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Spontaneous alternation and spatial learning in Dab1scm (scrambler) mutant mice
Brain Research Bulletin 87 (2012) 383–386
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Spontaneous alternation and spatial learning in Dab1scm (scrambler) mutant mice C. Jacquelin a , C. Strazielle a , R. Lalonde b,∗ a Université Henri Poincaré, Nancy I, Laboratoire de Nutrition Génétique et Exposition aux Risques Environnementaux, INSERM U954, Service de Microscopie Electronique, Faculté de Médecine, 54500 Vandoeuvre-les-Nancy, France b Université de Rouen, Faculté des Sciences, Dépt. Psychologie, 76821 Mont-Saint-Aignan Cedex, France
a r t i c l e
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Article history: Received 13 September 2011 Received in revised form 20 December 2011 Accepted 1 January 2012 Available online 8 January 2012 Keywords: Reelin Cerebellum Hippocampus Spontaneous alternation Morris water maze Spatial learning
a b s t r a c t Homozygous Dab1scm mutants with cell ectopias in cerebellar cortex, hippocampus, and neocortex were compared with non-ataxic heterozygous and wild-type controls in spontaneous alternation and Morris water maze tests. Although there were no group differences in alternation rates, wild-type and heterozygote groups alternated above chance levels, whereas homozygous Dab1scm mutants did not. In the Morris water maze, Dab1scm mutants were impaired in both hidden and visible platform subtests. The deficits in spontaneous alternation and water maze measures reproduce the phenotype previously described in Relnrl–Orl mutants, attributed to disturbance of the same molecular pathway involving reelin. © 2012 Elsevier Inc. All rights reserved.
Reelin is disrupted in the spontaneous autosomal recessive reeler mutation of the Reln gene, encoding an extracellular matrix protein involved in neural adhesion and migration [7,9,20], of which two alleles exist: Edinburg (Relnrl–Ed ) and Orleans (Relnrl–Orl ) [7]. In each case, reeler mutants display architectonic disorganization and cell ectopias in cerebellum, inferior olive, hippocampus, and neocortex, but with preserved anatomical connections between these regions and others [3,8,14,15,21,31,42,43]. In the cerebellum, granule cells are the most severely depleted, Purkinje cells to a lesser extent [21], along with the inferior olive [2,41]. Despite cell ectopias, the zonal pattern of climbing fiber projections to cerebellum is maintained [3], though with multiple as opposed to the normal monoinnervation of Purkinje cells [31,32]. The spontaneous autosomal recessive scrambler mutation of the Dab1 gene situated on chromosome 4 causes a deficiency of the gene product, disabled-1, involved in reelin signaling [38,44]. As a result, homozygous Dab1scm mutants possess a loss-of-function reeler-like phenotype with cell malpositioning in cerebellar cortex, hippocampus, and neocortex [18,38,40,44,46]. The same cell ectopias occur in the Dab1 knockout [12,22]. Like Relnrl [30], and despite normal Relnrl mRNA levels [17], Purkinje and granule cell degeneration in Dab1scm mutants results in ataxia and deficits in motor coordination [28], indicating that disabled-1 acts
∗ Corresponding author. Tel.: +33 02 35 14 61 08; fax: +33 02 35 14 63 49. E-mail address: [email protected] (R. Lalonde). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2012.01.001
downstream of reelin. Despite Purkinje cell degeneration, the deep cerebellar nuclei appear intact [6]. The mutant Purkinje cells are not rescued by wild-type Purkinje cells in Dab1scm chimeras [47]. In addition to ataxia and motor deficits, Relnrl mutants were deficient in two spatial-related tasks: spontaneous alternation and the hidden platform version of the Morris water maze [4,30]. Low spontaneous alternation rates may be due to poor short-term spatial retention or to altered inhibitory responses, as the animal perseverates arm choices in contrast to the normal rodent response of exploring a novel maze arm [27]. Moreover, Relnrl–Orl mutants were impaired in the visible platform subtest of the Morris maze, often described as a visuomotor deficit, whereby the animal has a difficulty in coordinating whole-body movements towards a specific target. In the present study, we examined whether Dab1scm mutants display the same phenotype as Relnrl in these two tests. Since Reln and Dab1 are involved in the same molecular pathway, we expected Dab1scm mutants to be deficient relative to heterozygotes and wildtype controls in both. 1. Materials and methods 1.1. Mice Dab1scm /+ breeders on the A/A (agouti) genetic background were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and crossed to obtain ataxic Dab1scm /Dab1scm mutants (n = 9, 4 males, 5 females), non-ataxic Dab1scm /+ heterozygotes (n = 6, 2 males, 4 females), and +/+ wild-type (n = 7, 3 males, 4 females), determined by genetic history. To facilitate survival of mutant homozygotes, food pellets were spread on the cage floor and the overhead bin. The mice were tested at
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Table 1 Cumulative spontaneous alternation rate (%) and choice latencies (s) over 5 or 10 days in Dab1scm , heterozygous, and wild-type mice. Days and measures First 5 days Alternations (%) Latencies (s) Full 10 days Alternations (%) Latencies (s) * **
Statistical parameters
Wild-type
Heterozygous
Dab1scm
Mean ± SEM Median (range) Mean ± SEM
60 ± 8** 60 (20–80) 35 ± 9
70 ± 9* 80 (40–100) 45 ± 17
54 ± 8 40 (20–80) 48 ± 8
Mean ± SEM Median (range) Mean ± SEM
63 ± 6 50 (50–90) 66 ± 17
72 ± 6** 80 (50–90) 76 ± 19
58 ± 6 60 (30–80) 101 ± 14
p = 0.05 vs chance level, Mann–Whitney U test. p < 0.05 vs chance level, Mann–Whitney U test.
about 10 months of age (range: 8–12 months) in a protocol adhering to guidelines of the European Council Directive (86/609/EEC) and animal care regulations at the local university. 1.2. Methods and procedures Spontaneous alternation was tested in a T-maze made of plywood, containing a central stem and 2 side-arms, 30 cm in length and 10 cm in width, surrounded by 20 cm-high walls. Naive mice with no previous experience in the maze but accustomed to handling were used. On the initial trial, the mice were placed in the stem with the right arm blocked by a plastic barrier (forced choice). After entering the left arm (4-paw criterion), the mice were kept inside for 1 min, retrieved, and placed back in the stem for a free-choice trial. On the following 9 days, the same procedure was repeated, except that the blocked arm on the initial trial was changed alternatively from right to left each day. The number of alternations and the latencies before responding were measured in 1-min trials. To obtain a response after the cut-off period had expired, the mice were briefly prodded from behind, usually not more than once and only when situated far from the choice-point. The Morris water maze consisted of a basin (diameter: 86 cm, wall height: 30 cm) made of white plastic and filled with water (22 ◦ C) at a height of 21 cm. Yellow plastic beads were evenly spread over the water surface to camouflage the escape platform (diameter: 8 cm) made of white plastic and covered with a yellow wiremesh grid to ensure a firm grip. The pool was contained in a room with several extramaze visual cues, such as light fixtures and laboratory instruments. The mice were placed next to and facing the wall successively in north (N), east (E), south (S), and west (W) positions, with the escape platform hidden 1 cm beneath water level in the middle of the NW quadrant. An experimenter followed their swimming trajectories on a videomonitor, on which the image of the pool was separated into 4 equally spaced quadrants. The quadrant entries (4-paw criterion) and escape latencies were measured in 4-trial sessions for 5 consecutive days with a 15-min intertrial interval. The mice remained on the escape platform for at least 5 s. Whenever the mice failed to reach the platform within the 1 min cut-off period, they were retrieved from the pool and placed on it for 5 s. After their swim, the mice were kept dry in a plastic holding cage filled with paper towels. The day after the acquisition phase, a probe trial was conducted by removing the platform and placing the mouse next to and facing the N side. The time spent in the previously correct quadrant was measured in a single 1-min trial. One hour later, the visible platform version was evaluated, with the escape platform lifted 1 cm above water level and shifted to the SE quadrant. A 13-cm high pole was inserted on top of the escape platform as a viewing aid. As with the place learning task, quadrant entries and escape latencies were measured for 4 trials, the animals stayed on the platform for 5 s, and a 1-min cut-off period was imposed with a 15-min intertrial interval, except that the test was conducted in a single session.
heterozygotes [U = 3, p < 0.05] and Dab1scm mutants [U = 27, p > 0.05], but wild-type mice wound up only at borderline significance [U = 14, p = 0.06]. In contrast, there was no intergroup difference in choice latencies at either the midway point or throughout the testing period [p > 0.05]. During acquisition of the hidden platform version of the Morris water maze (Fig. 1), there was a main group effect [quadrants: F2,19 = 60.43, p < 0.001; latencies: F2,19 = 76.13, p < 0.001], but no day effect [quadrants: F4,76 = 0.43, p > 0.05; latencies: F4,76 = 0.29, p > 0.05] or group × day interaction [quadrants: F4,76 = 0.93, p > 0.05; latencies: F4,76 = 0.88, p > 0.05]. Despite the relatively poor performance of both control groups as manifested by the lack of a day effect, Dab1scm mutants had higher quadrant entries and escape
1.3. Statistical analyses Intergroup differences were estimated by analyses of variance (ANOVAs) and Fisher’s least significant difference test. For spontaneous alternation and probe tests, the groups were compared with the Kruskal–Wallis test and each group was compared by the Mann–Whitney U test to a theoretical group performing at chance (50% for alternations, 15/60 s for probe) with zero variance.
2. Results Although there were no intergroup differences in spontaneous alternation rates [Krusal–Wallis, p > 0.05], the more sensitive method of comparing each group to chance reached significance. Indeed, during the first 5 days of spontaneous alternation (Table 1), wild-type [U = 7, p < 0.05] and heterozygote [U = 5, p = 0.05] mice alternated above chance, whereas Dab1scm mutants did not [U = 36, p > 0.05]. This pattern held up for all 10 days of testing in
Fig. 1. Hidden platform acquisition in the Morris water maze as measured by quadrant entries and escape latencies (s); cumulative score over 4 trials during 5 days of training in Dab1scm , heterozygous, and wild-type mice (means ± SEM). *p < 0.01, Dab1scm mutants vs either heterozygotes or wild-type.
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p < 0.01; latencies: F2,19 = 14.55, p < 0.001], caused by higher quadrant entries and escape latencies in Dab1scm mutants than either heterozygotes or wild-type [p < 0.05].
3. Discussion
Fig. 2. Time (s) spent in the target quadrant during the probe trial and cumulative visible platform performance over 4 trials for quadrants and escape latencies (s) in Dab1scm , heterozygous, and wild-type mice (means ± SEM and individual values). *p < 0.05, Dab1scm mutants vs either heterozygotes or wild-type Fisher’s exact test.
latencies than either heterozygotes or wild-type during all five days of training [p < 0.01, Fisher test]. In the probe trial (Fig. 2), wild-type [U = 0, p < 0.01] and heterozygote [U = 0, p < 0.01] mice stayed in the target quadrant above chance, but Dab1scm mutants did not [U = 27, p > 0.05]. However, there was no intergroup difference [F2,19 = 0.96, p > 0.05]. Although the homozygous mutants had comparable means, their intragroup variances were high, seemingly precluding significance when compared to a theoretical group performing at chance. The results found in the hidden platform substask were reproduced in visible platform testing (Fig. 2), as there was a significant group effect [quadrants: F2,19 = 8.68,
The tendency of alternating arm choices in a T-maze was evaluated in Dab1scm mutant mice relative to two non-ataxic control groups. There was no intergroup difference in alternation rates, but significant results emerged with the more sensitive method of comparing each group to chance. Unlike either wild-type or heterozygotes, homozygous mutants did not alternate significantly above chance during the first 5 days of evaluation. When the test was prolonged to a maximum of 10 days, Dab1scm mutants continued to perseverate while heterozygotes continued to alternate, whereas the rate of wild-type mice only approached significance. Overall, unlike most other mouse strains [1], the background A/A line used here showed relatively low alternation levels. Nevertheless, the deficiency in spontaneous alternation found in Dab1scm mutants reproduces the phenotype previously reported in Relnrl–Orl mutants on the Balb/c background [4,30]. The deficit in both mutants can be ascribed in part to cell ectopias and degeneration in the hippocampus. The importance of the medial temporal lobe in spontaneous alternation is underlined by findings of reduced spontaneous alternation rates in rats with lesions of the hippocampus relative to sham-operated controls [10,25,33,39]. The impairment can also be attributed to cell ectopias in neocortex, since lesions of frontal association cortex also reduced spontaneous alternation rates relative to sham-operated controls [11]. Likewise, the role of the cerebellum on spontaneous alternation is supported by findings of similar deficits in several murine mutants with cerebellar atrophy in the absence of overt pathology in either hippocampus or neocortex, such as Grid2Lc (Lurcher) [27,29]. It is unlikely to be attributed to ataxia, since turning left or right does not require fine motor control. Moreover, it is unlikely to be attributed to motor slowing, since choice latencies did not differ between the groups. Instead, the deficit may be caused by poorer working memory, an hypothesis that should be further explored in other paradigms, such as learned left-right alternation. A second explanation is that such lesions alter inhibitory processes, since hippocampal, prefrontal, and cerebellar lesioned animals all show disinhibitory tendencies in several paradigms [24,26,27,45]. The test is dependent on optimal arousal levels, neither too high nor too low, as fearful, inhibited animals also alternate less frequently [34]. In addition to spontaneous alternation, Dab1scm mutants were impaired in hidden and visible platform versions of the Morris water maze. This occurred despite the relatively poor performance of the control groups on the A/A background, as shown by a lack of significance on the day factor during acquisition of the hidden platform phase. The deficit of Dab1scm mutants on hidden and visible platform versions reproduces the phenotype previously reported in Relnrl–Orl mutants [30], as well as Grid2Lc mutants [5,29,37] and hemicerebellectomized rats [36]. The deficit in the visible platform subtest may be ascribed to poor visuomotor control, as defined by an inability to coordinate swimming movements towards a specific target despite the presence of visual cues. It can also ascribed to a procedural deficit [36], as defined by an inability to acquire skills underlying task solution, such as abandoning the potent tendency of swimming along the walls of the pool. In contrast to the pattern displayed by Dab1scm , Relnrl–Orl , and Grid2Lc mutants, a dissociation between hidden and visible platform subtests was obtained in the Agtpbp1pcd (Purkinje cell degeneration) mutant [19], as well as rats with selective lesions of the dentate nuclei [23,35], attributable in part to ascending
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cerebellar pathways towards the parietal cortex [13]. Overall, these results point towards a role for separate cerebellar pathways on spatial learning and visuomotor control or procedural learning. Despite poor performance during acquisition, Dab1scm mutants had comparable means to the control groups on the probe trial, indicating no specific impairment in long-term memory relative to the other two groups, though the only group not performing significantly above chance in target location, most likely due at least in part to a high intragroup variance. Instead, their maze deficit can best be interpreted as a difficulty in coordinating swimming movements towards a visible goal or learning appropriate procedural skills. We noted that, unlike the other two groups, most mutants tended to swim in circles far from the walls, whose circumference comprised one or more quadrants, a stereotyped response that could explain to a certain extent both elevated quadrant entries and escape latencies. They also appear rarely at rest, so that intergroup differences seem unlikely to be caused by freezing responses. It remains to be determined to what extent the deficits in Dab1scm mutants can be generalized to other spatial learning tasks such as the radial arm maze, described as being impaired in the Relnrl mutant [16]. This would best be examined in a within-study design with two or more mutants. Acknowledgment This study was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to RL. References [1] J.Y. Bertholet, W.E. Crusio, Spatial and non-spatial spontaneous alternation and hippocampal mossy fibre distribution in nine inbred mouse strains, Behav. Brain Res. 4 (1991) 197–202. [2] G.J. Blatt, L.M. Eisenman, A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice, J. Comp. Neurol. 232 (1985) 117–128. [3] G.J. Blatt, L.M. Eisenman, Topographic and zonal organization of the olivocerebellar projection in the reeler mutant mouse, J. Comp. Neurol. 267 (1988) 603–615. [4] T.V. Bliss, M.L. Errington, Reeler mutant mice fail to show spontaneous alternation, Brain Res. 124 (1977) 168–170. [5] J. Cendelin, I. Korelusova, F. Vozeh, The effect of repeated rotarod training on motor skills and spatial learning ability in Lurcher mutant mice, Behav. Brain Res. 189 (2008) 65–74. [6] S. Chung, Y. Zhang, F. Van Der Hoorn, R. Hawkes, The anatomy of the cerebellar nuclei in the normal and scrambler mouse as revealed by the expression of the microtubule-associated protein kinesin light chain 3, Brain Res. 1140 (2007) 120–131. [7] G. D’Arcangelo, Reelin mouse mutants as models of cortical development disorders, Epilepsy Behav. 8 (2006) 81–90. [8] G. D’Arcangelo, T. Curran, Reeler: new tales on an old mutant mouse, Bioessays 20 (1998) 235–244. [9] G. D’Arcangelo, G.G. Miao, S.C. Chen, H.D. Soares, J.I. Morgan, T. Curran, A protein related to extracellular matrix proteins deleted in the mouse mutant reeler, Nature 374 (1995) 719–723. [10] G.M. Dillon, X. Qu, J.N. Marcus, J.C. Dodart, Excitotoxic lesions restricted to the dorsal CA1 field of the hippocampus impair spatial memory and extinction learning in C57BL/6 mice, Neurobiol. Learn. Mem. 90 (2008) 426–433. [11] I. Divac, R.G.E. Wikmark, A. Gade, Spontaneous alternation in rats with lesions in the frontal lobe: extension of the frontal lobe syndrome, Physiol. Behav. 3 (1975) 39–42. [12] E. Gallagher, B.W. Howell, P. Soriano, J.A. Cooper, R. Hawkes, Cerebellar abnormalities in the disabled (mdab1-1) mouse, J. Comp. Neurol. 402 (1998) 238–251. [13] S. Giannetti, M. Molinari, Cerebellar input to the posterior parietal cortex in the rat, Brain Res. Bull. 58 (2002) 481–489. [14] A.M. Goffinet, The embryonic development of the cerebellum in normal and reeler mutant mice, Anat. Embryol. 168 (1983) 73–86. [15] A.M. Goffinet, The embryonic development of the inferior olivary complex in normal and reeler (rlOrl ) mutant mice, J. Comp. Neurol. 219 (1983) 10–24. [16] D. Goldowitz, J. Koch, Performance of normal and neurological mutant mice on radial arm maze and active avoidance tasks, Behav. Neural Biol. 46 (1986) 216–226. [17] D. Goldowitz, R.C. Cushing, E. Laywell, G. D’Arcangelo, M. Sheldon, H.O. Sweet, M. Davisson, D. Steindler, T. Curran, Cerebellar disorganization characteristic of reeler in scrambler mutant mice despite presence of reelin, J. Neurosci. 17 (1987) 8767–8777.
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Contents lists available at SciVerse ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Spontaneous alternation and spatial learning in Dab1scm (scrambler) mutant mice C. Jacquelin a , C. Strazielle a , R. Lalonde b,∗ a Université Henri Poincaré, Nancy I, Laboratoire de Nutrition Génétique et Exposition aux Risques Environnementaux, INSERM U954, Service de Microscopie Electronique, Faculté de Médecine, 54500 Vandoeuvre-les-Nancy, France b Université de Rouen, Faculté des Sciences, Dépt. Psychologie, 76821 Mont-Saint-Aignan Cedex, France
a r t i c l e
i n f o
Article history: Received 13 September 2011 Received in revised form 20 December 2011 Accepted 1 January 2012 Available online 8 January 2012 Keywords: Reelin Cerebellum Hippocampus Spontaneous alternation Morris water maze Spatial learning
a b s t r a c t Homozygous Dab1scm mutants with cell ectopias in cerebellar cortex, hippocampus, and neocortex were compared with non-ataxic heterozygous and wild-type controls in spontaneous alternation and Morris water maze tests. Although there were no group differences in alternation rates, wild-type and heterozygote groups alternated above chance levels, whereas homozygous Dab1scm mutants did not. In the Morris water maze, Dab1scm mutants were impaired in both hidden and visible platform subtests. The deficits in spontaneous alternation and water maze measures reproduce the phenotype previously described in Relnrl–Orl mutants, attributed to disturbance of the same molecular pathway involving reelin. © 2012 Elsevier Inc. All rights reserved.
Reelin is disrupted in the spontaneous autosomal recessive reeler mutation of the Reln gene, encoding an extracellular matrix protein involved in neural adhesion and migration [7,9,20], of which two alleles exist: Edinburg (Relnrl–Ed ) and Orleans (Relnrl–Orl ) [7]. In each case, reeler mutants display architectonic disorganization and cell ectopias in cerebellum, inferior olive, hippocampus, and neocortex, but with preserved anatomical connections between these regions and others [3,8,14,15,21,31,42,43]. In the cerebellum, granule cells are the most severely depleted, Purkinje cells to a lesser extent [21], along with the inferior olive [2,41]. Despite cell ectopias, the zonal pattern of climbing fiber projections to cerebellum is maintained [3], though with multiple as opposed to the normal monoinnervation of Purkinje cells [31,32]. The spontaneous autosomal recessive scrambler mutation of the Dab1 gene situated on chromosome 4 causes a deficiency of the gene product, disabled-1, involved in reelin signaling [38,44]. As a result, homozygous Dab1scm mutants possess a loss-of-function reeler-like phenotype with cell malpositioning in cerebellar cortex, hippocampus, and neocortex [18,38,40,44,46]. The same cell ectopias occur in the Dab1 knockout [12,22]. Like Relnrl [30], and despite normal Relnrl mRNA levels [17], Purkinje and granule cell degeneration in Dab1scm mutants results in ataxia and deficits in motor coordination [28], indicating that disabled-1 acts
∗ Corresponding author. Tel.: +33 02 35 14 61 08; fax: +33 02 35 14 63 49. E-mail address: [email protected] (R. Lalonde). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2012.01.001
downstream of reelin. Despite Purkinje cell degeneration, the deep cerebellar nuclei appear intact [6]. The mutant Purkinje cells are not rescued by wild-type Purkinje cells in Dab1scm chimeras [47]. In addition to ataxia and motor deficits, Relnrl mutants were deficient in two spatial-related tasks: spontaneous alternation and the hidden platform version of the Morris water maze [4,30]. Low spontaneous alternation rates may be due to poor short-term spatial retention or to altered inhibitory responses, as the animal perseverates arm choices in contrast to the normal rodent response of exploring a novel maze arm [27]. Moreover, Relnrl–Orl mutants were impaired in the visible platform subtest of the Morris maze, often described as a visuomotor deficit, whereby the animal has a difficulty in coordinating whole-body movements towards a specific target. In the present study, we examined whether Dab1scm mutants display the same phenotype as Relnrl in these two tests. Since Reln and Dab1 are involved in the same molecular pathway, we expected Dab1scm mutants to be deficient relative to heterozygotes and wildtype controls in both. 1. Materials and methods 1.1. Mice Dab1scm /+ breeders on the A/A (agouti) genetic background were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and crossed to obtain ataxic Dab1scm /Dab1scm mutants (n = 9, 4 males, 5 females), non-ataxic Dab1scm /+ heterozygotes (n = 6, 2 males, 4 females), and +/+ wild-type (n = 7, 3 males, 4 females), determined by genetic history. To facilitate survival of mutant homozygotes, food pellets were spread on the cage floor and the overhead bin. The mice were tested at
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Table 1 Cumulative spontaneous alternation rate (%) and choice latencies (s) over 5 or 10 days in Dab1scm , heterozygous, and wild-type mice. Days and measures First 5 days Alternations (%) Latencies (s) Full 10 days Alternations (%) Latencies (s) * **
Statistical parameters
Wild-type
Heterozygous
Dab1scm
Mean ± SEM Median (range) Mean ± SEM
60 ± 8** 60 (20–80) 35 ± 9
70 ± 9* 80 (40–100) 45 ± 17
54 ± 8 40 (20–80) 48 ± 8
Mean ± SEM Median (range) Mean ± SEM
63 ± 6 50 (50–90) 66 ± 17
72 ± 6** 80 (50–90) 76 ± 19
58 ± 6 60 (30–80) 101 ± 14
p = 0.05 vs chance level, Mann–Whitney U test. p < 0.05 vs chance level, Mann–Whitney U test.
about 10 months of age (range: 8–12 months) in a protocol adhering to guidelines of the European Council Directive (86/609/EEC) and animal care regulations at the local university. 1.2. Methods and procedures Spontaneous alternation was tested in a T-maze made of plywood, containing a central stem and 2 side-arms, 30 cm in length and 10 cm in width, surrounded by 20 cm-high walls. Naive mice with no previous experience in the maze but accustomed to handling were used. On the initial trial, the mice were placed in the stem with the right arm blocked by a plastic barrier (forced choice). After entering the left arm (4-paw criterion), the mice were kept inside for 1 min, retrieved, and placed back in the stem for a free-choice trial. On the following 9 days, the same procedure was repeated, except that the blocked arm on the initial trial was changed alternatively from right to left each day. The number of alternations and the latencies before responding were measured in 1-min trials. To obtain a response after the cut-off period had expired, the mice were briefly prodded from behind, usually not more than once and only when situated far from the choice-point. The Morris water maze consisted of a basin (diameter: 86 cm, wall height: 30 cm) made of white plastic and filled with water (22 ◦ C) at a height of 21 cm. Yellow plastic beads were evenly spread over the water surface to camouflage the escape platform (diameter: 8 cm) made of white plastic and covered with a yellow wiremesh grid to ensure a firm grip. The pool was contained in a room with several extramaze visual cues, such as light fixtures and laboratory instruments. The mice were placed next to and facing the wall successively in north (N), east (E), south (S), and west (W) positions, with the escape platform hidden 1 cm beneath water level in the middle of the NW quadrant. An experimenter followed their swimming trajectories on a videomonitor, on which the image of the pool was separated into 4 equally spaced quadrants. The quadrant entries (4-paw criterion) and escape latencies were measured in 4-trial sessions for 5 consecutive days with a 15-min intertrial interval. The mice remained on the escape platform for at least 5 s. Whenever the mice failed to reach the platform within the 1 min cut-off period, they were retrieved from the pool and placed on it for 5 s. After their swim, the mice were kept dry in a plastic holding cage filled with paper towels. The day after the acquisition phase, a probe trial was conducted by removing the platform and placing the mouse next to and facing the N side. The time spent in the previously correct quadrant was measured in a single 1-min trial. One hour later, the visible platform version was evaluated, with the escape platform lifted 1 cm above water level and shifted to the SE quadrant. A 13-cm high pole was inserted on top of the escape platform as a viewing aid. As with the place learning task, quadrant entries and escape latencies were measured for 4 trials, the animals stayed on the platform for 5 s, and a 1-min cut-off period was imposed with a 15-min intertrial interval, except that the test was conducted in a single session.
heterozygotes [U = 3, p < 0.05] and Dab1scm mutants [U = 27, p > 0.05], but wild-type mice wound up only at borderline significance [U = 14, p = 0.06]. In contrast, there was no intergroup difference in choice latencies at either the midway point or throughout the testing period [p > 0.05]. During acquisition of the hidden platform version of the Morris water maze (Fig. 1), there was a main group effect [quadrants: F2,19 = 60.43, p < 0.001; latencies: F2,19 = 76.13, p < 0.001], but no day effect [quadrants: F4,76 = 0.43, p > 0.05; latencies: F4,76 = 0.29, p > 0.05] or group × day interaction [quadrants: F4,76 = 0.93, p > 0.05; latencies: F4,76 = 0.88, p > 0.05]. Despite the relatively poor performance of both control groups as manifested by the lack of a day effect, Dab1scm mutants had higher quadrant entries and escape
1.3. Statistical analyses Intergroup differences were estimated by analyses of variance (ANOVAs) and Fisher’s least significant difference test. For spontaneous alternation and probe tests, the groups were compared with the Kruskal–Wallis test and each group was compared by the Mann–Whitney U test to a theoretical group performing at chance (50% for alternations, 15/60 s for probe) with zero variance.
2. Results Although there were no intergroup differences in spontaneous alternation rates [Krusal–Wallis, p > 0.05], the more sensitive method of comparing each group to chance reached significance. Indeed, during the first 5 days of spontaneous alternation (Table 1), wild-type [U = 7, p < 0.05] and heterozygote [U = 5, p = 0.05] mice alternated above chance, whereas Dab1scm mutants did not [U = 36, p > 0.05]. This pattern held up for all 10 days of testing in
Fig. 1. Hidden platform acquisition in the Morris water maze as measured by quadrant entries and escape latencies (s); cumulative score over 4 trials during 5 days of training in Dab1scm , heterozygous, and wild-type mice (means ± SEM). *p < 0.01, Dab1scm mutants vs either heterozygotes or wild-type.
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p < 0.01; latencies: F2,19 = 14.55, p < 0.001], caused by higher quadrant entries and escape latencies in Dab1scm mutants than either heterozygotes or wild-type [p < 0.05].
3. Discussion
Fig. 2. Time (s) spent in the target quadrant during the probe trial and cumulative visible platform performance over 4 trials for quadrants and escape latencies (s) in Dab1scm , heterozygous, and wild-type mice (means ± SEM and individual values). *p < 0.05, Dab1scm mutants vs either heterozygotes or wild-type Fisher’s exact test.
latencies than either heterozygotes or wild-type during all five days of training [p < 0.01, Fisher test]. In the probe trial (Fig. 2), wild-type [U = 0, p < 0.01] and heterozygote [U = 0, p < 0.01] mice stayed in the target quadrant above chance, but Dab1scm mutants did not [U = 27, p > 0.05]. However, there was no intergroup difference [F2,19 = 0.96, p > 0.05]. Although the homozygous mutants had comparable means, their intragroup variances were high, seemingly precluding significance when compared to a theoretical group performing at chance. The results found in the hidden platform substask were reproduced in visible platform testing (Fig. 2), as there was a significant group effect [quadrants: F2,19 = 8.68,
The tendency of alternating arm choices in a T-maze was evaluated in Dab1scm mutant mice relative to two non-ataxic control groups. There was no intergroup difference in alternation rates, but significant results emerged with the more sensitive method of comparing each group to chance. Unlike either wild-type or heterozygotes, homozygous mutants did not alternate significantly above chance during the first 5 days of evaluation. When the test was prolonged to a maximum of 10 days, Dab1scm mutants continued to perseverate while heterozygotes continued to alternate, whereas the rate of wild-type mice only approached significance. Overall, unlike most other mouse strains [1], the background A/A line used here showed relatively low alternation levels. Nevertheless, the deficiency in spontaneous alternation found in Dab1scm mutants reproduces the phenotype previously reported in Relnrl–Orl mutants on the Balb/c background [4,30]. The deficit in both mutants can be ascribed in part to cell ectopias and degeneration in the hippocampus. The importance of the medial temporal lobe in spontaneous alternation is underlined by findings of reduced spontaneous alternation rates in rats with lesions of the hippocampus relative to sham-operated controls [10,25,33,39]. The impairment can also be attributed to cell ectopias in neocortex, since lesions of frontal association cortex also reduced spontaneous alternation rates relative to sham-operated controls [11]. Likewise, the role of the cerebellum on spontaneous alternation is supported by findings of similar deficits in several murine mutants with cerebellar atrophy in the absence of overt pathology in either hippocampus or neocortex, such as Grid2Lc (Lurcher) [27,29]. It is unlikely to be attributed to ataxia, since turning left or right does not require fine motor control. Moreover, it is unlikely to be attributed to motor slowing, since choice latencies did not differ between the groups. Instead, the deficit may be caused by poorer working memory, an hypothesis that should be further explored in other paradigms, such as learned left-right alternation. A second explanation is that such lesions alter inhibitory processes, since hippocampal, prefrontal, and cerebellar lesioned animals all show disinhibitory tendencies in several paradigms [24,26,27,45]. The test is dependent on optimal arousal levels, neither too high nor too low, as fearful, inhibited animals also alternate less frequently [34]. In addition to spontaneous alternation, Dab1scm mutants were impaired in hidden and visible platform versions of the Morris water maze. This occurred despite the relatively poor performance of the control groups on the A/A background, as shown by a lack of significance on the day factor during acquisition of the hidden platform phase. The deficit of Dab1scm mutants on hidden and visible platform versions reproduces the phenotype previously reported in Relnrl–Orl mutants [30], as well as Grid2Lc mutants [5,29,37] and hemicerebellectomized rats [36]. The deficit in the visible platform subtest may be ascribed to poor visuomotor control, as defined by an inability to coordinate swimming movements towards a specific target despite the presence of visual cues. It can also ascribed to a procedural deficit [36], as defined by an inability to acquire skills underlying task solution, such as abandoning the potent tendency of swimming along the walls of the pool. In contrast to the pattern displayed by Dab1scm , Relnrl–Orl , and Grid2Lc mutants, a dissociation between hidden and visible platform subtests was obtained in the Agtpbp1pcd (Purkinje cell degeneration) mutant [19], as well as rats with selective lesions of the dentate nuclei [23,35], attributable in part to ascending
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cerebellar pathways towards the parietal cortex [13]. Overall, these results point towards a role for separate cerebellar pathways on spatial learning and visuomotor control or procedural learning. Despite poor performance during acquisition, Dab1scm mutants had comparable means to the control groups on the probe trial, indicating no specific impairment in long-term memory relative to the other two groups, though the only group not performing significantly above chance in target location, most likely due at least in part to a high intragroup variance. Instead, their maze deficit can best be interpreted as a difficulty in coordinating swimming movements towards a visible goal or learning appropriate procedural skills. We noted that, unlike the other two groups, most mutants tended to swim in circles far from the walls, whose circumference comprised one or more quadrants, a stereotyped response that could explain to a certain extent both elevated quadrant entries and escape latencies. They also appear rarely at rest, so that intergroup differences seem unlikely to be caused by freezing responses. It remains to be determined to what extent the deficits in Dab1scm mutants can be generalized to other spatial learning tasks such as the radial arm maze, described as being impaired in the Relnrl mutant [16]. This would best be examined in a within-study design with two or more mutants. Acknowledgment This study was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to RL. References [1] J.Y. Bertholet, W.E. Crusio, Spatial and non-spatial spontaneous alternation and hippocampal mossy fibre distribution in nine inbred mouse strains, Behav. Brain Res. 4 (1991) 197–202. [2] G.J. Blatt, L.M. Eisenman, A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice, J. Comp. Neurol. 232 (1985) 117–128. [3] G.J. Blatt, L.M. Eisenman, Topographic and zonal organization of the olivocerebellar projection in the reeler mutant mouse, J. Comp. Neurol. 267 (1988) 603–615. [4] T.V. Bliss, M.L. Errington, Reeler mutant mice fail to show spontaneous alternation, Brain Res. 124 (1977) 168–170. [5] J. Cendelin, I. Korelusova, F. Vozeh, The effect of repeated rotarod training on motor skills and spatial learning ability in Lurcher mutant mice, Behav. Brain Res. 189 (2008) 65–74. [6] S. Chung, Y. Zhang, F. Van Der Hoorn, R. Hawkes, The anatomy of the cerebellar nuclei in the normal and scrambler mouse as revealed by the expression of the microtubule-associated protein kinesin light chain 3, Brain Res. 1140 (2007) 120–131. [7] G. D’Arcangelo, Reelin mouse mutants as models of cortical development disorders, Epilepsy Behav. 8 (2006) 81–90. [8] G. D’Arcangelo, T. Curran, Reeler: new tales on an old mutant mouse, Bioessays 20 (1998) 235–244. [9] G. D’Arcangelo, G.G. Miao, S.C. Chen, H.D. Soares, J.I. Morgan, T. Curran, A protein related to extracellular matrix proteins deleted in the mouse mutant reeler, Nature 374 (1995) 719–723. [10] G.M. Dillon, X. Qu, J.N. Marcus, J.C. Dodart, Excitotoxic lesions restricted to the dorsal CA1 field of the hippocampus impair spatial memory and extinction learning in C57BL/6 mice, Neurobiol. Learn. Mem. 90 (2008) 426–433. [11] I. Divac, R.G.E. Wikmark, A. Gade, Spontaneous alternation in rats with lesions in the frontal lobe: extension of the frontal lobe syndrome, Physiol. Behav. 3 (1975) 39–42. [12] E. Gallagher, B.W. Howell, P. Soriano, J.A. Cooper, R. Hawkes, Cerebellar abnormalities in the disabled (mdab1-1) mouse, J. Comp. Neurol. 402 (1998) 238–251. [13] S. Giannetti, M. Molinari, Cerebellar input to the posterior parietal cortex in the rat, Brain Res. Bull. 58 (2002) 481–489. [14] A.M. Goffinet, The embryonic development of the cerebellum in normal and reeler mutant mice, Anat. Embryol. 168 (1983) 73–86. [15] A.M. Goffinet, The embryonic development of the inferior olivary complex in normal and reeler (rlOrl ) mutant mice, J. Comp. Neurol. 219 (1983) 10–24. [16] D. Goldowitz, J. Koch, Performance of normal and neurological mutant mice on radial arm maze and active avoidance tasks, Behav. Neural Biol. 46 (1986) 216–226. [17] D. Goldowitz, R.C. Cushing, E. Laywell, G. D’Arcangelo, M. Sheldon, H.O. Sweet, M. Davisson, D. Steindler, T. Curran, Cerebellar disorganization characteristic of reeler in scrambler mutant mice despite presence of reelin, J. Neurosci. 17 (1987) 8767–8777.
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