimal view of exploration. Only use software with nose-point detection for accurate analysis. If using software, capture a background image with the objects in place prior to starting. In the software, set the area of exploration to be approximately 2 - 3 cm around the object.
6. To reduce experimenter interference, record the trial and score it later. Blind the scorer to the experimental conditions. If manually scoring during the experiment, ensure that the experimenter is more than 1 meter from the arena, and not visible to the mouse.
   NOTE: Any time the mouse spends sitting on the object without active vibrissae sweeping or sniffing does not count as exploration time.
7. Define exploration as when the mouse's noise is pointed towards the object and within 2 - 3 cm of the object, with active vibrissae sweeping or sniffing. Do not count any time sitting on the object without indication of active exploration.

2. Necessary Pilot Experiments

1. Testing for induced preference
   1. For habituation, remove the mouse from its home cage and place it in the middle of the open arena. Allow the mouse to freely explore for 5 min.
   2. On Training Day (T1), place 2 different objects in opposite quadrants of the apparatus, (i.e., NW and SE corners). Remove the mouse from its home cage and place it in the middle of the open arena. Allow free exploration for 10 min.
   3. Calculate the discrimination index. If there is no induced preference, the discrimination index should be at or near zero. Any objects that show preference should not be used for ORT.
2. Testing for discrimination ability
   1. For habituation, remove mouse from its home cage and place it in the middle of the open arena. Allow the mouse to freely explore for 5 min.
   2. On Training Session (T1), place 2 identical objects in opposite quadrants. Place the mouse in the center of the arena and allow the mouse to explore for 10 min.
   3. On Testing Session (T2), place one of the object used during T1 and one novel object in opposite quadrants (i.e., NW and SE corners). Sixty min after T1, place the mouse in the center of the arena and allow free exploration for 10 min.
   4. Calculate the discrimination index. At a 60 min retention interval, the discrimination index should be above 0.25.

3. Experimental Procedure

1. Habituation
   1. Remove the mouse from its home cage and place it in the middle of the open, empty arena. Allow for free exploration of the arena for 5 min. Once the home cage is empty, save it for use as a holding cage the next day.
   2. At the end of 5 min, remove the mouse and place in a holding cage. Do not return the mouse to its original cage, or this may affect the behavior of the remaining mice to be tested.
   3. Thoroughly clean the apparatus between mice using 70% vol/vol ethanol.
      NOTE: During habituation, anxiety-like behavior can be assessed by calculating time spent in the center (see Prut & Belzung 2003³¹). This is a useful metric when considering the length of time for T1. Higher anxiety mice may require a 10 min session to reach the minimum exploration criterion.
2. Training (T1)
   1. Place two identical objects in opposite quadrants of the arena (i.e., NE corner and SW corner).
   2. 24 h after habituation, remove the mouse from its home cage and place it in the center of the arena, equidistant from the 2 identical objects.
   3. Allow free exploration for a minimum of 5 min. If using a strain of mice that are known to have low locomotor or exploration activity, (i.e., most mice do not reach a minimum of 20 s exploration of both objects by 5 min, as noted in the pilot experiments or the literature), extend the trial to 10 min for all mice in the cohort.
   4. At the end of the trial, remove the mouse and place in the holding cage. Once the home cage is empty, save it for use as the holding cage on testing day.
   5. Thoroughly clean the apparatus and objects between mice using 70% vol/vol ethanol.
3. Testing (T2)
   1. Place one object used during T1 (i.e., the familiar object) and one novel object in opposite quadrants of the arena. Use the same locations as used during T1 for each mouse.
   2. At the T1 to T2 interval of choosing, remove mouse from its home cage and place it in the center arena, equidistant from the familiar object and the novel object.
      NOTE: At a retention interval of 24 h, most mice will not be able to discriminate between the familiar and novel object (usually -0.2 < d2 < 0.2, as compared to a positive control³²). If testing for nootropic effects, use this time point to probe for memory enhancement. To probe for memory deficits, use a shorter retention interval of anywhere between 20 min to 4 h, depending on the strain of mouse.
   3. Allow free exploration for 10 min. At the end of the trial, remove the mouse and place in the holding cage.
4. For both T1 and T2, score the first 5 min. If the mouse does not meet the minimum exploration time of 20 s for both objects, continue scoring past 5 min until total exploration exceeds 20 s.

4. Data Analysis

1. Exclusion Criteria
   1. During both training (T1) and testing (T2), calculate the total exploration time for both objects for each session (e1 and e2). Most mice should reach a minimum exploration total for both objects of 20 s by 5 min.
   2. Extend T1 and T2 time to 10 min for strains of mice that have low exploration and do not meet this minimum criterion by 5 min, as observed during pilot testing.
   3. Score behavior for 5 min or beyond 5 min until they reach the 20 s minimum criterion.
   4. If mice do not reach a 20 s minimum of exploration for both objects for either T1 or T2 at 10 min, exclude from analysis, as it cannot be confirmed they spent enough time exploring to learn/discriminate.
2. Absolute vs Relative Analysis
   NOTE: Different formulas for analysis and their relationship to one another can be seen in Table 1.
   1. Calculate e1 as the total exploration time during training for 2 identical objects, where a1 and a2 are the identical objects.
      e1 = a1 + a2
   2. Calculate e2 as the total exploration time during testing for the familiar object (a) and the novel object (b).
      e2 = a+b
   3. Calculate d1 as simply the time spent exploring the novel object minus time spent exploring the familiar object. The absolute discrimination measure (d1) does not take into account differences in exploration time between mice or treatment groups, though in certain circumstances, it may be a more sensitive measure².
      d1 = b-a
   4. Calculate d2 as the time spent exploring the novel object minus the time spent exploring the familiar object divided by total exploration time. The most commonly used measure is a relative discrimination value often referred to as the discrimination index (d2), which is not influenced by differences in exploration time. This means all values will fall between -1 and +1.
      d2 = d1/e2
   5. Alternatively, calculate the recognition or preference index (d3)³. This is the time spent exploring the novel object divided by the total time. This means all values will fall between 0 and 1. It is often multiplied by 100 and used as percentage value.
      d3 = b/e2*100
3. Statistical Analysis
   1. Using the mean discrimination values for each group, determine memory performance using one-way ANOVA. For further analysis, make two-way post hoc comparisons with the treated vs vehicle condition and positive/negative control groups.

Results

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...

Discussion

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 analysis^2,3,5,

Materials

Name	Company	Catalog Number	Comments
Open Field Box	Panlab/Harvard Apparatus	LE800SC	Available in grey, white, or black
ANY-maze	Stoelting Co.	60000	Behavior tracking system
EthoVisionXT 12	Noldus		Behavior tracking system; requires 3 point tracking
Video Camera	Any		Video camera should be mounted directly overhead of the apparatus
70% Ethanol	Fisher Scientific	BP2818-4	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.