Abdelkader Ennaceur

One-trial object recognition in rats and mice: methodological and theoretical issues.

The one-trial object recognition task involves memory of a familiar object in parallel with the detection and encoding of a novel object. It provides the basis for the study of a wide range of cognitive and neuropsychological functions and processes in rats and mice. However, unlike in humans, primate and pigeon studies, object recognition in rats and mice has been mostly limited to memory while little is known about object perception, affordances and acquisition of a representation of an object. In the present paper, we addressed some of these issues. We also described novelty preference models and hypotheses that account for one-trial object recognition and question the validity of the novelty preference concept. In addition, we discussed whether one-trial object recognition involves working memory and how it involves memory of an episode.

Detailed analysis of the behavior of lister and wistar rats in anxiety, object recognition and object location tasks

The present study, examines some issues in the measure and analysis of behavior in animals. Two strains of rats of both genders were used to illustrate and discuss these issues. We examined to what extent various behavioral measures reflect different or identical emotional or cognitive factors and, how sensitive are the various parameters of a task to differences between strains and genders. Wistar and Lister males and females rats were tested in an anxiety test then in the object recognition task followed by the object location task. Taking advantage of a simple computer program it is possible to: (1) record several parameters of theses tasks and examine the pattern of animal responses toward novelty and/or familiarity; (2) examine whether different measurements of the same response would reflect anxiety response to novelty and, can they discriminate between novelty and familiarity responses to objects; and (3) examine if changes in the pattern of animal responses are reflected by these measurements and, whether anxiety or discrimination is evident mainly during the first minute of the test. The results on the anxiety test show that different measures of the same response proved concordant and revealed significant differences between Lister males and Wistar males. Lister males approached more frequently an object and spent more time on an object in each approach compared to Wistar males in the first 5 min of test and in the total 10 min. They have also shorter latencies between approaches compared to Wistar males. The examination of performance over different time bins was significant with the measure of frequency. Lister male rats approached less frequently the object in the last 5 min of the test compared to the first 5 min. Their performance, however, did not differ from that of the other groups in this last 5 min. In the memory tasks, the measure of the frequency of approaches suggests that Lister male rats were able to discriminate between novel and familiar objects and, between novel and familiar location of objects. The measure of latency of first approach shows that Wistar female rats were able to discriminate between objects only in the spatial memory test. Discrimination in the object recognition task was observed in the first and second minute, and in the total 3 min sessions. Discrimination in the object location task was observed with the measure of frequency of approaches, in the first minute, and in the total 3 min sessions. Results from the total 3 min sessions were more concordant between the different measures of discrimination than results from separate 1 min bins. The results from the two memory tasks show that novelty prevented habituation to re-exposure to the testing environment. In many cases, novelty increased exploration of the objects in the choice phase compared to the sample phase. However, this lack of habituation or increased exploration in the choice phase is not concordant with most results of discrimination between novelty and familiarity from the same type of measurements.

Emotional responses and memory performance of middle-aged CD1 mice in a 3D maze: Effects of low infrared light

Non-thermal near infra-red (IR) has been shown to have many beneficial photobiological effects on a range of cell types, including neurons. In the present study, a pretreatment with a daily 6 min exposure to IR1072 for 10 days yielded a number of significant behavioral effects on middle-aged female CD-1 mice (12-months) tested in a 3D-maze. Middle-aged mice show significant deficits in a working memory test and IR treatment reversed this deficit. Interestingly, the IR treated middle-aged group despite making less memory errors than sham middle-aged group spent longer time in different parts of the maze than both the young group (3-months) and sham-middle-aged group (12-months). Young mice appeared more anxious than middle-aged mice in the first sessions of the test. Exposure to IR appeared to have no significant effects upon exploratory activity or anxiety responses. However, it elicited significant effects on working memory, with the IR middle-aged mice being more considerate in their decision making, which results in an overall improved cognitive performance which is comparable to that of young CD-1 mice. The present study describes a novel method for assessing emotional responses and memory performance in a 3D spatial navigation task and demonstrates the validity of our new all-in-one test and its sensitivity to ageing and non-invasive beneficial IR treatment.

Effects of exposure to extremely low-frequency magnetic field of 2 G intensity on memory and corticosterone level in rats

In the present study, we examined the effects of chronic exposure (1 and 2 weeks) to an extremely low-frequency magnetic field (ELFMF) of 2 G intensity on memory in rats using an object recognition task. Comparable groups of rats were exposed for 1, 2 or 4 weeks to ELFMF and the following day blood samples were collected from each rat for the measurement of corticosterone level. Our results demonstrate that exposure to ELFMF induces a significant increase in the level of corticosterone in blood plasma and is associated with impairment in discrimination between familiar and novel objects.

Tests of unconditioned anxiety — Pitfalls and disappointments

The plus-maze, the light-dark box and the open-field are the main current tests of unconditioned anxiety for mice and rats. Despite their disappointing achievements, they remain as popular as ever and seem to play an important role in an ever-growing demand for behavioral phenotyping and drug screening. Numerous reviews have repeatedly reported their lack of consistency and reliability but they failed to address the core question of whether these tests do provide unequivocal measures of fear-induced anxiety, that these measurements are not confused with measures of fear-induced avoidance or natural preference responses - i.e. discriminant validity. In the present report, I examined numerous issues that undermine the validity of the current tests, and I highlighted various flaws in the aspects of these tests and the methodologies pursued. This report concludes that the evidence in support of the validity of the plus-maze, the light/dark box and the open-field as anxiety tests is poor and methodologically questionable.

Models of anxiety: Responses of mice to novelty and open spaces in a 3D maze.

The present report describes the emotional responses of different strains of mice to exposure to a novel open space model of anxiety using a 3D spatial navigation task. The 3D maze is modification of the radial maze with flexible arms that can be raised above or lowered below the horizontal level of a central platform. To access the arms animals need to cross a bridge linking the arms to the central platform. In this model, mice are exposed to novelty in an unfamiliar open space setting with no safe alternative. Fear from novelty is compounded with the need to explore. The drive to escape and the drive to approach are intermingled making this open space model radically different from the current models of anxiety which provide animals with the choice between safe and anxiogenic spaces. In a series of experiments, we examined the behaviour of different groups of mice from C57, C3H, CD1 and Balb/c strains. In the first experiment, different groups of C57 mice were tested in one of the three arms configurations. In the second experiment, C57 mice were compared to C3H mice. In the third experiment, C57 mice were compared to CD1 and Balb/c mice in the raised arm configuration over three successive sessions. In the fourth experiment, we examined the behaviour of C57 mice in the lowered arm configuration with an open and an enclosed central. In the final experiment, we examined the difference between C57 and C3H mice of both genders. Using several spatio-temporal parameters of the transition responses between central platform, bridges and arms, we have been able to show consistent results demonstrating significant differences between C57 and C3H mice, and between Balb/c and both C57 and CD1 mice. C3H appear more anxious than C57 mice, and Balb/c mice seem more anxious than C57 and CD1 mice. We also observed significant differences between sexes in C3H mice but not in C57 mice. C3H male mice appear more anxious than C3H female mice and than both C57 male and female mice. In the lowered arm configuration with an enclosed central platform, C57 mice took longer time to make a first entry to an arm, made more visits to bridges before first entry to an arm and required longer time between re-entries to arms, spent longer time on the central platform and shorter time on arms compared to mice in the other arm configurations. They also made frequent entries to the centre and bridges compared to mice in the lowered arm with an open central platform. These results demonstrate not only the sensitivity of the parameters of the test but also the consistencies and concordances of the results which make this 3D maze a valuable new tool in the study of the underlying neural mechanisms of anxiety responses in addition to learning and memory, and in assessing the effects of potential anxiolytic drugs. In this report we examine methodological issues related to the design of animal behavioural paradigms and question the value and the construct validity of the current models of human anxiety.

Do rats really express neophobia towards novel objects? Experimental evidence from exposure to novelty and to an object recognition task in an open space and an enclosed space.

Three set of experiments were performed in an enclosed space (open-field) and in an open space (elevated platform). The surface of the open-field and the elevated platform were divided in nine equal squares. Rats were exposed (without previous habituation) in a unique session (experiment 1) or three consecutive sessions (experiment 2) either to an open-field (enclosed space) or to an elevated platform (open space) with and without an object on the centre of the field. In experiment 3, rats were exposed (without previous habituation) either to an enclosed or an open space on five consecutive sessions, one session a day. They were tested in an object recognition test in sessions 1, 3 and 5. In sessions 2 and 4, no objects were present. In experiment 1, we recorded the latency, frequency and duration of entries into different areas of the field. In experiment 3, we recorded the latency, frequency and duration of contacts with objects in addition to entries into different areas of the field. The first experiment demonstrates that rats exposed for the first time to an enclosed or an open space do not express neophobia toward novel objects in the absence of walls that surround an open-field. They crossed frequently into and spent more time in areas occupied with an object than in unoccupied areas. After two sessions of habituation to an empty open space or an empty enclosed space, the latency of first approach to a novel object is reduced while the frequency and duration of approaches are increased. The third experiment on object recognition confirmed that rats do not avoid novel objects; they made frequent visit and spent more time in the corner of the field occupied with an object than in empty corners. Recording of crossings provided detailed information about the patterns of exploratory behavior of rats but failed to reveal discrimination between novel and familiar objects which was evident in both open and enclosed space with recording of contacts with objects on the fifth exposure.

Are benzodiazepines really anxiolytic?: Evidence from a 3D maze spatial navigation task

The effects of diazepam and chlordiazepoxide were assessed in a 3D maze which is a modification of an 8-arm radial maze. Each arm of the maze is attached to a bridge radiating from a central platform. Animals exposed for the first time to the maze do not venture beyond the line that separate a bridge from an arm. The prime criteria set for an anxiolytic effect is whether mice would increase the frequency of entries onto arms and increase arm/bridge entries ratio. C57 mice readily cross the line on first exposure and make more than 8 arm visits onto arms on second exposure, while other strains (CD-1 and Balb/c) hold back and rarely cross the line on first exposure and require more sessions to make more than 8 arm entries. An anxiolytic drug is expected to encourage intermediate (CD-1) and high (Balb/c) anxiety mice to adventure onto the arms of the maze and make more visits to the arms to comparable levels seen with low anxiety c57 mice. In the present report, administration of different doses of diazepam (0.625, 1.25, 2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (5, 10 and 15 mg kg(-1) i.p.) did not reduce anxiety in animals, with the lowest dose of diazepam increasing motor activity in Balb/c and increasing anxiety in c57 mice while the highest doses of both diazepam (2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (15 mg kg(-1) i.p.) induced mild sedation. Our results raise some concerns about the methodological foundations in the current assessment of anxiety and anxiolytic compounds both in animal and human studies.

Anxiety responses in Balb/c, c57 and CD-1 mice exposed to a novel open space test

In the present report we describe the behavior of two albinos (BALB/c and CD-1) and one pigmented (c57/BL6) strains of mice exposed to a novel open space anxiety test in a single 12 min session. The test is based on exposure of mice to an unfamiliar elevated platform which is extended on two opposite sides with steep slopes presented downward or upward. In the first experiment, the behavior of mice was examined on the elevated platform at two different heights (75 and 100 cm) with downward slopes. In the second experiment, we examined the behavior of mice on the platform at the lowest height (75 cm) but with upward slopes which lead to a stand. In the third experiment, we examined the behavior of Balb/c mice on the platform at the lowest height (75 cm) with downward slopes, and a hub enclosure providing a protected space located in the centre of the platform. The least anxious strain of mice was expected to take risks and cross onto the slopes (experiments 1 and 3) and onto the stands (experiment 2). The results of experiment 1 show that Balb/c mice did not cross onto the slope, and CD-1 mice made more crossings into and spent more time on the slopes than c57 mice. The increase in the heights of the platform reduced the number of crossings on the platform in all three strains of mice, and decreased the time spent on the platform before first entry onto a slope in c57 and in CD-1 mice. It also decreased the number of entries and duration of entries onto the slopes in CD-1 mice. In experiment 2, Balb/c mice did cross onto the upward slopes but significantly less than c57 and CD-1 mice but they did not cross onto the stands attached to the end of the slopes. CD-1 mice made more entries onto and spent more time on the stands than c57 mice. In the third experiment, Balb/c and c57 mice spent most of their time inside a protective space (cylinder) placed in the centre of the platform demonstrating strong avoidance responses of the outer area of the platform, and only three c57 mice crossed onto the slopes for a very brief duration in one or two entries. In all three experiments, mice entered more frequently and spent more time in the outer areas than in the inner areas of the platform, particularly in the areas adjacent to slopes than in the areas adjacent to a void space. CD-1 mice appears the least anxious taking more risks by venturing onto the slopes and onto the stands while Balb/c appears the most anxious spending a large amount of time in the areas adjacent to the slopes. The different configurations of the test apparatus (experiments 1 and 2) seem to provide different incentives for the drive to explore and escape which may account for differences in anxiety responses whereas the presence of a protective space (experiment 3) appears to encourage avoidance responses.

Detailed analysis of the behavior and memory performance of middle-aged male and female CD-1 mice in a 3D maze.

Fifty percent of CD-1 mice from both sex die by the end of 2 years. The survival rate is higher in females than in males. This high mortality rate is associated to the high susceptibility of this strain of mice to some immuno-pathologies and the high incidence of systemic amyloidosis. It is therefore possible that premature cognitive deficits can be observed in CD-1 mice. In the present study, we describe a novel method for assessing emotional responses and memory performance of young (4 months) and middle-aged (12 months) CD-1 mice of both sexes in a 3D spatial navigation task. Animals are introduced to the maze without preliminary habituation and trained in a working memory test. As expected CD-1 mice have a low number of entries to arms on their first exposure to the maze which confirm our previous report on the anxious trait of this strain compared to C57/BL6 mice. The measure of arm/bridge ratio suggests that anxiety induced by exposure to the maze persists much longer in middle-aged male mice compared to middle-aged female mice and compared to both young male and female mice. The measure of memory revealed that young female mice made significantly less arm repeats and more unique arm visits before first arm repeat than middle-aged female and male mice. There are also significant differences between young female and young male mice with the former committing fewer errors than the latter.