The basic procedure of the NOR and its modifications
The NOR task evaluates the rodents’ ability to recognize a novel object in the environment. Basically, in the NOR task, there are no positive or negative reinforcers, and this methodology assesses the natural preference for novel objects displayed by rodents. The task procedure consists of three phases: habituation, familiarization, and test phase. In the habituation phase, each animal is allowed freely exploring the open-field arena in the absence of objects. The animal is then removed from the arena and placed in its holding cage. During the familiarization phase, a single animal is placed in the open-field arena containing two identical sample objects (
A +
A), for a few minutes. To prevent coercion to explore the objects, rodents are released against the center of the opposite wall with its back to the objects. The experimental context is not drastically different during the familiarization and the test phase. After a retention interval, during the test phase, the animal is returned to the open-field arena with two objects, one is identical to the sample and the other is novel (
A +
B) (Ennaceur
2010; Ennaceur and Delacour
1988; Gaskin et al.
2010; Hammond et al.
2004; Taglialatela et al.
2009). During both the familiarization and the test phase, objects are located in opposite and symmetrical corners of the arena and location of novel versus familiar object is counterbalanced (Hammond et al.
2004). Normal rats spend more time exploring the novel object during the first few minutes of the test phase, and when this bias is observed, the animal could remember the sample object. However, if animal repeats brief exposures to the sample object over a period of a few days, it can discriminate the sample from a novel object after delays of several weeks (Mumby et al.
2002). The strongest novel object preference scores tend to occur early in the test phase; while the novel object is still relatively novel, since in the course of time, the novel object became familiar (Broadbent et al.
2010). Despite animals spent more time exploring the novel object, the recognition performance varies according to the delay between the familiarization and the test phase, as well as the time of exploration of the sample during the familiarization phase (Ennaceur and Delacour
1988).
The procedure described above is the basis of the NOR. However, taking into account the objective of each investigation, some modifications can be made to the original method. In the study of Hale and Good (
2005), for a half of rats, the sample object was
A and the novel object was
B, while for the other half, the sample object was
B and the novel object
A. These modifications were made to reduce object and place preference effects. The objects apparently had no natural significance for rats and had never been associated with reinforcement.
A modification in the number of objects presented in the familiarization and the test phase could be also observed. It is noted in Oliveira et al. (
2010), Sarkisyan and Hedlund (
2009) and Benice et al. (
2006) works, where three distinct objects were presented during the familiarization phase. In the test phase, three objects were also presented, but one of them had a novel spatial location. Also, in Benice and Raber’s study (
2008), three objects were used. In the test phase, the location of objects did not change, but one of them was replaced by a novel one.
In the study of Hale and Good (
2005), the number of objects was different. They used four different objects placed in the center of the four squares of the arena. In the familiarization phase, animals contacted with four objects. In the test phase, two objects were placed in the same position remaining two objects switched positions. The object position alteration occurred in a diagonal plane. During the familiarization, an object was placed in the top left, while in the test phase, it was placed in the lower right, or vice versa.
Piterkin et al. (
2008) who evaluated the role of the hippocampus in the modulation of novel object preference made a modification in the test phase. Animals explored sample objects in one context and, after a retention interval, they returned to either the same context or to a different one, where they encountered sample objects paired with novel objects. However, this different context was also familiar. Only local features proximal to the object changed between sample exposure and test, whereas global features of the context did not change.
In Williams et al.’s study (
2007), in the familiarization phase, two identical objects were placed in the open-field arena. After delay, in the test phase, two identical objects used in the familiarization phase were placed in open-field arena, but one of them was displaced 90° from the original position.
Another modification can be observed in the study of Burke et al. (
2010). They tested object recognition memory in the aged rats and developed three experimental conditions. The first one was the basis of the NOR. The second experience could be considered as a modification of the NOR and involved the simultaneous presentation of two identical objects during both the familiarization phase and the test phase. As such, in familiarization phase, two identical novel objects (C + C) were simultaneously presented, but during the test phase, either two different novel objects (
D +
D) were presented (“novel condition”) or the objects from familiarization phase were presented again (“repeat condition”). Here, each animal executed two trials of recognition testing; one trial was the novel condition, while the other was the repeat condition. In this test, to promote more exploration, the position of the objects in the open-field arena was also changed. The order of trials and stimuli presented were counterbalanced across animal groups. After familiarization phase and retention interval, animals that participated in the repeat condition were exposed to the novel condition, while animals that participated in the novel condition were exposed to the repeat condition. This modification allowed direct comparison of exploration time in the test phase relative to exploration time in the sample phase, because both phases involved exploration of a pair of identical objects. The third experiment could be also considered as a modification of the NOR. It evaluated the NOR test with context change. For this, an open-field arena
A was used for both familiarization phases, while the both test phases occurred in open-field arena
B. Here, each animal participated in two object familiarization and test phases.
Dere et al. (
2005) assessed the long-term memory for different objects, their spatial location, and their order of presentation in a familiar open field. Authors designed a three-trial object exploration task in which different versions of the novelty preference paradigm, the memory for spatial locations in which objects were explored, and the temporal order memory for object presentations were combined to examine whether mice could simultaneously encode and subsequently remember the “what,”-“where,”-and -“when” components of a unique episode, during two sample trials separated by 50 min, and remembered during a single test trial applied after another delay of 50 min. According to the behavioral criteria for episodic-like memory in animals, these results showed that during a single test trial the mice were able to recognize previously explored objects, remember the location in which particular objects were previously encountered, as well as to discriminate the relative recency in which different objects were presented.
A different test phase of NOR was made by Weible et al. (
2009), as they worked with two sample objects in the first two test sessions. In the third and fourth test sessions, a sample and a novel object were used. And finally, in the fifth and sixth test sessions, objects had a novel location. Each test phase lasted 10 min, 6 min apart.
This way, we can observe an amount of features that could be evaluated with single modifications of a method of the NOR task, although all modifications of this test are always based on three steps: habituation, familiarization, and test phase.
Animals
The NOR test is widely used to evaluate object recognition memory in rodents and lead itself well cross-species generalization (Gaskin et al.
2010; Reger et al.
2009). Thus, it is important to understand what kind of animals has been used in the NOR test and which are their features (details presented in Table
1). Sometimes animals’ models with specific modifications were necessary. In Taglialatela et al.’s study (
2009), transgenic animals to study Alzheimer’s disease (AD) have been used, since these mice suffer from progressive decline in several forms of declarative memory including fear conditioning and novel object recognition. For the same purpose, this kind of mice was also used in Hale and Good research (
2005), once they studied the effect of a human amyloid precursor protein mutation that results in an autosomal dominant familial form of AD on processes supporting recognition memory, including object location memory.
Table 1
Animals used in the NOR test
Gender
|
Rats | Aggleton et al. 2010; Albasser et al. 2009; Aubele et al. 2008; Bevins et al. 2002; Broadbent et al. 2010; Burke et al. 2010; Clark et al. 2000; Ennaceur and Delacour 1988; Frumberg et al. 2007; Gaskin et al. 2010; Goulart et al. 2010; Herring et al. 2008; Nanfaro et al. 2010; Piterkin et al. 2008; Reger et al. 2009; Silvers et al. 2007
|
Mice | Benice and Raber 2008; Benice et al. 2006; Bilsland et al. 2008; Botton et al. 2010; Clarke et al. 2010; Dere et al. 2005; Hale and Good 2005; Hammond et al. 2004; Oliveira et al. 2010; Schindler et al. 2010; Wang et al. 2007; Weible et al. 2009
|
Sex
|
Males | Aggleton et al. 2010; Aubele et al. 2008; Bevins et al. 2002; Botton et al. 2010; Burke et al. 2010; Clark et al. 2000; Dere et al. 2005; Ennaceur and Delacour 1988; Frumberg et al. 2007; Gaskin et al. 2010; Goulart et al. 2010; Hammond et al. 2004; Herring et al. 2008; Nanfaro et al. 2010; Oliveira et al. 2010; Piterkin et al. 2008; Reger et al. 2009; Silvers et al. 2007; Schindler et al. 2010; Wang et al. 2007
|
Both males and females | Bilsland et al. 2008; Hale and Good 2005
|
Age
|
Animals 2–4 months old | Clark et al. 2000; Clarke et al. 2010; Dere et al. 2005; Frumberg et al. 2007; Goulart et al. 2010; Hammond et al. 2004; Mumby et al. 2002; Oliveira et al. 2010; Nanfaro et al. 2010; Piterkin et al. 2008; Walf et al. 2009
|
Immature, i.e., 20–23 days (weanling), 29–40 days (juvenile), and more than 50 days (young adulthood) old | |
Aged, i.e., 7–9 months and 24–25 months old | |
Housing
|
Individual cage | Broadbent et al. 2010; Burke et al. 2010; Ennaceur and Delacour 1988; Gaskin et al. 2010; Mumby et al. 2002; Oliveira et al. 2010; Piterkin et al. 2008
|
In groups of 2–5/cage | Aggleton et al. 2010; Botton et al. 2010; Goulart et al. 2010; Herring et al. 2008; Nanfaro et al. 2010
|
Feeding |
Ad libitum | Aggleton et al. 2010; Albasser et al. 2009; Aubele et al. 2008; Bevins et al. 2002; Bilsland et al. 2008; Botton et al. 2010; Broadbent et al. 2010; Burke et al. 2010; Clark et al. 2000; Clarke et al. 2010; Goulart et al. 2010; Hammond et al. 2004; Herring et al. 2008; Nanfaro et al. 2010; Oliveira et al. 2010; Reger et al. 2009; Sarkisyan and Hedlund 2009; Silvers et al. 2007; Schindler et al. 2010; Wang et al. 2007
|
Access restricted, 25–30 g/day | |
Although most of studies have used rats or mice, we found two works where monkeys have been used to recognize objects. The first one was developed by Buckmaster et al. (
2004), who applied the DNMS test and the object discrimination acquisition and retention test. They worked with nine feral-born, experimentally naive, male cynomolgus monkeys (
Macaca fascicularis), weighing 4.2–6.0 kg at the beginning of the experiment, estimated to be about 5–7.5 years old. As rodents, they were maintained on a 12-h light/dark cycle, housed individually and fed with primate chow twice per day in an amount that supported reliable performance, and received a daily multivitamin and water available ad libitum
. The second one was developed by Peissig et al. (
2007), who distinguished between the effect of long-term object familiarity and that due to short-term repletion, but object familiarity was only measured over the course of several days or even weeks. In their research, they used 2 adult male rhesus macaque monkeys (
Macaca mulatta), weighing between 9 and 13 kg.
According to the European Community Council Directive for the Care and Use of Laboratory Animals of 24 November 1986 (86/609/EEC), all procedures and the place where animals live must be controlled with respect to variables temperature (19°–23°C ± 1°C), humidity (45–60%), and light (12/12-h light/dark cycle). Regarding the form of animal feeding, during the most of experiments, animals had free access to food (laboratory chow) and water in their home cages. However, sometimes animals received reduced daily ratio. It is important to note that in all studies with animals, the procedures were designed to minimize the potential discomfort during behavioral tests. Depending on the laboratory and country, experiments were performed in a set of standards that researches must respect and comply in order to protect the rights of animals and minimize suffering. It is important to note that in most articles, this issue was approached.
We may notice extensive modifications that were made to the work in respect of animals used, their features, and characteristics. Collectively, the findings cited above suggest some differences in preference for novelty, dependent upon gender, strain, sex, and especially age. It is important to remember that in the first research of Ennaceur and Delacour (
1988), these authors used a total of 220 male Wistar rats weighing 200–250 g, housed in individual cages, with 12 h of light–dark cycle (7.00 a.m. to 7.00 p.m.), the ambient temperature 23 ± 1°C, and free access to food and water.
Exploration concept
A concept that needs to be clarified is the “exploration.” Ennaceur and Delacour (
1988) defined as exploration of an object the directing the nose at a distance ≥2 cm to the object and/or touching it with the nose, while turning around or sitting on the object was not considered as an exploration. For most studies, exploration was defined as the orientation of animal’s snout toward the object, sniffing or touching with snout, while running around the object, sitting or climbing on it was not recorded as exploration (Aggleton et al.
2010; Aubele et al.
2008; Bilsland et al.
2008; Broadbent et al.
2010; Clark et al.
2000; Clarke et al.
2010; Goulart et al.
2010; Hale and Good
2005; Nanfaro et al.
2010; Reger et al.
2009; Silvers et al.
2007; Schindler et al.
2010). Sometimes, when animal’s head was oriented within 45° of the object, it can also be viewed as exploration (Gaskin et al.
2010; Mumby et al.
2002; Piterkin et al.
2008). However, the major difference between studies was the distance from the snout to the object that each one considered as exploration, basically within 1–4 cm (Aggleton et al.
2010; Botton et al.
2010; Broadbent et al.
2010; Burke et al.
2010; Clark et al.
2000; Ennaceur and Delacour
1988; Gaskin et al.
2010; Hale and Good
2005; Mumby et al.
2002; Nanfaro et al.
2010; Piterkin et al.
2008; Sarkisyan and Hedlund
2009; Silvers et al.
2007; Wang et al.
2007; Williams et al.
2007).
However, in Aubele et al.’s research (
2008), three behaviors were evaluated during the experiment. They were categorized as ambulating, rearing, or remaining stationary; times spent on these three activities were measured separately. Thus, they defined “Ambulation” as the crossing of at least 1 floor grid line within a 3-s period; “Stationary” was when the animal remained unmoving at least during 3 s; “Rearing” was defined as a lifting of the forelimbs and sitting back upon the haunches. The behaviors were quantified from digital recordings. In several studies, when animals showed lack of exploration activity, they were excluded from the experiment (Bilsland et al.
2008; Clarke et al.
2010; Ennaceur and Delacour
1988; Reger et al.
2009; Taglialatela et al.
2009).
Habituation, familiarization, and test delays
The NOR test consists of the habituation phase, the familiarization phase, and finally the test phase. The time that animal spent during each of these phases as well as the delay between them can differ from study to study (for details, see Table
2).
Table 2
Habituation, familiarization, and test phase in the NOR paradigm
Habituation phase
|
1 day, with different duration and number of sessions |
One session: 3 min (Aubele et al. 2008), 5 min (Goulart et al. 2010; Oliveira et al. 2010; Walf et al. 2009), 6 min (Silvers et al. 2007), 10 min (Bevins et al. 2002; Botton et al. 2010; Gaskin et al. 2010; Hale and Good 2005; Wang et al. 2009); two sessions: 10 min (Taglialatela et al. 2009), four sessions: ~20–30 min (Piterkin et al. 2008) |
2–5 consecutive days with different duration |
2 days: 5 min (Albasser et al. 2009; Hammond et al. 2004), 10 min (Bevins et al., Gaskin et al. 2010; Burke et al. 2010), 3 days: 5, 10 or 30 min (Benice and Raber 2006; Herring et al. 2008; Reger et al. 2009; Sarkisyan and Hedlund 2009), 4 days, 20 min (Clarke et al. 2010), 10 min (Williams et al. 2007), 5 days, 5 min (Clark et al. 2000; Oliveira et al. 2010) |
Familiarization phase
|
1 day, with different duration and number of sessions | 3 or 5 min (Ennaceur and Delacour 1988), 3 min (Aubele et al. 2008; Ennaceur and Delacour 1988; Reger et al. 2009; Nanfaro et al. 2010; Walf et al. 2009), 4 min (Burke et al. 2010), 5 min (Clarke et al. 2010; Ennaceur and Delacour 1988; Reger et al. 2009), 10 min (Botton et al. 2010; Frumberg et al. 2007; Hale and Good 2005; Taglialatela et al. 2009; Wang et al. 2007), 15 min (Nanfaro et al. 2010), three consecutive 10-min trials (Benice and Raber 2008) |
2–5 consecutive days with different duration | 2 days (Ennaceur 2010, Silvers et al. 2007), 3 days (Benice et al. 2006—5 min; Sarkisyan and Hedlund 2009—5 min; Schindler et al. 2010—6 min), 5 days (Weible et al. 2009—10 min) |
Time of contact with an object | 20 s (Ennaceur and Delacour 1988; Stemmelin et al. 2008), 30 s (Buckmaster et al. 2004; Clark et al. 2010, Herring et al. 2008; Goulart et al. 2010; Williams et al. 2007), 38 s (Hammond et al. 2004), 5 min (Stemmelin et al. 2008), 10 min (Hammond et al. 2004; Williams et al. 2007), 20 min (Goulart et al. 2010) |
Delay between the familiarization and the test phase | 10 s (Clark et al. 2000), 1 min (Ennaceur 2010; Ennaceur and Delacour 1988), 2 min (Hale and Good 2005; Taglialatela et al. 2009), 5 min (Hammond et al. 2004), 15 min (Gaskin et al. 2010; Reger et al. 2009; Piterkin et al. 2008), 10 min (Clark et al. 2000), 30 min (Hale and Good 2005), 1 h (Clark et al. 2000; Piterkin et al. 2008; Reger et al. 2009; Stemmelin et al. 2008; Williams et al. 2007), 3 h (Gaskin et al. 2010), 4 h (Aubele et al. 2008; Frumberg et al. 2007; Taglialatela et al. 2009; Walf et al. 2009), 24 h (Albasser et al. 2009; Bevins et al. 2002; Botton et al. 2010; Burke et al. 2010; Clark et al. 2000; Clarke et al. 2010; Ennaceur and Delacour 1988; Gaskin et al. 2010; Goulart et al. 2010; Hale and Good 2005; Herring et al. 2008; Nanfaro et al. 2010; Reger et al. 2009; Wang et al. 2007), 48 h (Ennaceur and Delacour 1988) |
Test phase
|
1 day, with different duration and number of sessions | 3 min (Aubele et al. 2008; Clarke et al. 2010; Ennaceur 2010; Ennaceur and Delacour 1988; Reger et al. 2009; Nanfaro et al. 2010; Stemmelin et al. 2008), 4 min (Burke et al. 2010), 5 min (Clarke et al. 2010; Frumberg et al. 2007; Gaskin et al. 2010; Goulart et al. 2010; Mumby et al. 2002), 6 min (Silvers et al. 2007; Schindler et al. 2010), 10 min (Bevins et al. 2002; Hale and Good 2005; Taglialatela et al. 2009; Wang et al. 2007), 15 min (Oliveira et al. 2010), two consecutive 10-min trials (Benice and Raber 2008, Benice et al. 2006) |
2–6 consecutive days with different duration | 2 days: 3 or 5 min (Ennaceur and Delacour 1988; Sarkisyan and Hedlund 2009), 6 days: 5 min (Weible et al. 2009) |
Starting from the first study that used the NOR test (Ennaceur and Delacour
1988), five experiments were developed. Animals were randomly allocated to four groups. In experiment 1, animal explored, during 2 min, the empty open-field arena. Then, two testing sessions that comprised two trials were performed. In the first trial, i.e., a familiarization phase (T1), rats explored only one object during 5 min. After 1-min (group 1), 1-h (group 2), 4-h (group 3), and 24-h (group 4) delay, a test phase (T2) occured. Here, animals explored the familiar object and a novel one during 3 min. A repetition of this procedure was performed after 48-h delay. In experiment 2, authors fixed the time spent exploring object during T1, in order to make the test more sensitive to retention duration. Thus, animals remained in the apparatus until they explored the object during 20 s. In experiment 3, during T1, two identical objects were presented instead of one, in order to make T1 and T2 more comparable. Animals explored the empty open-field arena during 2 min. On the next day, rats had a familiarization phase (T1) when they explored two identical objects during 3 min. After 1-min (group 1) or 1-h (group 2) delay, during the test phase, they also explored two objects, the familiar and novel one. A repetition of this procedure was developed after 48-h delay. In experiment 4, experimental conditions and behavioral testing were similar to those described in experiment 3; however, all animals were submitted in a random sequence to 3 different intertrial delays (1 min, 1 h, and 24 h), one session per delay. The intersession interval was 48 h. This experiment had a purpose to understand the influence of the retention time. Lastly, in experiment 5, animals were exposed to experimental conditions for a 2-min session by day for 2 days. On the third day, only one session of the test began. Familiarization and test phase lasted 3 min, and the intertrial delay 1 min. There were three animal groups that were exposed to pair of objects identical to the sample (
A +
A), a pair of two identical new objects (
B +
B), or the sample and a new object (
A +
B) according to the groups. The aim of this experiment was to control the performance of rats by exposing them to a pair of familiar objects or a pair of new objects in T2 session.
We can note that both the habituation and the familiarization phases occurred during only 1 day with different duration and number of sessions or during 2–5 consecutive days from experiment to experiment. As in the study of Ennaceur and Delacour (
1988), some researchers fixed the time that animals should contact with the object, in order to make the test more sensitive to retention duration. In the most studies, there was a delay between the familiarization and the test phase which varied from study to study and allowed checking the retention capacity of animals (Table
2). It is important to note that in some studies, researchers often used different retention intervals from assay to assay or from animal group to animal group, in the same work.
Concerning the test phase, we also observe different contact times. As for the familiarization phase, authors defined in different manner the time that animal should contact directly with the object.
However, there are studies that are very special and deserve a different approach. It is the case of the study of Piterkin et al. (
2008), where they used a circular track apparatus divided into multiple compartments with the use of modular walls. During the first experiment, animals were habituated to the apparatus during four daily sessions of 20–30 min each. Here, the panel that separated the start and the end compartment was removed, and animal could explore the entire track freely. However, a door in each divider wall opened in only one direction; so after the rat left a compartment, it could not return. Traveling around the track in that direction only, the rat became familiar with a different pair of matching sample objects in different compartments. For the test, the two objects in each compartment were replaced by a novel object and a copy of the sample, and the rat once again traveled around the track. These experiments assessed the performance of rats with hippocampal lesions, when the learning and test contexts were the same or when the contexts were different. In the first experiment, the context shift involved conducting the test phase in a second circular track that was located in a different room. Thus, rats with lesions and control groups were allowed exploring sample objects in one context, and after a retention interval, they returned to either the same context or to a different, but familiar context, where they encountered sample objects paired with novel objects. In the second experiment, there was only one circular track and one room, and objects were removed from one compartment of the apparatus during the sample exposure phase, to a different compartment for the test phase. Moreover, only local features proximal to the object changed between sample exposure and test, while global features of the context did not.
In the study of Bevins et al. (
2002), they associated the NOR test with place conditioning. They used an apparatus that had three compartments, one black, one white, and one, small, gray, each with different kind of flooring. In one of them, during the first 2 days, animals were placed in the center gray compartment for 10 min, and the non-preferred compartment, in which the least amount of time was spent, was defined. On the third day, 1 h before going to the apparatus, both Same and Novel groups contacted with an object in their home cage. Then, animals were placed in the non-preferred compartment, but while Novel group had accessed to a novel object, the Same group had accessed to a sample object, the same as it had in the home cage. Animals explored the place for 10 min. This procedure was repeated for 8 days. After 24-h delay, on day 11, each animal was placed in the center gray compartment and allowed free access to both end compartments for 10 min without objects. Authors concluded that animals that contacted a novel object, repeatedly paired with an environment (Group Novel), displayed a significant increase in preference for that environment while this shift was not seen if the object was familiar (Group Same). This way, they concluded that object novelty is required condition of an increase in preference for the non-preferred compartment.
At each stage of the NOR test (habituation, familiarization, and test phase), there were studies with particular characteristics. For example, in the study of Frumberg et al. (
2007), it is possible to observe a modification in the habituation phase. Here, animals were placed in the test room for at least 45 min to adjust to conditions. We can note that this phase was performed in relation to the room and not to the open-field arena.
In Broadbent et al.’s (
2010) study, it is possible to note a modification not only in the habituation phase but also in the design of all study. During the familiarization phase, each rat was allowed to explore the objects for 5 min on 12 different sessions (three times each day for 4 days). The authors calculated the total amount of time each rat spent exploring the objects during these 5-min periods. As there was a huge reduction in object exploration across the days of familiarization, authors aimed to further explore the relationship between amount of exploration during the familiarization phase and subsequent object recognition memory. Interestingly, these findings indicated that the less time animals spent exploring the objects during the familiarization phase, the stronger was the novel object preference during the test phase. The implication of these data was that animals that learned about the familiar objects more effectively became less interested in the objects across the multiple familiarization episodes than animals that learned about the objects less efficiently.
In one experiment from the Gaskin et al.’s study (
2010), animals were habituated to the open-field arena for 10 min, on 2 consecutive days. However, it was possible to observe a modification in familiarization and test phase, as they occurred in the form of intersession during 5 days. That is, on day 1, animals were familiarized with two identical copies of a sample object for 5 min in the open-field arena. Then, there were 2 h of retention interval. After that, animals were placed back in the open-field arena for 5 min, now with the third copy of the novel object. However, on day 2 to day 5, the same procedure was repeated, but the retention interval increased to 24 h.
This analysis allows us to understand that each study can differ in respect to time that each phase i.e., habituation, familiarization, and testing phase, takes and to the number of trials or duration of sessions.
Apparatus
It is important to understand what kind of apparatus is used in the NOR task, i.e., its size, shape, colors, materials, and how these parameters may differ.
Ennaceur and Delacour (
1988) used an open box made of wood 65 × 45 × 65 cm. However, in the course of time, other materials have appeared, and their size or shape also varied from experiments to experiments (for details, see Table
3). Concerning their shapes, most of them had a rectangular or quadrangular form, with different dimensions. Less common were the circular arenas, as for example in Piterkin et al.’s study (
2008) which is worth to be mentioned in details. These authors used a circular track divided into multiple compartments through the use of modular walls (more detailed procedure was already described in the previous paragraph). Concerning the size, this apparatus formed a circle with an extern diameter of 270 cm for one track, or 300 cm for the other, extended from the floor to a height of 40 cm. Both intern and extern walls had a slight concave curvature to give the animal inside the apparatus greater visual access to extra maze room cues. Divider walls separated the track into nine compartments, i.e., seven test compartments and a start and an end compartment. Each divider wall had a swinging door at the bottom center, 10 cm in diameter, that could be set to open in only one direction. Thus, when rat passed through it and into the adjacent compartment, it could not return to the previous one. These researchers used two apparatus with different visual, tactile, and olfactory properties of walls, where in one track animal explored in clockwise direction and in the other in counterclockwise direction.
Table 3
Apparatus used in the NOR paradigm
Material | Plywood (Hale and Good 2005; Goulart et al. 2010), acrylic (Botton et al. 2010), plastic (Aubele et al. 2008; Clarke et al. 2010; Hammond et al. 2004), plexiglas (Benice and Raber 2008; Clark et al. 2000; Reger et al. 2009; Sarkisyan and Hedlund 2009; Williams et al. 2007), acrylonitrile butadiene styrene (ABS) (Hammond et al. 2004), polyvinyl chloride plastic (PVC), (Clarke et al. 2010; Gaskin et al. 2010; Mumby et al. 2002), and wood (Albasser et al. 2009; Burke et al. 2010; Oliveira et al. 2010; Nanfaro et al. 2010) |
Shape | Rectangular (Aubele et al. 2008; Benice and Raber 2008; Botton et al. 2010; Broadbent et al. 2010; Clarke et al. 2010; Gaskin et al. 2010; Goulart et al. 2010; Oliveira et al. 2010; Nanfaro et al. 2010; Stemmelin et al. 2008; Walf et al. 2009) Quadrangular (Albasser et al. 2009; Benice and Raber 2008; Burke et al. 2010; Clark et al. 2000; Hale and Good 2005; Schindler et al. 2010; Taglialatela et al. 2009) Circular (Weible et al. 2009 (60 cm in diameter and 45 cm height), Williams et al. 2007 (91 cm in diameter and 51 cm height), Piterkin et al. 2008) |
Color | Black (Burke et al. 2010; Clark et al. 2000; Weible et al. 2009), opaque (Broadbent et al. 2010; Taglialatela et al. 2009), gray (Albasser et al. 2009; Bilsland et al. 2008; Gaskin et al. 2010; Mumby et al. 2002), white (Hale and Good 2005), and transparent (Benice and Raber 2008) |
However, an apparatus should take into account the objective of work and be adapted to the features of animals. It the study of Reger et al. (
2009), they had three sizes of arenas according to animals’ ages. The weanling arena that measured 32 × 52 × 30 cm
3 accommodated for animals that weighed at least 50 g, the juvenile arena that measured 52 × 52 × 30 cm
3 for animals that weighed at least 100 g, and adult arena that measured 70 × 70 × 30 cm
3 for animals of 200 g and more.
As it can be seen from Table
3, the color of apparatus was also a particularity of each study. The floor of apparatus can be covered with sawdust (Albasser et al.
2009; Gaskin et al.
2010; Goulart et al.
2010) or paper beddings (Wang et al.
2007). This cover could be agitated between trials or regularly replaced. In Gaskin et al. (
2010), a stainless steel tray served as floor and was covered with sawdust; however, the floor was removed through a slot at the bottom of one wall to facilitate changing the sawdust between each trial.
An adaptation in the apparatus was made by Bevins et al. (
2002), as they used two place condition chambers to evaluate the NOR. Here, each chamber had rectangular dimensions of 31 × 24 × 45.5 cm; one of them had walls painted flat black, flooring made of 13 metal rods, and newspaper lining the litter tray, while the other had walls painted flat white, flooring made of hardware cloth, and pine wood chips lining the litter tray. Between these two chambers, there was a small chamber with inside dimensions of 15 × 24 × 45.5 cm, gray walls, and an aluminum floor. The walls of this compartment were raised 11 cm during preference test to allow an animal to move freely between compartments.
Yet, Aggleton et al. (
2010) used a bow tie–shaped maze to develop the NOR task. This maze was made of opaque Perspex, it was 120 cm long, 50 cm wide and 50 cm high, and both ends were triangular. There was an opaque door in the center of the corridor that could be raised by the researcher. The far wall of each triangle contained two recessed food wells, 3.5 cm in diameter and 2 cm depth. The food wells were divided by a short, opaque dividing wall that protruded 15 cm from the center of the end wall. These food wells were covered by objects in the experiment proper.
Kind of objects
Objects that have been used in the NOR test vary widely in shapes, sizes, textures, materials, colors, and appearance. From the familiarization to the test phase, object features change when a novel object that is somehow different from the familiar one is presented. For instance, it can be observed in Nanfaro et al.’s study (
2010), where during the familiarization phase, animal contacted with two pink truncated pyramids (familiar object) while in the test phase with a gray opaque candlestick (novel, unfamiliar object) and a pink truncated pyramid. Thus, novel and familiar objects had different colors, shape, and size which allowed recognizing them as novelty. It is also important to know whether object eliciting abnormally high levels of spontaneous investigation does not influence the outcome of experiments. Thus, Gaskin et al. (
2010) preselected novel/sample object pairs on the basis that each object in the pairs elicited the same amount of spontaneous investigation.
Many objects have been used in this test. For instance, cans, bottles, tins, glasses, pots, pyramids, candlestick, tower, cylinder, box, Playmobil toys (man, woman, monkey, horse, and cow), Lego toys, coffee mugs, teacups, socks, PVC pipe, a sheet of newspaper wadded into a ball, Styrofoam dome, tennis ball, bath loofah, shuttlecock, pet toys, and glass vase have been used (Albasser et al.
2009; Benice and Raber
2008; Bevins et al.
2002; Botton et al.
2010; Nanfaro et al.
2010; Sarkisyan and Hedlund
2009). The objects can be made of metal, glass, porcelain, glazed ceramic, rubber, durable nontoxic plastic, aluminum, or wood (Benice and Raber
2008; Broadbent et al.
2010; Burke et al.
2010; Clark et al.
2000; Ennaceur and Delacour
1988; Goulart et al.
2010; Hale and Good
2005; Oliveira et al.
2010; Piterkin et al.
2008; Reger et al.
2009; Mumby et al.
2002; Sarkisyan and Hedlund
2009; Schindler et al.
2010; Walf et al.
2009), i.e., materials that cannot be easily gnawed by animals and that can be easily cleaned. Concerning the object height, this was influenced by kind of object and varied between 4.5 and 24 cm (Aubele et al.
2008; Gaskin et al.
2010; Goulart et al.
2010; Mumby et al.
2002; Piterkin et al.
2008; Sarkisyan and Hedlund
2009; Silvers et al.
2007). However, concerning the weight, the object should be heavy enough that animals cannot move it, as well as height enough to unable animals climbing or resting on it during trials (Clark et al.
2000; Ennaceur and Delacour
1988; Hale and Good
2005; Gaskin et al.
2010). However, as safeguard in some studies, Velcro (Broadbent et al.
2010; Clark et al.
2000; Reger et al.
2009) or glue (Clarke et al.
2010) to fix object to the arena floor was used. The object copy number differed from work to work. While there were researchers who used three identical copies (Burke et al.
2010; Gaskin et al.
2010; Nanfaro et al.
2010; Piterkin et al.
2008), others used four copies of each object (Mumby et al.
2002). These copies were used interchangeably.
The aim of each research influenced the object choice. It can be observed in Reger et al.’s work (
2009), where they studied the developmental aspects of memory in weanling, juvenile, and adult rats. Here, object size needed to be age-appropriate, objects were no taller than twice the size of an animal, and they did not resemble living stimuli. Sometimes objects were carefully selected. Aubele et al. (
2008) placed an animal in the open-field arena with four objects belonging to four categories that were differentiated by size and shape. Thus, they defined criteria for different categories such as large (>18 cm tall), small (<12 cm tall), smooth (having a regular, cylindrical shape), and complex (having sharp angles, curves, or extending features). Then, the object categories were as follows: small/smooth objects (e.g., small bowl); large/smooth objects (e.g., soda can); small/complex objects (e.g., teacup); and large/complex objects (e.g., coffee mug).
The kind of familiar or novel object as well as the relative position should be counterbalanced and randomly permuted for each experimental animal.