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Assessment of spatial learning abilities of mice in a new circular maze
Physiology & Behavior 79 (2003) 683 – 693
Assessment of spatial learning abilities of mice in a new circular maze Guido Koopmansa, Arjan Bloklandb,*, Petra van Nieuwenhuijzena, Jos Prickaertsa,1 b
a Department of Psychiatry and Neuropsychology, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands Section of Neurocognition, Faculty of Psychology, Brain and Behaviour Institute, Maastricht University, P.O. Box 616, Maastricht 6200 MD, The Netherlands
Received 30 October 2002; received in revised form 29 April 2003; accepted 14 May 2003
Abstract In the present study, we tested the spatial learning behavior of four different mouse strains (129/Sv, BALB/c, C57BL and Swiss) in a newly developed circular maze. The maze was based on the circular Barnes maze, which was initially developed for rats. Since mice do not readily enter holes in floor, additional reinforcers (positive and negative) or pretraining procedures have been used to train the animals. Because these methods are not always desirable, we examined whether mice are more willing to enter escape holes (12), which were located in the rim of the apparatus. C57BL mice appeared to improve their performance on three different measures of spatial learning: latency to find escape hole, distance to escape hole and errors (visit to other holes). The other strains also improved their performance although this was only seen for one parameter (i.e. 129/Sv and BALB/c on latency, and Swiss on distance). When the animals were trained to find another location, it was found that only the performance of the C57BL mice was transiently impaired. The C57BL mice were also very efficient in improving their performance in a repeated acquisition paradigm (six trials per day on four successive days). Applying a probe trial procedure, a clear preference for the goal location was found. These findings indicate that these mice used a spatial search strategy. Although this circular maze can be used as an additional tool to assess spatial learning in (genetically modified) mice, it is noted that strain differences in spatial learning seem to be independent of task. Further, our data with different strains indicate that different measures of behavior should be evaluated to assess the spatial learning performance of mice. D 2003 Elsevier Inc. All rights reserved. Keywords: Spatial; Learning; Memory; Mice; Strain differences; Phenotyping
1. Introduction The rapid development of transgenic and knockout mice has been a challenging field for behavioral neuroscience. Most animal models of learning and memory were designed to test the cognitive abilities of rats. However, the assessment of cognitive functions may not always be comparable between rats and mice (e.g. Ref. [1]). Thus, mice may use different behavioral strategies to solve cognitive tasks which may directly affect the validity of parameters that were initially based on rat studies. The Morris water escape task has been a favorite task to test the spatial reference memory in (mutant) mice (but see
* Corresponding author. Fax: +31-43-3884125. E-mail address: [email protected] (A. Blokland). 1 Present address: CNS Research, Johnson and Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgium. 0031-9384/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0031-9384(03)00171-9
Ref. [2]). Although mice readily acquire the Morris task, it has been mentioned that this task may be too stressful for mice. Such a possible confounding effect of stress was clearly demonstrated in a study using apoE knockout mice [3]. Tests that require no strong aversive stimuli (i.e. water or shock) are preferred since the performance is less likely to be influenced by the factor stress. Different tests are available to assess the cognitive performance of mice which are based on the spontaneous behavior of these animals. For example, the T-maze or Ymaze assesses the tendency of mice to alternate left –right choices (e.g. Refs. [4,5]), and the object recognition test measures the tendency of mice to explore novel objects (e.g. Ref. [6]). Recently, the Barnes maze, which was originally developed for rats [7], has become more popular to assess spatial reference memory in mice. In this task, the mice escape from a hole in the floor to get access to their home cage. The great advantage of this type of tasks is that no strong aversive stimuli or deprivation procedures have to be
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used. However, weak aversive stimulation must be applied to increase the motivation to escape from the circular platform (e.g., buzzer, light, fan; e.g. Ref. [8]). Although the Barnes maze does not require strong stressful stimuli, the training of mice in this task is not always successful (personal communications). It appears that mice often ‘hesitate’ to enter the escape hole and start inspecting other holes. It is clear that such a behavior has a direct effect on latency and distance measures. For example, control mice needed at least 1 min to find the escape hole in this task [8,9], which is relatively long when compared to the Morris task performance (< 20 s) in which the diameter of the apparatus is almost similar. One factor that may contribute to this observation could be that whiskers of mice are bitten off when they are group housed, which makes them more anxious to enter a hole. Positive reinforcement (i.e. sugar in escape cage) appears to improve the level of performance of mice on the latency measure to about 20 s [3]. In the present study, we introduce a modified Barnes maze. Instead of making a hole in the floor, 12 escape holes were located in the wall that surrounded the circular arena, one of them giving access to the home cage. The essential difference is that mice do not have to climb down into a hole, which appeared to be a nonpreferred behavior of mice in the circular Barnes maze. Further, since it is well known that the performance of mice in spatial tasks is strain dependent [10 – 16], we assessed the performance of four different mouse strains that are frequently used as background for genetic manipulations in this task. In a second experiment, we used a repeated acquisition paradigm to further evaluate the learning performance of mice in this task using a different testing protocol. This second experiment was conducted after the first experiment was evaluated, and therefore we only tested C57BL mice in this experiment. In a final experiment, we again tested the acquisition of C57BL mice and evaluated the spatial bias of the animals in a ‘probe trial’. This was done to examine whether the mice used a spatial orientation strategy.
lon cages (Type II) on sawdust bedding in an airconditioned room (about 20 °C) under a 12/12-h light/dark cycle (lights off from 1800 to 0600 h) and had free access to food and water. They were housed individually 2 weeks before the start of testing and the animals were housed in the same room as they were tested. The mice were tested (between 1000 and 1500 h) during the light phase of the light/dark cycle. 2.1.2. Apparatus and procedures A schematic drawing of the circular maze is shown in Fig. 1. The apparatus used consisted of a circular arena with a diameter of 950 mm and was made of polyvinylchloride. The colour of the floor was grey (RAL 7035), which allowed to detect black and white mice using a video tracking system. In the rim (250 mm high) of the arena, 12 equally spaced holes (diameter 50 mm, 5 mm above the floor) gave access to horizontal, L-shaped, exit tunnels (100 mm long). The L-shaped exit tunnels (also made from polyvinylchloride) were turned 45° facing down with their open end. Only one exit gave access to the home cage, by way of a removable polyvinylchloride tunnel extension (250 mm long). A video camera, mounted in the centre above the circular arena, provided a picture of the maze on a TV monitor. The movements of a mouse were registered (five samples per second) and stored in a personal computer using the video tracking system EthoVision (Noldus equipment, Wageningen, NL). The mice were tested under light conditions (65 lx on floor of apparatus). Abundant visual cues were available. A mouse was placed into a start box, which consisted of an open-ended tube, and was positioned in the centre of the
2. Materials and methods 2.1. Experiment 1 2.1.1. Subjects All experimental procedures were approved by the research ethics committee of Maastricht University for animal experiments and met governmental guidelines. Male mice of four different strains were used: 129/Sv (129/SvPAS@Ico; n = 10), BALB/c (BALB/CBYIco; n = 9), C57BL (C57BL/6Jlco; n = 10) and Swiss (OF1 Ico; n = 11). All mice were supplied by Ifa Credo. At the start of the experiment, the animals were 3 months old. The animals were housed individually in standard Makro-
Fig. 1. Schematic drawing of the circular maze.
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circular arena. Directly after lifting the start box, a trial was started. A trial was terminated when the mouse had entered the hole that gave access to the home cage or when 4 min had elapsed, whichever occurred first. The position of the escape tunnel was the same for an animal during the training trials but was different for individual mice. This was done to prevent the use of odour cues. If an animal did not find the escape hole within 4 min (which only occurred rarely), it was picked up by the experimenter and placed into the escape hole. Once the animal entered the escape tunnel, the tunnel was closed at the side of entrance with an object so it could not re-enter the circular arena. After the mouse was confined in the escape hole, the escape tunnel was connected to the escape hole and the home cage was placed under the escape tunnel. A mouse always returned to its home cage within a few seconds. The apparatus was cleaned with a damp sponge and the next subject was tested. The mice received four trials per day. The intertrial period for a mouse was about 15– 20 min. First, the mice were trained on four consecutive days to a total of 16 trials. Subsequently, the mice were subjected to eight trials (four trials per day). For each mouse, a new escape hole for the reversal training was assigned. All mice (except one Swiss mouse which had to be excluded from Experiment 1) did escape from the arena via the escape hole within the maximum trial duration (4 min). This was also the case in the second and third experiments. The following four parameters were measured:
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performance from the last trial block of the acquisition to the first trial block of the acquisition of the new escape position, a repeated-measures analysis was performed over these two blocks per strain. Finally, interdependency between the measures registered in the maze were analysed using Pearson’s correlation coefficient. One Swiss mouse was excluded from the analysis because it did not enter the escape hole in many trials. 2.2. Experiment 2 2.2.1. Subjects We used eight male C57BL (C57BL/6Jlco) mice. Housing conditions were similar to those described for Experiment 1. 2.2.2. Apparatus and procedures The same apparatus was used as described in Experiment 1.
Escape latency: Time (s) needed to find and enter the escape tunnel with its head. Distance travelled: The total distance moved (cm) before entering the escape tunnel. Error: Total number of head deflections (scored when an animal moved its complete head into a hole) into incorrect tunnels. Running speed: The average speed (cm s 1) of a mouse during a trial. 2.1.3. Statistical analysis 2.1.3.1. Acquisition. For each parameter, the average of the four daily trials was calculated. The data were analysed using an ANOVA (GLM) with the factors Strain and Days, with Days as a repeated-measures factor. In addition, the performance was also analysed for each strain separately (one-factorial ANOVA) to examine the performance of individual strains. 2.1.3.2. Acquisition of new escape position. For each parameter, the average of two trials was calculated. The data were analysed using an ANOVA (GLM) with the factors Strain, Days and Trial blocks, with Days and Trial blocks as repeated measures. The performance of each strain was evaluated using a two-factorial within-subjects design (Days and Trial blocks). To examine the change in
Fig. 2. Performance of four mouse strains in the maze during acquisition in Experiment 1 (average of four trials (per day) are presented): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the enclosed holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean ( ± S.E.M.).
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Trial procedures were similar to those described in Experiment 1. However, the mice received six trials per day on four successive days and the escape position was changed every day. Each mouse of the group was given a different escape hole position and the position was different per day for each mouse. This was done to prevent the use of odour cues and to prevent the use of a familiar escape position. The intertrial period for a mouse was about 15 –20 min. 2.2.3. Statistical analysis For each parameter, the average of two trials was calculated (three trial blocks per day). The data were analysed using an ANOVA (GLM) with the factors Days and Trial blocks, with Days and Trial blocks as repeated measures. 2.3. Experiment 3 2.3.1. Subjects We used six male C57BL (C57BL/6Jlco) mice. Housing conditions were similar to those described for Experiment 1. 2.3.2. Apparatus and procedures The same apparatus was used as described in Experiment 1. Trial procedures were similar to those described in Experiment 1. However, the mice received six trials per day on five successive days and the escape position was the same on every day. Each mouse of the group was given a different escape hole position. This was done to prevent the use of odour cues. The intertrial period for a
Table 1 Statistical analysis of the acquisition of the maze of four different mouse strains
Fig. 3. Performance of four mouse strains in the maze during the phase in which the position of the escape hole was changed in Experiment 1 (average of two trials (per day) are presented): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean (± S.E.M.).
Strain
Parameter
Days
129/Sv
Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed
F(3,27) = 7.74** F(3,27) = 2.37 + F(3,27) = 1.09 F(3,27) = 18.34** F(3,24) = 10.51** F(3,24) = 0.97 F(3,24) = 4.62* F(3,24) = 4.28* F(3,27) = 11.56** F(3,27) = 4.56* F(3,27) = 5.50** F(3,27) = 0.57 F(3,27) = 2.25 F(3,27) = 4.23* F(3,27) = 0.15 F(3,27) = 4.47*
mouse was about 15 –20 min. Fifteen minutes after the last trial on the fifth day, the mice were left in the arena for 3 min and the time the mice spent in the different zones was measured. For this purpose, the arena was divided in 12 equal zones (i.e., one per escape hole). The layout of the borders of the zones was like sparks of a wheel. When a mouse entered the escape hole there was no possibility to escape from the hole via an escape tunnel, i.e. all 12 holes were identical. Further, in this experiment an error was scored when a mouse poked its nose in a hole in the rim, instead of its complete head as in the first two experiments.
Column ‘‘Days’’ tabulates the results of the within-subjects factor Days. The degrees of freedom, F values and associated probabilities are shown. * P < .05. ** P < .01. + 0.05 < P < .10.
2.3.3. Statistical analysis For each parameter, the average of six trials was calculated (one trial block per day). The data were analysed using an ANOVA (GLM) with the factor Days as
BALB/c
C57BL
Swiss
G. Koopmans et al. / Physiology & Behavior 79 (2003) 683–693 Table 2 Statistical analysis of the acquisition of a new position of the escape hole in maze of four different mouse strains Strain
Parameter
Factor Days
129/Sv
BALB/c
C57BL
Swiss
Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed
F(1,9) = 0.58 F(1,9) = 1.98 F(1,9) = 0.65 F(1,9) = 0.20 F(1,8) = 0.99 F(1,8) = 0.13 F(1,8) = 1.68 F(1,8) = 2.11 F(1,9) = 5.77* F(1,9) = 3.74+ F(1,9) = 5.17* F(1,9) = 0.18 F(1,9) = 0.00 F(1,9) = 0.28 F(1,9) = 0.71 F(1,9) = 0.12
Days Trials
Trials +
F(1,9) = 5.05 F(1,9) = 0.18 F(1,9) = 0.01 F(1,9) = 0.81 F(1,8) = 2.48 F(1,8) = 0.01 F(1,8) = 3.85+ F(1,8) = 6.92* F(1,9) = 8.13* F(1,9) = 7.51* F(1,9) = 4.29+ F(1,9) = 1.26 F(1,9) = 0.37 F(1,9) = 0.14 F(1,9) = 0.77 F(1,9) = 5.83*
F(1,9) = 0.05 F(1,9) = 0.36 F(1,9) = 0.63 F(1,9) = 3.56+ F(1,8) = 0.19 F(1,8) = 0.12 F(1,8) = 0.20 F(1,8) = 0.01 F(1,9) = 2.90 F(1,9) = 1.29 F(1,9) = 1.33 F(1,9) = 1.33 F(1,9) = 0.28 F(1,9) = 0.92 F(1,9) = 0.88 F(1,9) = 0.37
Columns ‘‘Days,’’ ‘‘Trials’’ and ‘‘Days Trials’’ tabulate the results of the within-subjects factors. The degrees of freedom, F values and associated probabilities are shown. * P < .05. + 0.05 < P < .10.
repeated measures. The data of the probe trial were analysed using an ANOVA (GLM) with the factor Zone as repeated measures. To further examine the difference the mice spent in the different zones, a post hoc LSD test was used to test whether the animals spent more time in the goal zone (i.e. zone where the escape hole was positioned).
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3. Results 3.1. Experiment 1 3.1.1. Acquisition 3.1.1.1. Escape latency. There was a clear effect of Days [ F(3,105) = 23.60, P < .01; see Fig. 2A], indicating that the mice became faster in finding the escape tunnel in the course of training. There was a clear effect on strain on this parameter [Strain: F(3,35) = 3.18, P < .05], but the decrease was comparable in all strains [Strain Days: F(9,105) =1.83, P>.05]. In Table 1, it can be seen that all strains, except the Swiss mice, improved their performance in the course of training. 3.1.1.2. Distance travelled. The total distance travelled decreased in the course of training [ F(3,105) = 3.36, P < .05; see Fig. 2B]. The performance on this variable was different for the mouse strains [Strain and Strain Days: F’s>3.64, P < .01]. Analysis within each strain indicated that the distance travelled decreased in the C57BL and the Swiss mice, whereas no change in performance was found for the BALB/c strain. There was a weak statistical support that the 129/Sv increased the total distance travelled in the course of training. 3.1.1.3. Errors. When analysing the performance of all animals, the total number of errors did not change in the course of training [ F(3,105) = 0.99, n.s.; see Fig. 2C]. However, as can be seen from Fig. 2C, there were clear strain differences [Strain and Strain Days: F’s>2.38, P < .05]. In
Table 3 Pearson’s correlation coefficients between the four variables of the maze
Light grey squares indicate correlation coefficients between different parameters on the same trial block. Bold/italic entries indicate P < .05.
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Table 1, it can be seen that the number of errors only decreased in the C57BL mice. In contrast, the number of errors of the BALB/c mice increased in the course of training. The performance of the 129/Sv and Swiss mice did not change during training.
but not Days [ F(1,35) = 0.15, n.s.; see Fig. 3D]. The running speed of the strains was comparable [ F(3,35) = 2.23, n.s.] and no interaction effects were found [Strain Days and Strain Trial blocks interactions: F’s < 2.38, n.s.]. The BALB/c, and to a less extent, the Swiss mice, increased their running speed on each day (see Table 2).
3.1.1.4. Running speed. There was a clear effect of training on the mean running speed of the mice [ F(3,105) = 13.59, P < .01; see Fig. 2D]. Again, there were differences between the four strains [Strain and Strain Days: F’s>4.87, P < .01]. As can be seen in Fig. 2D and Table 1, the mean running speed increased in the 129/Sv, BALB/c and Swiss mice, whereas there was no change in the running speed in the C57BL strain. 3.1.2. Acquisition of new escape position 3.1.2.1. Escape latency. Although there was weak support for an improvement over Days [ F(1,35) = 3.16, 0.05 < P < .10], there was improvement across Trial blocks [ F(1,35) = 11.00, P < .01; see Fig. 3A]. There were no Strain interaction effects [Strain Days and Strain Trial blocks interactions: F’s < 0.73, n.s.]. However, there was a clear effect of Strain on the variable escape latency in the acquisition of a new escape position [ F(3,35) = 4.64, P < .01]. Analysis of the individual strains revealed that only the C57BL mice improved their performance in the test with the new escape position (see Table 2). There was only a weak support for an improvement across trials in the 129/Sv mice. 3.1.2.2. Distance travelled. There was a tendency that the distance travelled decreased from Day 1 to Day 2 [Days: F(1,35) = 3.81, 0.05 < P < .10; see Fig. 3B]. No Strain interaction effects were found [Strain Days and Strain Trial blocks interactions: F’s < 2.17, n.s.]. On the other hand, there was a clear effect of Strain [ F(3,35) = 8.76, P < .01]. Withinstrain analysis revealed that the performance of the 129/Sv, BALB/c and Swiss mice did not change in the trials in which the escape hole position was changed (see Table 2). However, the performance of the C57BL mice improved across the trials of the acquisition of the new position. 3.1.2.3. Errors. Both for the factor Day and Trial blocks, a tendency was found that the number of errors decreased [Day: F(1,35) = 3.34, 0.05 < P < .10; Trial blocks: F(1,35) = 3.24, 0.05 < P < .10; see Fig. 3C]. A Strain effect was found [ F(3,36) = 8.58, P < .01], but no interactions with Days or Trial blocks [Strain Days and Strain Trial blocks interactions: F’s < 1.57, n.s.]. From Table 2, it can be seen that this effect was mainly due to the improved performance of the C57BL mice and to a modest improvement of the BALB/c mice. There was no change in performance in the 129/Sv and Swiss mice. 3.1.2.4. Running speed. There was a clear increase of running speed over Trial blocks [ F(1,35) = 13.27, P < .01],
Fig. 4. Performance of C57BL mice in the circular maze on four successive days in Experiment 2 (six trials per day): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean (± S.E.M.) of two trials.
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3.1.2.5. Last block acquisition vs. first block acquisition new position. It was found that the performance of the 129/Sv, BALB/c and Swiss mice on the latency, distance and error measures did not change from the last block of the acquisition to the first block of trials of the task with the new escape position [all F’s < 1.74, n.s.]. There was only a tendency that the escape latency was increased for the 129/Sv mice in the first block of the new acquisition test [ F(1,9) = 4.33, 0.05 < P < .10]. The performance of the C57BL mice on the first block of the new acquisition was worse than at the end of the first acquisition phase on all measures except the running speed [latency, distance, error: F’s>6.70, P < .05]. 3.1.3. Correlations In Table 3, the correlations between the parameters of the maze are listed. As can be seen, there was a strong correlation between successive days for each of the measures. Examination of the correlation between measures revealed that there was a good interrelation between the three indices of learning, especially between the distance travelled and the number of errors made on the same day. The measure running speed was found to have a negative correlation with escape latency on the first two days but not on Days 3 and 4. The running speed also correlated with the distance travelled, almost at every day, although the correlation was positive. The error measure poorly correlated with running speed.
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find the escape hole decreased similarly on each day [Trial blocks Days: F(6,42) = 1.46, n.s.]. 3.2.2. Distance travelled There was a clear indication that the distance travelled to find the escape hole decreased with repeated testing [Days: F(3,21) = 4.56, P < .05; Trial blocks: F(2,14) = 25.67, P < .01; see Fig. 4B]. The improvement of performance across trials differed per day [Trial blocks Days: F(6,42) = 3.11, P < .05]. This effect was due to a sharp improvement on Day 1 and only a modest improvement on Days 3 and 4. 3.2.3. Errors There was no indication that the number of errors decreased across days [Days: F(3,21) = 1.36, n.s.; see Fig. 4C]. On the other hand, performance was improved on individual days [Trial blocks: F(2,14) = 4.52, P < .05] and was similar on each day [Trial blocks Days: F(6,42) = 0.57, n.s.]. 3.2.4. Running speed During the 4 days of training, the running speed of the animals remained stable [Days: F(3,21) = 1.36, n.s.; see Fig. 4D]. The running speed of the animals increased on each day [Trial blocks: F(2,14) = 5.97, P < .05], but the increase was similar on each day [Trial blocks Days: F(6,42) = 1.58, n.s.]. 3.3. Experiment 3
3.2. Experiment 2 3.2.1. Escape latency The C57BL mice improved their performance across trials and days [Days: F(3,21) = 4.56, P < .05; Trial blocks: F(2,14) = 25.67, P < .01; see Fig. 4A]. The time needed to
3.3.1. Acquisition Similar to the first two experiments, the C57BL mice improved their performance on the different measures in the course of training [ F’s>4.94, P < .01; see Fig. 5A – C].
Fig. 5. Performance of C57BL mice in the circular maze on five successive days in Experiment 3 (six trials per day): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) Mean number of errors defined as nose pokes; (D) Time spent in the different zones of the arena (TZ: target zone; 2 – 12: other zones in clockwise order). The dotted line represents the chance level that mice spent in each zone. Values represent mean (± S.E.M.) of two trials.
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3.3.2. Probe trial In the probe trial, the mice spent an unequal amount of time in the different zones [Zone: F(11,55) = 5.62, P < .01]. As can be seen in Fig. 5D, the mice spent more time in the zone (about 35 s) where the escape hole was positioned during the acquisition trials when compared to the other zones (about 10– 15 s). Post hoc analysis revealed that the time spent in the different zones was statistically lower when compared with the time spent in the target zone, except for one zone (i.e. Zone 9).
4. Discussion 4.1. The task In the present study, we assessed the spatial learning behavior of four different mouse strains in a circular maze. This maze is an adaptation of the circular Barnes maze that was originally developed for rats [7]. Instead of making escape holes in the floor, 12 escape holes were made in the rim that surrounded a circular arena. This set-up is comparable with a task that combines the Morris and Barnes maze task [17]. In this task, escape holes were also made in the rim of the wall of a pool with water. A clear effect of hippocampal lesions was found suggesting that this was a spatial discrimination task. In our study, it was found that the C57BL mice readily acquired the task and the acquisition of a new position of the escape hole showed that the performance was transiently impaired. In the probe trial procedure, it was found that the C57BL mice spent significantly more time in the zone (1/12 of total arena) where the escape hole was positioned during training. This finding indicates that these mice used a spatial strategy to locate the position of the escape hole. No extra pretraining or additional (negative or positive) reinforcements were required to train the C57BL mice in this task which can be considered as an advantage when compared with the circular maze with holes in the floor. Further, the apparatus can easily be built and the behavior of the animals can be measured using a video tracking system. Correlation analysis revealed that the three measures of learning performance (i.e. latency, distance and errors) were interrelated. This suggests that these parameters all measure a similar underlying process. The running speed appeared independent of the escape latency (only a low correlation on the first two trial blocks) and errors made. On the other hand, there was a more consistent correlation between running speed and the measure distance travelled. These findings suggest that differences in running speed could affect the measure distance travelled, but are less likely to affect the latency and error measure. In general, the number of errors made by the mice was relatively low. This is best explained by the manner in which an error was scored, i.e. when a mouse entered a hole with its complete head. When it poked its nose in a hole, it
was not scored as an error. This was done to make a visit to the escape hole and other holes comparable. Probably, it is better to use two error scores. One error as defined in this study (i.e. complete head), and a second error score defined as a nose poke. This additional scoring may give a better insight of the commitment of mice to visit a hole, viz. making an error. This was done in the third experiment and it was found that the number of errors was higher when applying this way of scoring (i.e. nose poke). Even after 5 days of training, the mice needed about 40 s to enter the escape hole and travelled about 2 m. From this it can be inferred that the mice did not move along a straight line to the escape hole. But this appears also to be the case for the Morris water escape task, in which mice take about 10 s to find the hidden platform (e.g. Refs. [10,18]). Apparently, mice do not always directly go to an escape position. Further, the need to escape from the water may be greater than the need to escape from the circular maze in the present study. This may explain that in the present study the escape latencies are higher than those observed in the Morris task. 4.2. Strain differences in acquisition The statistical analysis did not reveal Strain Days interactions, suggesting that the rate of speed of learning of the strains was not different. It should be noted that all strains were included in the statistical analysis and that the relative high variance of the different strains may have masked the interaction effect. A separate analysis between the C57BL and 129/Sv strains revealed a clear Strain Days interaction effect on the measures distance and errors, indicating a difference in the learning rate of these strains. Strain differences were observed that were dependent on the behavioral measure (i.e. escape latency, distance travelled, errors, running speed). With respect to the escape latency, it appeared that the C57BL strain acquired the task efficiently, i.e. the time to escape gradually decreased during the acquisition of the task. The mice of this strain entered the escape hole within 40 s on the fourth day of training. On the other hand, the other three strains (i.e. 129/Sv, BALB/c and Swiss) only reduced their escape latency from the first to the second day and there was no apparent improvement from Day 2 to Day 4. For the measure distance travelled, a different picture emerged. Although the C57BL mice again showed a clear improvement over days, the BALB/c and the 129/Sv mice did not improve their performance on this measure. Although the apparent increase in distance travelled was not supported statistically, the 129/Sv mice seemed to get worse in the course of training. In contrast to the latency measure, the Swiss mice improved their performance from day to day, but the distance travelled was greater than those of the other strains. The measure ‘number of errors’ showed a different picture for the four mouse strains. As in the previous two
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measures, the C57BL mice showed a consistent decrease of the number of errors. The Swiss and 129/Sv mice did not change their performance during training. The Swiss mice made the most errors of all strains. Conversely, the 129/Sv mice made the lowest number of errors. Remarkably, the BALB/c mice made more errors in the course of training, suggesting a negative learning curve. The running speed of the mice was also different between strains. The 129/Sv mice started as the slowest animals, whereas the Swiss mice were the fastest. The BALB/c mice started intermediate but travelled as fast as the Swiss mice at the end of the four training days. The running speed of the C57BL mice did not change across the acquisition trials. Taken together, it is clear that there were significant strain differences on the four measures that were assessed in the present study. The picture of these strain differences was different for each measure. The C57BL mice appeared to show the most consistent learning on all three learning measures. Also, the running speed of this strain did not change, which can be considered as an advantage since a change in this measure across training blocks may affect the learning parameters (as discussed above). The performance of the other three strains is more difficult to interpret. Thus, a mouse strain that showed an improvement on one measure did not show an improvement on another measure (e.g. BALB/c improved on latency and got worse on errors, and the Swiss only improved on distance). It depends on what measure is assumed to be the best parameter for learning to indicate which mouse strains acquire this task. The good performance of the C57BL mice is in accordance with previous studies in which this mouse strain was tested in the Morris water escape task (e.g. Refs. [10,15,19]), the radial maze (e.g. Ref. [13,20]) and the circular Barnes maze (e.g. Ref. [3,19]). Apparently, the C57BL strain is suitable for testing spatial abilities in mice. However, it must be mentioned that various C57BL substrains exist, which may differ in their learning performance (like the 129/Sv substrains; see Ref. [21]). On the other hand, the BALB/c strain does not seem to be suitable for testing the spatial abilities in the maze, which was based on the finding that the number of errors increased and the distance to find the hole remained constant in the course of training. This poor performance of this strain was also demonstrated in other studies [10,20], in which it was concluded that BALB/c mice do not use spatial cues to guide their behavior in spatial tasks. However, it has been suggested that the poor performance of the BALB/c strain in the Morris task [18,22,23] is related to a high light intensity. Thus, it has been shown that the performance of BALB/c mice is relatively good when they are tested in a dimly lit room [24]. In our study, the animals were trained in a relative bright room, which may explain the poor performance of these mice in our test.
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The performance of the 129/Sv mice was different for the three learning measures. There was an improvement on the latency measure, no change on the error measure and a tendency for an increase on the distance measure. These findings do not support the notion that these mice acquired the task. It has been reported before that the 129/Sv mice are worse than C57BL mice in acquiring a spatial Morris task, but do acquire this task [25,26]. It should be mentioned that there are various 129 substrains and that a good Morris task performance has been reported for the 129S6/SvEvTac, but that the performance is poor for the 129/SvJ and 129/J mice [21]. In the present study, we used the 129/Sv strain from Iffa Credo. Only a few studies have used Swiss mice to evaluate spatial learning and memory performance. In one study, it was concluded that Swiss mice do not reliably find the escape platform in a learning set paradigm in the Morris task [27]. Interestingly, another study showed that Swiss mice acquired the task, although no probe trial was performed [28]. Interestingly, in the same study, the Swiss mice were impaired in a cued version of the Morris task when compared with a Down’s syndrome transgenic mouse. This latter finding questions the ability of the Swiss strain to acquire the spatial task. In the maze, the Swiss mice only reduced the mean distance to find the escape hole, but no changes in escape latency or number of errors were found. These findings indicate that Swiss mice do not acquire this spatial task. If only one parameter was chosen per strain, it could be concluded that all strains acquired the task. Although the correlation analysis revealed that the three learning parameters were correlated, there appears to be no straightforward relation between these measures between strains when the learning component of these measures was considered. This could, for example, be related to behavioral strategies or other behavioral tendencies of mice of the different strains. Therefore, we argue that it is safest to include all measures in the behavioral analysis before drawing conclusions about the learning performance of mice in this task. A further point that should be mentioned with respect to strain differences is that the findings of the present study only relate to the test conditions used in this study. As mentioned above, the lighting conditions may affect the learning behavior of the BALB/c mice. It is clear that the effect of test conditions on genetic differences in learning needs to be investigated in further studies. Although the change in the position of the escape holes resulted in a greater preparedness of the mice to enter the escape hole (i.e. no pretraining or additional reinforcers were required), the strain differences in our study are similar to those observed in previous studies, including the Barnes maze. This may suggest that the spatial learning performance of mice is independent of the task used, and may, for example, be related to the morphology of the hippocampus [29,30].
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4.3. Acquisition of a new escape position
4.6. Behavioral strategies
After the mice were given 16 trials, they were given a test with a new escape position, which can provide information whether the animals used a spatial strategy. Thus, if an animal uses a spatial strategy, the performance should get worse if the position of the escape hole is changed. This procedure has also been applied in the Morris task to assess the spatial search strategies of subjects. In our study, a clear effect of changing the escape hole was found in the C57BL mice, indicating that these mice adopted a spatial search strategy. Further, the C57BL mice improved their performance in the trials in which they had to locate a new position. In contrast, there was no clear indication that the performance of the other strains changed from the last block of the acquisition to the first block of the new acquisition test. This finding confirms that the circular maze can be considered as a spatial reference memory task, at least for C57BL mice. However, it cannot be excluded that (spatial) search strategies may go unnoticed using this procedure (see below).
There were some qualitative observations that could explain some of the findings described above. The Swiss mice showed a very active behavior in the maze. They were continuously running in the maze albeit in a specific manner. Typically, it was found that they moved to the rim of the apparatus and started checking the escape holes (anti)clockwise. This search strategy leads to a relative high number of errors. Although the Swiss mice did not find the escape hole faster, it was found that the distance to the escape hole was decreased across trials. This latter finding may indicate that the search strategy of the mice improved with repeated testing. This may be due to learning the location of the escape hole, although the mice did not directly go to this position. It could be argued that the rim of the apparatus increased the thigmotaxis behavior of this strain. However, it seems that this was only the case for the Swiss mice because the other strains did not show this typical thigmotaxis. The 129/Sv mice were very inactive, especially in the first trials. This may be related to a high anxiety level in this mouse strain, which has also been reported in other studies [15,16]. Interestingly, it was found that the distance travelled by the 129/Sv mice tended to increase in the course of training. Although no clear explanation can be given for this observation, it could be argued that this may be related to an increase in exploratory behavior when the anxiety level decreased. The decrease in the escape latency across trials can be explained by the increased running speed of the 129/ Sv mice. No apparent behavioral strategy could be detected for the C57BL and BALB/c mice. In the third experiment, it was shown that C57BL mice used a spatial orientation strategy in this task.
4.4. Repeated acquisition In the second experiment of this study, we tested C57BL mice in the circular maze using a different test procedure. We only tested the C57BL mice because Experiment 1 indicated that only these mice used an allocentric spatial orientation strategy in this task. The mice were tested to find a new escape hole position every day. This repeated acquisition procedure may be considered as a learning set paradigm [27,31], although it has to be noted that a learning set is defined as an improvement in learning using similar procedures but with different stimuli (e.g. new environment in this task) or samples. It was found that the performance on the measures latency and distance was improved on each day. However, this was not found for the error measure. This lack of effect could be due to the scoring of an error as was defined earlier (see above). There was also a relative constant drop in performance when comparing the last block of trials of a day with the first block of the next day. This finding further supports the use of a spatial orientation strategy used by C57BL mice. 4.5. Probe trial Similar to the Morris task, we used a probe trial to assess the spatial bias of the mice after they had acquired the task. For this purpose, the arena was divided into 12 equal zones that corresponded with the 12 escape holes. The mice showed a clear preference for the location where the escape hole was during the acquisition of the task. These data strongly suggest that the mice had developed a spatial orientation strategy to find the escape hole in the course of training.
4.7. Conclusions The circular maze was developed on the basis of the circular Barnes maze for rats. It appears that mice do not readily enter escape holes in the floor like rats do. As an alternative, escape holes were made in the rim of the apparatus. We found that C57BL mice acquired the task since the performance on three parameters of learning was improved with repeated testing. These mice appeared to use a spatial orientation strategy as shown in a probe trial procedure. No extra pretraining of mice or additional reinforcers were required, suggesting that the escape via a hole in the rim is more adapted to mice than a hole in the floor of the apparatus. However, 129/Sv, BALB/c and Swiss mice did not acquire the task to a same level under these testing conditions, and a spatial learning improvement was only seen on one parameter. On basis of this study, it was concluded that: (1) the circular maze can be used as an alternative tool to assess spatial learning in (C57BL) mice; (2) at least three parameters (i.e. latency,
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distance, errors) should be evaluated to assess spatial learning in this task.
[16]
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Assessment of spatial learning abilities of mice in a new circular maze Guido Koopmansa, Arjan Bloklandb,*, Petra van Nieuwenhuijzena, Jos Prickaertsa,1 b
a Department of Psychiatry and Neuropsychology, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands Section of Neurocognition, Faculty of Psychology, Brain and Behaviour Institute, Maastricht University, P.O. Box 616, Maastricht 6200 MD, The Netherlands
Received 30 October 2002; received in revised form 29 April 2003; accepted 14 May 2003
Abstract In the present study, we tested the spatial learning behavior of four different mouse strains (129/Sv, BALB/c, C57BL and Swiss) in a newly developed circular maze. The maze was based on the circular Barnes maze, which was initially developed for rats. Since mice do not readily enter holes in floor, additional reinforcers (positive and negative) or pretraining procedures have been used to train the animals. Because these methods are not always desirable, we examined whether mice are more willing to enter escape holes (12), which were located in the rim of the apparatus. C57BL mice appeared to improve their performance on three different measures of spatial learning: latency to find escape hole, distance to escape hole and errors (visit to other holes). The other strains also improved their performance although this was only seen for one parameter (i.e. 129/Sv and BALB/c on latency, and Swiss on distance). When the animals were trained to find another location, it was found that only the performance of the C57BL mice was transiently impaired. The C57BL mice were also very efficient in improving their performance in a repeated acquisition paradigm (six trials per day on four successive days). Applying a probe trial procedure, a clear preference for the goal location was found. These findings indicate that these mice used a spatial search strategy. Although this circular maze can be used as an additional tool to assess spatial learning in (genetically modified) mice, it is noted that strain differences in spatial learning seem to be independent of task. Further, our data with different strains indicate that different measures of behavior should be evaluated to assess the spatial learning performance of mice. D 2003 Elsevier Inc. All rights reserved. Keywords: Spatial; Learning; Memory; Mice; Strain differences; Phenotyping
1. Introduction The rapid development of transgenic and knockout mice has been a challenging field for behavioral neuroscience. Most animal models of learning and memory were designed to test the cognitive abilities of rats. However, the assessment of cognitive functions may not always be comparable between rats and mice (e.g. Ref. [1]). Thus, mice may use different behavioral strategies to solve cognitive tasks which may directly affect the validity of parameters that were initially based on rat studies. The Morris water escape task has been a favorite task to test the spatial reference memory in (mutant) mice (but see
* Corresponding author. Fax: +31-43-3884125. E-mail address: [email protected] (A. Blokland). 1 Present address: CNS Research, Johnson and Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgium. 0031-9384/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0031-9384(03)00171-9
Ref. [2]). Although mice readily acquire the Morris task, it has been mentioned that this task may be too stressful for mice. Such a possible confounding effect of stress was clearly demonstrated in a study using apoE knockout mice [3]. Tests that require no strong aversive stimuli (i.e. water or shock) are preferred since the performance is less likely to be influenced by the factor stress. Different tests are available to assess the cognitive performance of mice which are based on the spontaneous behavior of these animals. For example, the T-maze or Ymaze assesses the tendency of mice to alternate left –right choices (e.g. Refs. [4,5]), and the object recognition test measures the tendency of mice to explore novel objects (e.g. Ref. [6]). Recently, the Barnes maze, which was originally developed for rats [7], has become more popular to assess spatial reference memory in mice. In this task, the mice escape from a hole in the floor to get access to their home cage. The great advantage of this type of tasks is that no strong aversive stimuli or deprivation procedures have to be
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used. However, weak aversive stimulation must be applied to increase the motivation to escape from the circular platform (e.g., buzzer, light, fan; e.g. Ref. [8]). Although the Barnes maze does not require strong stressful stimuli, the training of mice in this task is not always successful (personal communications). It appears that mice often ‘hesitate’ to enter the escape hole and start inspecting other holes. It is clear that such a behavior has a direct effect on latency and distance measures. For example, control mice needed at least 1 min to find the escape hole in this task [8,9], which is relatively long when compared to the Morris task performance (< 20 s) in which the diameter of the apparatus is almost similar. One factor that may contribute to this observation could be that whiskers of mice are bitten off when they are group housed, which makes them more anxious to enter a hole. Positive reinforcement (i.e. sugar in escape cage) appears to improve the level of performance of mice on the latency measure to about 20 s [3]. In the present study, we introduce a modified Barnes maze. Instead of making a hole in the floor, 12 escape holes were located in the wall that surrounded the circular arena, one of them giving access to the home cage. The essential difference is that mice do not have to climb down into a hole, which appeared to be a nonpreferred behavior of mice in the circular Barnes maze. Further, since it is well known that the performance of mice in spatial tasks is strain dependent [10 – 16], we assessed the performance of four different mouse strains that are frequently used as background for genetic manipulations in this task. In a second experiment, we used a repeated acquisition paradigm to further evaluate the learning performance of mice in this task using a different testing protocol. This second experiment was conducted after the first experiment was evaluated, and therefore we only tested C57BL mice in this experiment. In a final experiment, we again tested the acquisition of C57BL mice and evaluated the spatial bias of the animals in a ‘probe trial’. This was done to examine whether the mice used a spatial orientation strategy.
lon cages (Type II) on sawdust bedding in an airconditioned room (about 20 °C) under a 12/12-h light/dark cycle (lights off from 1800 to 0600 h) and had free access to food and water. They were housed individually 2 weeks before the start of testing and the animals were housed in the same room as they were tested. The mice were tested (between 1000 and 1500 h) during the light phase of the light/dark cycle. 2.1.2. Apparatus and procedures A schematic drawing of the circular maze is shown in Fig. 1. The apparatus used consisted of a circular arena with a diameter of 950 mm and was made of polyvinylchloride. The colour of the floor was grey (RAL 7035), which allowed to detect black and white mice using a video tracking system. In the rim (250 mm high) of the arena, 12 equally spaced holes (diameter 50 mm, 5 mm above the floor) gave access to horizontal, L-shaped, exit tunnels (100 mm long). The L-shaped exit tunnels (also made from polyvinylchloride) were turned 45° facing down with their open end. Only one exit gave access to the home cage, by way of a removable polyvinylchloride tunnel extension (250 mm long). A video camera, mounted in the centre above the circular arena, provided a picture of the maze on a TV monitor. The movements of a mouse were registered (five samples per second) and stored in a personal computer using the video tracking system EthoVision (Noldus equipment, Wageningen, NL). The mice were tested under light conditions (65 lx on floor of apparatus). Abundant visual cues were available. A mouse was placed into a start box, which consisted of an open-ended tube, and was positioned in the centre of the
2. Materials and methods 2.1. Experiment 1 2.1.1. Subjects All experimental procedures were approved by the research ethics committee of Maastricht University for animal experiments and met governmental guidelines. Male mice of four different strains were used: 129/Sv (129/SvPAS@Ico; n = 10), BALB/c (BALB/CBYIco; n = 9), C57BL (C57BL/6Jlco; n = 10) and Swiss (OF1 Ico; n = 11). All mice were supplied by Ifa Credo. At the start of the experiment, the animals were 3 months old. The animals were housed individually in standard Makro-
Fig. 1. Schematic drawing of the circular maze.
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circular arena. Directly after lifting the start box, a trial was started. A trial was terminated when the mouse had entered the hole that gave access to the home cage or when 4 min had elapsed, whichever occurred first. The position of the escape tunnel was the same for an animal during the training trials but was different for individual mice. This was done to prevent the use of odour cues. If an animal did not find the escape hole within 4 min (which only occurred rarely), it was picked up by the experimenter and placed into the escape hole. Once the animal entered the escape tunnel, the tunnel was closed at the side of entrance with an object so it could not re-enter the circular arena. After the mouse was confined in the escape hole, the escape tunnel was connected to the escape hole and the home cage was placed under the escape tunnel. A mouse always returned to its home cage within a few seconds. The apparatus was cleaned with a damp sponge and the next subject was tested. The mice received four trials per day. The intertrial period for a mouse was about 15– 20 min. First, the mice were trained on four consecutive days to a total of 16 trials. Subsequently, the mice were subjected to eight trials (four trials per day). For each mouse, a new escape hole for the reversal training was assigned. All mice (except one Swiss mouse which had to be excluded from Experiment 1) did escape from the arena via the escape hole within the maximum trial duration (4 min). This was also the case in the second and third experiments. The following four parameters were measured:
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performance from the last trial block of the acquisition to the first trial block of the acquisition of the new escape position, a repeated-measures analysis was performed over these two blocks per strain. Finally, interdependency between the measures registered in the maze were analysed using Pearson’s correlation coefficient. One Swiss mouse was excluded from the analysis because it did not enter the escape hole in many trials. 2.2. Experiment 2 2.2.1. Subjects We used eight male C57BL (C57BL/6Jlco) mice. Housing conditions were similar to those described for Experiment 1. 2.2.2. Apparatus and procedures The same apparatus was used as described in Experiment 1.
Escape latency: Time (s) needed to find and enter the escape tunnel with its head. Distance travelled: The total distance moved (cm) before entering the escape tunnel. Error: Total number of head deflections (scored when an animal moved its complete head into a hole) into incorrect tunnels. Running speed: The average speed (cm s 1) of a mouse during a trial. 2.1.3. Statistical analysis 2.1.3.1. Acquisition. For each parameter, the average of the four daily trials was calculated. The data were analysed using an ANOVA (GLM) with the factors Strain and Days, with Days as a repeated-measures factor. In addition, the performance was also analysed for each strain separately (one-factorial ANOVA) to examine the performance of individual strains. 2.1.3.2. Acquisition of new escape position. For each parameter, the average of two trials was calculated. The data were analysed using an ANOVA (GLM) with the factors Strain, Days and Trial blocks, with Days and Trial blocks as repeated measures. The performance of each strain was evaluated using a two-factorial within-subjects design (Days and Trial blocks). To examine the change in
Fig. 2. Performance of four mouse strains in the maze during acquisition in Experiment 1 (average of four trials (per day) are presented): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the enclosed holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean ( ± S.E.M.).
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Trial procedures were similar to those described in Experiment 1. However, the mice received six trials per day on four successive days and the escape position was changed every day. Each mouse of the group was given a different escape hole position and the position was different per day for each mouse. This was done to prevent the use of odour cues and to prevent the use of a familiar escape position. The intertrial period for a mouse was about 15 –20 min. 2.2.3. Statistical analysis For each parameter, the average of two trials was calculated (three trial blocks per day). The data were analysed using an ANOVA (GLM) with the factors Days and Trial blocks, with Days and Trial blocks as repeated measures. 2.3. Experiment 3 2.3.1. Subjects We used six male C57BL (C57BL/6Jlco) mice. Housing conditions were similar to those described for Experiment 1. 2.3.2. Apparatus and procedures The same apparatus was used as described in Experiment 1. Trial procedures were similar to those described in Experiment 1. However, the mice received six trials per day on five successive days and the escape position was the same on every day. Each mouse of the group was given a different escape hole position. This was done to prevent the use of odour cues. The intertrial period for a
Table 1 Statistical analysis of the acquisition of the maze of four different mouse strains
Fig. 3. Performance of four mouse strains in the maze during the phase in which the position of the escape hole was changed in Experiment 1 (average of two trials (per day) are presented): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean (± S.E.M.).
Strain
Parameter
Days
129/Sv
Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed
F(3,27) = 7.74** F(3,27) = 2.37 + F(3,27) = 1.09 F(3,27) = 18.34** F(3,24) = 10.51** F(3,24) = 0.97 F(3,24) = 4.62* F(3,24) = 4.28* F(3,27) = 11.56** F(3,27) = 4.56* F(3,27) = 5.50** F(3,27) = 0.57 F(3,27) = 2.25 F(3,27) = 4.23* F(3,27) = 0.15 F(3,27) = 4.47*
mouse was about 15 –20 min. Fifteen minutes after the last trial on the fifth day, the mice were left in the arena for 3 min and the time the mice spent in the different zones was measured. For this purpose, the arena was divided in 12 equal zones (i.e., one per escape hole). The layout of the borders of the zones was like sparks of a wheel. When a mouse entered the escape hole there was no possibility to escape from the hole via an escape tunnel, i.e. all 12 holes were identical. Further, in this experiment an error was scored when a mouse poked its nose in a hole in the rim, instead of its complete head as in the first two experiments.
Column ‘‘Days’’ tabulates the results of the within-subjects factor Days. The degrees of freedom, F values and associated probabilities are shown. * P < .05. ** P < .01. + 0.05 < P < .10.
2.3.3. Statistical analysis For each parameter, the average of six trials was calculated (one trial block per day). The data were analysed using an ANOVA (GLM) with the factor Days as
BALB/c
C57BL
Swiss
G. Koopmans et al. / Physiology & Behavior 79 (2003) 683–693 Table 2 Statistical analysis of the acquisition of a new position of the escape hole in maze of four different mouse strains Strain
Parameter
Factor Days
129/Sv
BALB/c
C57BL
Swiss
Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed Latency Distance Error Speed
F(1,9) = 0.58 F(1,9) = 1.98 F(1,9) = 0.65 F(1,9) = 0.20 F(1,8) = 0.99 F(1,8) = 0.13 F(1,8) = 1.68 F(1,8) = 2.11 F(1,9) = 5.77* F(1,9) = 3.74+ F(1,9) = 5.17* F(1,9) = 0.18 F(1,9) = 0.00 F(1,9) = 0.28 F(1,9) = 0.71 F(1,9) = 0.12
Days Trials
Trials +
F(1,9) = 5.05 F(1,9) = 0.18 F(1,9) = 0.01 F(1,9) = 0.81 F(1,8) = 2.48 F(1,8) = 0.01 F(1,8) = 3.85+ F(1,8) = 6.92* F(1,9) = 8.13* F(1,9) = 7.51* F(1,9) = 4.29+ F(1,9) = 1.26 F(1,9) = 0.37 F(1,9) = 0.14 F(1,9) = 0.77 F(1,9) = 5.83*
F(1,9) = 0.05 F(1,9) = 0.36 F(1,9) = 0.63 F(1,9) = 3.56+ F(1,8) = 0.19 F(1,8) = 0.12 F(1,8) = 0.20 F(1,8) = 0.01 F(1,9) = 2.90 F(1,9) = 1.29 F(1,9) = 1.33 F(1,9) = 1.33 F(1,9) = 0.28 F(1,9) = 0.92 F(1,9) = 0.88 F(1,9) = 0.37
Columns ‘‘Days,’’ ‘‘Trials’’ and ‘‘Days Trials’’ tabulate the results of the within-subjects factors. The degrees of freedom, F values and associated probabilities are shown. * P < .05. + 0.05 < P < .10.
repeated measures. The data of the probe trial were analysed using an ANOVA (GLM) with the factor Zone as repeated measures. To further examine the difference the mice spent in the different zones, a post hoc LSD test was used to test whether the animals spent more time in the goal zone (i.e. zone where the escape hole was positioned).
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3. Results 3.1. Experiment 1 3.1.1. Acquisition 3.1.1.1. Escape latency. There was a clear effect of Days [ F(3,105) = 23.60, P < .01; see Fig. 2A], indicating that the mice became faster in finding the escape tunnel in the course of training. There was a clear effect on strain on this parameter [Strain: F(3,35) = 3.18, P < .05], but the decrease was comparable in all strains [Strain Days: F(9,105) =1.83, P>.05]. In Table 1, it can be seen that all strains, except the Swiss mice, improved their performance in the course of training. 3.1.1.2. Distance travelled. The total distance travelled decreased in the course of training [ F(3,105) = 3.36, P < .05; see Fig. 2B]. The performance on this variable was different for the mouse strains [Strain and Strain Days: F’s>3.64, P < .01]. Analysis within each strain indicated that the distance travelled decreased in the C57BL and the Swiss mice, whereas no change in performance was found for the BALB/c strain. There was a weak statistical support that the 129/Sv increased the total distance travelled in the course of training. 3.1.1.3. Errors. When analysing the performance of all animals, the total number of errors did not change in the course of training [ F(3,105) = 0.99, n.s.; see Fig. 2C]. However, as can be seen from Fig. 2C, there were clear strain differences [Strain and Strain Days: F’s>2.38, P < .05]. In
Table 3 Pearson’s correlation coefficients between the four variables of the maze
Light grey squares indicate correlation coefficients between different parameters on the same trial block. Bold/italic entries indicate P < .05.
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Table 1, it can be seen that the number of errors only decreased in the C57BL mice. In contrast, the number of errors of the BALB/c mice increased in the course of training. The performance of the 129/Sv and Swiss mice did not change during training.
but not Days [ F(1,35) = 0.15, n.s.; see Fig. 3D]. The running speed of the strains was comparable [ F(3,35) = 2.23, n.s.] and no interaction effects were found [Strain Days and Strain Trial blocks interactions: F’s < 2.38, n.s.]. The BALB/c, and to a less extent, the Swiss mice, increased their running speed on each day (see Table 2).
3.1.1.4. Running speed. There was a clear effect of training on the mean running speed of the mice [ F(3,105) = 13.59, P < .01; see Fig. 2D]. Again, there were differences between the four strains [Strain and Strain Days: F’s>4.87, P < .01]. As can be seen in Fig. 2D and Table 1, the mean running speed increased in the 129/Sv, BALB/c and Swiss mice, whereas there was no change in the running speed in the C57BL strain. 3.1.2. Acquisition of new escape position 3.1.2.1. Escape latency. Although there was weak support for an improvement over Days [ F(1,35) = 3.16, 0.05 < P < .10], there was improvement across Trial blocks [ F(1,35) = 11.00, P < .01; see Fig. 3A]. There were no Strain interaction effects [Strain Days and Strain Trial blocks interactions: F’s < 0.73, n.s.]. However, there was a clear effect of Strain on the variable escape latency in the acquisition of a new escape position [ F(3,35) = 4.64, P < .01]. Analysis of the individual strains revealed that only the C57BL mice improved their performance in the test with the new escape position (see Table 2). There was only a weak support for an improvement across trials in the 129/Sv mice. 3.1.2.2. Distance travelled. There was a tendency that the distance travelled decreased from Day 1 to Day 2 [Days: F(1,35) = 3.81, 0.05 < P < .10; see Fig. 3B]. No Strain interaction effects were found [Strain Days and Strain Trial blocks interactions: F’s < 2.17, n.s.]. On the other hand, there was a clear effect of Strain [ F(3,35) = 8.76, P < .01]. Withinstrain analysis revealed that the performance of the 129/Sv, BALB/c and Swiss mice did not change in the trials in which the escape hole position was changed (see Table 2). However, the performance of the C57BL mice improved across the trials of the acquisition of the new position. 3.1.2.3. Errors. Both for the factor Day and Trial blocks, a tendency was found that the number of errors decreased [Day: F(1,35) = 3.34, 0.05 < P < .10; Trial blocks: F(1,35) = 3.24, 0.05 < P < .10; see Fig. 3C]. A Strain effect was found [ F(3,36) = 8.58, P < .01], but no interactions with Days or Trial blocks [Strain Days and Strain Trial blocks interactions: F’s < 1.57, n.s.]. From Table 2, it can be seen that this effect was mainly due to the improved performance of the C57BL mice and to a modest improvement of the BALB/c mice. There was no change in performance in the 129/Sv and Swiss mice. 3.1.2.4. Running speed. There was a clear increase of running speed over Trial blocks [ F(1,35) = 13.27, P < .01],
Fig. 4. Performance of C57BL mice in the circular maze on four successive days in Experiment 2 (six trials per day): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) The total number of errors, defined as complete head in one of the holes; (D) The average speed (cm/s) of the animals during a trial. Values represent mean (± S.E.M.) of two trials.
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3.1.2.5. Last block acquisition vs. first block acquisition new position. It was found that the performance of the 129/Sv, BALB/c and Swiss mice on the latency, distance and error measures did not change from the last block of the acquisition to the first block of trials of the task with the new escape position [all F’s < 1.74, n.s.]. There was only a tendency that the escape latency was increased for the 129/Sv mice in the first block of the new acquisition test [ F(1,9) = 4.33, 0.05 < P < .10]. The performance of the C57BL mice on the first block of the new acquisition was worse than at the end of the first acquisition phase on all measures except the running speed [latency, distance, error: F’s>6.70, P < .05]. 3.1.3. Correlations In Table 3, the correlations between the parameters of the maze are listed. As can be seen, there was a strong correlation between successive days for each of the measures. Examination of the correlation between measures revealed that there was a good interrelation between the three indices of learning, especially between the distance travelled and the number of errors made on the same day. The measure running speed was found to have a negative correlation with escape latency on the first two days but not on Days 3 and 4. The running speed also correlated with the distance travelled, almost at every day, although the correlation was positive. The error measure poorly correlated with running speed.
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find the escape hole decreased similarly on each day [Trial blocks Days: F(6,42) = 1.46, n.s.]. 3.2.2. Distance travelled There was a clear indication that the distance travelled to find the escape hole decreased with repeated testing [Days: F(3,21) = 4.56, P < .05; Trial blocks: F(2,14) = 25.67, P < .01; see Fig. 4B]. The improvement of performance across trials differed per day [Trial blocks Days: F(6,42) = 3.11, P < .05]. This effect was due to a sharp improvement on Day 1 and only a modest improvement on Days 3 and 4. 3.2.3. Errors There was no indication that the number of errors decreased across days [Days: F(3,21) = 1.36, n.s.; see Fig. 4C]. On the other hand, performance was improved on individual days [Trial blocks: F(2,14) = 4.52, P < .05] and was similar on each day [Trial blocks Days: F(6,42) = 0.57, n.s.]. 3.2.4. Running speed During the 4 days of training, the running speed of the animals remained stable [Days: F(3,21) = 1.36, n.s.; see Fig. 4D]. The running speed of the animals increased on each day [Trial blocks: F(2,14) = 5.97, P < .05], but the increase was similar on each day [Trial blocks Days: F(6,42) = 1.58, n.s.]. 3.3. Experiment 3
3.2. Experiment 2 3.2.1. Escape latency The C57BL mice improved their performance across trials and days [Days: F(3,21) = 4.56, P < .05; Trial blocks: F(2,14) = 25.67, P < .01; see Fig. 4A]. The time needed to
3.3.1. Acquisition Similar to the first two experiments, the C57BL mice improved their performance on the different measures in the course of training [ F’s>4.94, P < .01; see Fig. 5A – C].
Fig. 5. Performance of C57BL mice in the circular maze on five successive days in Experiment 3 (six trials per day): (A) The average latency (s) to enter the escape hole; (B) The average distance travelled (cm) to find the escape hole; (C) Mean number of errors defined as nose pokes; (D) Time spent in the different zones of the arena (TZ: target zone; 2 – 12: other zones in clockwise order). The dotted line represents the chance level that mice spent in each zone. Values represent mean (± S.E.M.) of two trials.
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3.3.2. Probe trial In the probe trial, the mice spent an unequal amount of time in the different zones [Zone: F(11,55) = 5.62, P < .01]. As can be seen in Fig. 5D, the mice spent more time in the zone (about 35 s) where the escape hole was positioned during the acquisition trials when compared to the other zones (about 10– 15 s). Post hoc analysis revealed that the time spent in the different zones was statistically lower when compared with the time spent in the target zone, except for one zone (i.e. Zone 9).
4. Discussion 4.1. The task In the present study, we assessed the spatial learning behavior of four different mouse strains in a circular maze. This maze is an adaptation of the circular Barnes maze that was originally developed for rats [7]. Instead of making escape holes in the floor, 12 escape holes were made in the rim that surrounded a circular arena. This set-up is comparable with a task that combines the Morris and Barnes maze task [17]. In this task, escape holes were also made in the rim of the wall of a pool with water. A clear effect of hippocampal lesions was found suggesting that this was a spatial discrimination task. In our study, it was found that the C57BL mice readily acquired the task and the acquisition of a new position of the escape hole showed that the performance was transiently impaired. In the probe trial procedure, it was found that the C57BL mice spent significantly more time in the zone (1/12 of total arena) where the escape hole was positioned during training. This finding indicates that these mice used a spatial strategy to locate the position of the escape hole. No extra pretraining or additional (negative or positive) reinforcements were required to train the C57BL mice in this task which can be considered as an advantage when compared with the circular maze with holes in the floor. Further, the apparatus can easily be built and the behavior of the animals can be measured using a video tracking system. Correlation analysis revealed that the three measures of learning performance (i.e. latency, distance and errors) were interrelated. This suggests that these parameters all measure a similar underlying process. The running speed appeared independent of the escape latency (only a low correlation on the first two trial blocks) and errors made. On the other hand, there was a more consistent correlation between running speed and the measure distance travelled. These findings suggest that differences in running speed could affect the measure distance travelled, but are less likely to affect the latency and error measure. In general, the number of errors made by the mice was relatively low. This is best explained by the manner in which an error was scored, i.e. when a mouse entered a hole with its complete head. When it poked its nose in a hole, it
was not scored as an error. This was done to make a visit to the escape hole and other holes comparable. Probably, it is better to use two error scores. One error as defined in this study (i.e. complete head), and a second error score defined as a nose poke. This additional scoring may give a better insight of the commitment of mice to visit a hole, viz. making an error. This was done in the third experiment and it was found that the number of errors was higher when applying this way of scoring (i.e. nose poke). Even after 5 days of training, the mice needed about 40 s to enter the escape hole and travelled about 2 m. From this it can be inferred that the mice did not move along a straight line to the escape hole. But this appears also to be the case for the Morris water escape task, in which mice take about 10 s to find the hidden platform (e.g. Refs. [10,18]). Apparently, mice do not always directly go to an escape position. Further, the need to escape from the water may be greater than the need to escape from the circular maze in the present study. This may explain that in the present study the escape latencies are higher than those observed in the Morris task. 4.2. Strain differences in acquisition The statistical analysis did not reveal Strain Days interactions, suggesting that the rate of speed of learning of the strains was not different. It should be noted that all strains were included in the statistical analysis and that the relative high variance of the different strains may have masked the interaction effect. A separate analysis between the C57BL and 129/Sv strains revealed a clear Strain Days interaction effect on the measures distance and errors, indicating a difference in the learning rate of these strains. Strain differences were observed that were dependent on the behavioral measure (i.e. escape latency, distance travelled, errors, running speed). With respect to the escape latency, it appeared that the C57BL strain acquired the task efficiently, i.e. the time to escape gradually decreased during the acquisition of the task. The mice of this strain entered the escape hole within 40 s on the fourth day of training. On the other hand, the other three strains (i.e. 129/Sv, BALB/c and Swiss) only reduced their escape latency from the first to the second day and there was no apparent improvement from Day 2 to Day 4. For the measure distance travelled, a different picture emerged. Although the C57BL mice again showed a clear improvement over days, the BALB/c and the 129/Sv mice did not improve their performance on this measure. Although the apparent increase in distance travelled was not supported statistically, the 129/Sv mice seemed to get worse in the course of training. In contrast to the latency measure, the Swiss mice improved their performance from day to day, but the distance travelled was greater than those of the other strains. The measure ‘number of errors’ showed a different picture for the four mouse strains. As in the previous two
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measures, the C57BL mice showed a consistent decrease of the number of errors. The Swiss and 129/Sv mice did not change their performance during training. The Swiss mice made the most errors of all strains. Conversely, the 129/Sv mice made the lowest number of errors. Remarkably, the BALB/c mice made more errors in the course of training, suggesting a negative learning curve. The running speed of the mice was also different between strains. The 129/Sv mice started as the slowest animals, whereas the Swiss mice were the fastest. The BALB/c mice started intermediate but travelled as fast as the Swiss mice at the end of the four training days. The running speed of the C57BL mice did not change across the acquisition trials. Taken together, it is clear that there were significant strain differences on the four measures that were assessed in the present study. The picture of these strain differences was different for each measure. The C57BL mice appeared to show the most consistent learning on all three learning measures. Also, the running speed of this strain did not change, which can be considered as an advantage since a change in this measure across training blocks may affect the learning parameters (as discussed above). The performance of the other three strains is more difficult to interpret. Thus, a mouse strain that showed an improvement on one measure did not show an improvement on another measure (e.g. BALB/c improved on latency and got worse on errors, and the Swiss only improved on distance). It depends on what measure is assumed to be the best parameter for learning to indicate which mouse strains acquire this task. The good performance of the C57BL mice is in accordance with previous studies in which this mouse strain was tested in the Morris water escape task (e.g. Refs. [10,15,19]), the radial maze (e.g. Ref. [13,20]) and the circular Barnes maze (e.g. Ref. [3,19]). Apparently, the C57BL strain is suitable for testing spatial abilities in mice. However, it must be mentioned that various C57BL substrains exist, which may differ in their learning performance (like the 129/Sv substrains; see Ref. [21]). On the other hand, the BALB/c strain does not seem to be suitable for testing the spatial abilities in the maze, which was based on the finding that the number of errors increased and the distance to find the hole remained constant in the course of training. This poor performance of this strain was also demonstrated in other studies [10,20], in which it was concluded that BALB/c mice do not use spatial cues to guide their behavior in spatial tasks. However, it has been suggested that the poor performance of the BALB/c strain in the Morris task [18,22,23] is related to a high light intensity. Thus, it has been shown that the performance of BALB/c mice is relatively good when they are tested in a dimly lit room [24]. In our study, the animals were trained in a relative bright room, which may explain the poor performance of these mice in our test.
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The performance of the 129/Sv mice was different for the three learning measures. There was an improvement on the latency measure, no change on the error measure and a tendency for an increase on the distance measure. These findings do not support the notion that these mice acquired the task. It has been reported before that the 129/Sv mice are worse than C57BL mice in acquiring a spatial Morris task, but do acquire this task [25,26]. It should be mentioned that there are various 129 substrains and that a good Morris task performance has been reported for the 129S6/SvEvTac, but that the performance is poor for the 129/SvJ and 129/J mice [21]. In the present study, we used the 129/Sv strain from Iffa Credo. Only a few studies have used Swiss mice to evaluate spatial learning and memory performance. In one study, it was concluded that Swiss mice do not reliably find the escape platform in a learning set paradigm in the Morris task [27]. Interestingly, another study showed that Swiss mice acquired the task, although no probe trial was performed [28]. Interestingly, in the same study, the Swiss mice were impaired in a cued version of the Morris task when compared with a Down’s syndrome transgenic mouse. This latter finding questions the ability of the Swiss strain to acquire the spatial task. In the maze, the Swiss mice only reduced the mean distance to find the escape hole, but no changes in escape latency or number of errors were found. These findings indicate that Swiss mice do not acquire this spatial task. If only one parameter was chosen per strain, it could be concluded that all strains acquired the task. Although the correlation analysis revealed that the three learning parameters were correlated, there appears to be no straightforward relation between these measures between strains when the learning component of these measures was considered. This could, for example, be related to behavioral strategies or other behavioral tendencies of mice of the different strains. Therefore, we argue that it is safest to include all measures in the behavioral analysis before drawing conclusions about the learning performance of mice in this task. A further point that should be mentioned with respect to strain differences is that the findings of the present study only relate to the test conditions used in this study. As mentioned above, the lighting conditions may affect the learning behavior of the BALB/c mice. It is clear that the effect of test conditions on genetic differences in learning needs to be investigated in further studies. Although the change in the position of the escape holes resulted in a greater preparedness of the mice to enter the escape hole (i.e. no pretraining or additional reinforcers were required), the strain differences in our study are similar to those observed in previous studies, including the Barnes maze. This may suggest that the spatial learning performance of mice is independent of the task used, and may, for example, be related to the morphology of the hippocampus [29,30].
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4.3. Acquisition of a new escape position
4.6. Behavioral strategies
After the mice were given 16 trials, they were given a test with a new escape position, which can provide information whether the animals used a spatial strategy. Thus, if an animal uses a spatial strategy, the performance should get worse if the position of the escape hole is changed. This procedure has also been applied in the Morris task to assess the spatial search strategies of subjects. In our study, a clear effect of changing the escape hole was found in the C57BL mice, indicating that these mice adopted a spatial search strategy. Further, the C57BL mice improved their performance in the trials in which they had to locate a new position. In contrast, there was no clear indication that the performance of the other strains changed from the last block of the acquisition to the first block of the new acquisition test. This finding confirms that the circular maze can be considered as a spatial reference memory task, at least for C57BL mice. However, it cannot be excluded that (spatial) search strategies may go unnoticed using this procedure (see below).
There were some qualitative observations that could explain some of the findings described above. The Swiss mice showed a very active behavior in the maze. They were continuously running in the maze albeit in a specific manner. Typically, it was found that they moved to the rim of the apparatus and started checking the escape holes (anti)clockwise. This search strategy leads to a relative high number of errors. Although the Swiss mice did not find the escape hole faster, it was found that the distance to the escape hole was decreased across trials. This latter finding may indicate that the search strategy of the mice improved with repeated testing. This may be due to learning the location of the escape hole, although the mice did not directly go to this position. It could be argued that the rim of the apparatus increased the thigmotaxis behavior of this strain. However, it seems that this was only the case for the Swiss mice because the other strains did not show this typical thigmotaxis. The 129/Sv mice were very inactive, especially in the first trials. This may be related to a high anxiety level in this mouse strain, which has also been reported in other studies [15,16]. Interestingly, it was found that the distance travelled by the 129/Sv mice tended to increase in the course of training. Although no clear explanation can be given for this observation, it could be argued that this may be related to an increase in exploratory behavior when the anxiety level decreased. The decrease in the escape latency across trials can be explained by the increased running speed of the 129/ Sv mice. No apparent behavioral strategy could be detected for the C57BL and BALB/c mice. In the third experiment, it was shown that C57BL mice used a spatial orientation strategy in this task.
4.4. Repeated acquisition In the second experiment of this study, we tested C57BL mice in the circular maze using a different test procedure. We only tested the C57BL mice because Experiment 1 indicated that only these mice used an allocentric spatial orientation strategy in this task. The mice were tested to find a new escape hole position every day. This repeated acquisition procedure may be considered as a learning set paradigm [27,31], although it has to be noted that a learning set is defined as an improvement in learning using similar procedures but with different stimuli (e.g. new environment in this task) or samples. It was found that the performance on the measures latency and distance was improved on each day. However, this was not found for the error measure. This lack of effect could be due to the scoring of an error as was defined earlier (see above). There was also a relative constant drop in performance when comparing the last block of trials of a day with the first block of the next day. This finding further supports the use of a spatial orientation strategy used by C57BL mice. 4.5. Probe trial Similar to the Morris task, we used a probe trial to assess the spatial bias of the mice after they had acquired the task. For this purpose, the arena was divided into 12 equal zones that corresponded with the 12 escape holes. The mice showed a clear preference for the location where the escape hole was during the acquisition of the task. These data strongly suggest that the mice had developed a spatial orientation strategy to find the escape hole in the course of training.
4.7. Conclusions The circular maze was developed on the basis of the circular Barnes maze for rats. It appears that mice do not readily enter escape holes in the floor like rats do. As an alternative, escape holes were made in the rim of the apparatus. We found that C57BL mice acquired the task since the performance on three parameters of learning was improved with repeated testing. These mice appeared to use a spatial orientation strategy as shown in a probe trial procedure. No extra pretraining of mice or additional reinforcers were required, suggesting that the escape via a hole in the rim is more adapted to mice than a hole in the floor of the apparatus. However, 129/Sv, BALB/c and Swiss mice did not acquire the task to a same level under these testing conditions, and a spatial learning improvement was only seen on one parameter. On basis of this study, it was concluded that: (1) the circular maze can be used as an alternative tool to assess spatial learning in (C57BL) mice; (2) at least three parameters (i.e. latency,
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distance, errors) should be evaluated to assess spatial learning in this task.
[16]
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