Physiology&Behavior,Vol. 51, pp. 523-525, 1992
0031-9384192 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
Printed in the USA.
Motor Abnormalities in Lurcher Mutant Mice ROBERT
L A L O N D E , 1 M. I. B O T E Z , C. C. J O Y A L A N D M I C H E L E C A U M A R T I N
Hotel-Dieu Hospital Neurology Service, Montreal Canada H2 W 1T8, and University of Montreal, Department of Medicine, Montreal, Canada R e c e i v e d 20 M a y 1991 LALONDE, R., M. I. BOTEZ, C. C. JOYAL AND M, CAUMARTIN. Motor abnormalitiesin lurchermutant mice. PHYSIOL BEHAV 51(3) 523-525, 1992.--Lurcher mutants had deficits in equilibrium in the coat-hanger and grid tests but not the wooden beam test. Although the mutants had less hole-pokes when confronted with small holes in most conditions, they had more holepokes with larger holes, demonstrating perseverative behavior. There was no decrease in rearing responses. These results indicate that in spite of cerebellar degeneration and ataxia, lurcher mutants are not impaired in all tests measuring motor function. Lurcher
mutant mice
Cerebellum
Motor abnormalities
Equilibrium
Behavioral Tests
LURCHER mutant mice are characterized by a loss of neurons in the olivocerebellar system (2,10,1 l). Decreases in cell number are observed in the inferior olive and in the cerebellar granule and Purkinge cells, but not in the deep nuclei (2). In spite of massive degeneration of this system, the projection from the inferior olive to the cerebellar cortex is still present (4). At one month of age, lurcher mutants are distinguishable from normal controls by their lurching gait. A series of tests were performed for the purpose of quantifying the motor abnormalities of this mutant. Previous experimentation has indicated the usefulness of wooden beam and grid tests for distinguishing between cerebellar mutant mice (weaver and staggerer) and normal mice (5,6). In addition to these tests, a coat-hanger test was used, in which the mice were positioned on a thin horizontal bar and latencies till reaching a diagonal bar were determined. General exploratory behavior was analyzed by means of hole-poking in two main experimental conditions: the stem portion of a T-maze and a steel cage with a single hole in the center.
The wooden beam, grid, and coat-hanger tests were all performed on the same day a few days after the mice were received. Whenever possible, the experiments were rated blindly by a single investigator, although in some tests the ataxic gait of the mice precluded blindness in the strictest sense. In the wooden beam test, the mice were placed on top of a wall (width, 1 cm) of a hole-board measuring 70 × 70 cm (height of wall, 34 cm) with an adjacent chamber measuring 30 X 70 cm. The main advantage of using this set-up rather than a standard short bar is that the animals were able to move around more, instead of staying in one position, thereby maximizing either their equilibrium capabilities or rendering their deficits more evident. There were two trials (intertrial interval, 2-5 min), and the latency before the mice fell offthe beam (cut-off point, 60 s) was determined. Immediately after this test, the mice were placed in the middle of a stainless steel grid (25 × 18 cm) atop the same wall, and the number of falls from the grid was recorded for four consecutive trials (intertrial interval, 2-5 min). In the coat-hanger test, the mice were placed in the middle of a thin horizontal bar (length, 40 cm; diameter, 2 mm) of a coat hanger of triangular shape (length of side bars, 19 cm). The coat hanger was placed at a height of 82 cm from a table. Four different latencies were determined by means of a stopwatch. Latency I measured the time elapsed before the two front paws of the animal touched one of the side bars. Latencies 2 and 3 measured the time elapsed before three or all four paws, respectively, reached one of the side bars. Latency 4 measured the time elapsed before falling. Thus, low values for latencies 1-3 indicate superior equilibrium, whereas low values for latency 4 indicate inferior equilibrium. In addition, climbs to the top of the hanger were tabulated, referred to as successful climbs. There were two trials (intertrial interval, 60 s). Whenever an animal fell, maximal scores of 60 s were written for latencies 1-3.
METHOD
Subjects Lurcher mutant mice (Le/+) and normal littermate controls ( + / + ) ofthe B6CBACa-AW'J/A strain of either sex were obtained from Jackson Laboratory (Bar Harbor, ME). In equilibrium tests, 30 lurehers were age-matched to 12 controls (range, 5-9 weeks). In exploration tests, a separate group of I 1 lurchers and 11 controis were used. These mice were all born within 4 days of each other and were 2 months old. The mice were placed in group cages with food and water available at all times in a temperature and humidity controlled room with a 12-h light-dark cycle (lights off at 1830). Testing was performed during the afternoon.
1Requests for reprints should be addressed to Robert Lalonde, Hotel-Dieu Hospital, Neurology Service, 3840 St. Urban Street, Montreal, Canada, H2W ITS.
523
524
L A L O N I ) E E l At.,
In the exploration tests, during the first 2 days, hole-poking and rearing responses were tested in a restricted region of the stem of a T-maze (47.5 × 8.5 cm; height of walls, 10.2 cm) made of transparent plastic, The mice were already familiar with the T-maze. Two rubber corks (diameter, 4.1 cm; height, 2.5 cm) with a small hole (0.5 cm) pierced in the center were placed against a 8.5-cm wide wall at one end of the stem of the maze. Another hole, slightly larger (diameter, 1.1 cm), could be found on the floor of the maze at a distance of 13 cm from the corks. On day 1, the number of hole-pokes in the corks against the wall (referred to as wall hole-pokes) and on the floor were counted in a 4-min session. On day 2, the corks were rolled over (at a distance of 10 cm from the wall), so that the holes now faced upward. The mice had to lean their head downward in order to poke their snout into the hole (cork hole-pokes). The number of rears on the corks and the n u m b e r of pokes on the floor were measured on both days. On days 3-4, the mice were placed in another apparatus, a stainless steel cage (18 × 18 cm; height, 25 cm) mounted on an identical steel cage. There was a single hole, much larger (2.6 cm) than the holes of the previous apparatus, in the center of the floor of the cage. There were no visual stimuli in the hole, but there was black empty space at a 20 cm height from the table. For 2 days (2 sessions on day 3, and 1 on day 4), the number of hole-pokes was counted in the first 3 min and in the last 3 min of each 6 min session. In the cage, the floor and two of the walls were made of solid steel. On the third wall, a layer of plastic was placed against a steel grid in order to prevent the mice from climbing. The fourth side had no wall in order to enable the experimenters to see the mice. Parametric comparisons for homogeneous variances between independent groups were made by t-tests, whereas nonparametric measures were made by the Wilcoxon rank sum test (3). RESULTS
In the wooden beam test, lurcher mutants did not fall off more quickly than normal mice (Table l). On the other hand, in the grid test, lurcher mutants fell more often than normal mice, t(40) = 2.5, p < 0.01. In the coat-hanger test, lurcher mutants showed inferior performance on all measures of equilibrium. The mutants had higher values for latencies 1-3: l, Rl(10,30) = 72.5, p < 0.001; 2, R1(7,23) = 36.5, p < 0.001; 3, Rl(10,30) = 46, p < 0.001; and lower values for latency 4, Rl(10,30) = 99.5, p < 0.01. Moreover, normal mice had more successful climbs than lurcher mutants, t(28) = 3.38, p < 0.005. There were missing data for the first 3 normal mice and for 7 mutants for the latency 2 and successful climb measures because we did not think of measuring these behaviors until later shipments. In the stem of the T-maze, lurcher mutants had less wall hole-pokes, t(19) = 1.73, p < 0.05 and floor hole-pokes, t(19) = 2.64, p < 0.01. There was no group difference in terms of cork hole-pokes, t(19) = 1.68, 0.1 > p > 0.05. Moreover, the groups did not differ in the number of rears on the corks, t(19) = 0.7, p > 0.1. One of the iurcher mice froze in the apparatus and therefore was not included in the results. In the steel cage, both normal mice and lurcher mutants had less hole-pokes during the second half of the sessions than the first: normal mice, t(10) = 2.78, p < 0.01; lurcher mutants, t( l 0) = 1.87, p < 0.05. The lurchers had more hole-pokes during the first 3 min, t(20) = 3.19, p < 0.005 and the last 3 min, t(20) = 4.24, p < 0.001, than normal mice. DISCUSSION
Lurcher mutants were impaired in all aspects of the coathanger test. In comparison to normal mice, mutant mice took
TABLE 1 EQUILIBRIUM BEHAVIOR AND EXPLORATORY ACI'IVITY IN LURCHER MUTANTS AND NORMAL MICE
Tests
Coat hanger (latencies in s) I 2 3 4 Successful climbs Wooden beam (latency in s until fall) Grid (number of falls) Stem of T-maze Wall hole-pokes Cork hole-pokes Floor hole-pokes Rears Steel cage hole-pokes First 3 min Last 3 min
Normal Mice
Lurcher Mutants
17.2 ± 6.5 22.0 _+ 8.4 25.0 _+ 9.2 59.5 _+ 1.4 0.7 _+ 0.5 56.1 _+ 7.6 0 +_0
46.0 51.2 57.3 41.0 0.2 53.2 0.5
+_ 16.9" _+ 13.8* ± 6.4* ± 19.0t _+ 0At _+ 14.0 ± 0.7+
0.9 + 1.0 0.7 + 1.0 5.8 _+ 3.0 12.5 _+ 6.3
0.2 ± 0.2 +_ 2.8 _+ 10.8 ±
20.9 ± 6.3 16.4 ± 7.9
31.2 +__ 6.0127.0 _+ 5.4*
0.6~ 0.3 1.9~" 7.6
Mean _+ SD. 0.001. t p
0031-9384192 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
Printed in the USA.
Motor Abnormalities in Lurcher Mutant Mice ROBERT
L A L O N D E , 1 M. I. B O T E Z , C. C. J O Y A L A N D M I C H E L E C A U M A R T I N
Hotel-Dieu Hospital Neurology Service, Montreal Canada H2 W 1T8, and University of Montreal, Department of Medicine, Montreal, Canada R e c e i v e d 20 M a y 1991 LALONDE, R., M. I. BOTEZ, C. C. JOYAL AND M, CAUMARTIN. Motor abnormalitiesin lurchermutant mice. PHYSIOL BEHAV 51(3) 523-525, 1992.--Lurcher mutants had deficits in equilibrium in the coat-hanger and grid tests but not the wooden beam test. Although the mutants had less hole-pokes when confronted with small holes in most conditions, they had more holepokes with larger holes, demonstrating perseverative behavior. There was no decrease in rearing responses. These results indicate that in spite of cerebellar degeneration and ataxia, lurcher mutants are not impaired in all tests measuring motor function. Lurcher
mutant mice
Cerebellum
Motor abnormalities
Equilibrium
Behavioral Tests
LURCHER mutant mice are characterized by a loss of neurons in the olivocerebellar system (2,10,1 l). Decreases in cell number are observed in the inferior olive and in the cerebellar granule and Purkinge cells, but not in the deep nuclei (2). In spite of massive degeneration of this system, the projection from the inferior olive to the cerebellar cortex is still present (4). At one month of age, lurcher mutants are distinguishable from normal controls by their lurching gait. A series of tests were performed for the purpose of quantifying the motor abnormalities of this mutant. Previous experimentation has indicated the usefulness of wooden beam and grid tests for distinguishing between cerebellar mutant mice (weaver and staggerer) and normal mice (5,6). In addition to these tests, a coat-hanger test was used, in which the mice were positioned on a thin horizontal bar and latencies till reaching a diagonal bar were determined. General exploratory behavior was analyzed by means of hole-poking in two main experimental conditions: the stem portion of a T-maze and a steel cage with a single hole in the center.
The wooden beam, grid, and coat-hanger tests were all performed on the same day a few days after the mice were received. Whenever possible, the experiments were rated blindly by a single investigator, although in some tests the ataxic gait of the mice precluded blindness in the strictest sense. In the wooden beam test, the mice were placed on top of a wall (width, 1 cm) of a hole-board measuring 70 × 70 cm (height of wall, 34 cm) with an adjacent chamber measuring 30 X 70 cm. The main advantage of using this set-up rather than a standard short bar is that the animals were able to move around more, instead of staying in one position, thereby maximizing either their equilibrium capabilities or rendering their deficits more evident. There were two trials (intertrial interval, 2-5 min), and the latency before the mice fell offthe beam (cut-off point, 60 s) was determined. Immediately after this test, the mice were placed in the middle of a stainless steel grid (25 × 18 cm) atop the same wall, and the number of falls from the grid was recorded for four consecutive trials (intertrial interval, 2-5 min). In the coat-hanger test, the mice were placed in the middle of a thin horizontal bar (length, 40 cm; diameter, 2 mm) of a coat hanger of triangular shape (length of side bars, 19 cm). The coat hanger was placed at a height of 82 cm from a table. Four different latencies were determined by means of a stopwatch. Latency I measured the time elapsed before the two front paws of the animal touched one of the side bars. Latencies 2 and 3 measured the time elapsed before three or all four paws, respectively, reached one of the side bars. Latency 4 measured the time elapsed before falling. Thus, low values for latencies 1-3 indicate superior equilibrium, whereas low values for latency 4 indicate inferior equilibrium. In addition, climbs to the top of the hanger were tabulated, referred to as successful climbs. There were two trials (intertrial interval, 60 s). Whenever an animal fell, maximal scores of 60 s were written for latencies 1-3.
METHOD
Subjects Lurcher mutant mice (Le/+) and normal littermate controls ( + / + ) ofthe B6CBACa-AW'J/A strain of either sex were obtained from Jackson Laboratory (Bar Harbor, ME). In equilibrium tests, 30 lurehers were age-matched to 12 controls (range, 5-9 weeks). In exploration tests, a separate group of I 1 lurchers and 11 controis were used. These mice were all born within 4 days of each other and were 2 months old. The mice were placed in group cages with food and water available at all times in a temperature and humidity controlled room with a 12-h light-dark cycle (lights off at 1830). Testing was performed during the afternoon.
1Requests for reprints should be addressed to Robert Lalonde, Hotel-Dieu Hospital, Neurology Service, 3840 St. Urban Street, Montreal, Canada, H2W ITS.
523
524
L A L O N I ) E E l At.,
In the exploration tests, during the first 2 days, hole-poking and rearing responses were tested in a restricted region of the stem of a T-maze (47.5 × 8.5 cm; height of walls, 10.2 cm) made of transparent plastic, The mice were already familiar with the T-maze. Two rubber corks (diameter, 4.1 cm; height, 2.5 cm) with a small hole (0.5 cm) pierced in the center were placed against a 8.5-cm wide wall at one end of the stem of the maze. Another hole, slightly larger (diameter, 1.1 cm), could be found on the floor of the maze at a distance of 13 cm from the corks. On day 1, the number of hole-pokes in the corks against the wall (referred to as wall hole-pokes) and on the floor were counted in a 4-min session. On day 2, the corks were rolled over (at a distance of 10 cm from the wall), so that the holes now faced upward. The mice had to lean their head downward in order to poke their snout into the hole (cork hole-pokes). The number of rears on the corks and the n u m b e r of pokes on the floor were measured on both days. On days 3-4, the mice were placed in another apparatus, a stainless steel cage (18 × 18 cm; height, 25 cm) mounted on an identical steel cage. There was a single hole, much larger (2.6 cm) than the holes of the previous apparatus, in the center of the floor of the cage. There were no visual stimuli in the hole, but there was black empty space at a 20 cm height from the table. For 2 days (2 sessions on day 3, and 1 on day 4), the number of hole-pokes was counted in the first 3 min and in the last 3 min of each 6 min session. In the cage, the floor and two of the walls were made of solid steel. On the third wall, a layer of plastic was placed against a steel grid in order to prevent the mice from climbing. The fourth side had no wall in order to enable the experimenters to see the mice. Parametric comparisons for homogeneous variances between independent groups were made by t-tests, whereas nonparametric measures were made by the Wilcoxon rank sum test (3). RESULTS
In the wooden beam test, lurcher mutants did not fall off more quickly than normal mice (Table l). On the other hand, in the grid test, lurcher mutants fell more often than normal mice, t(40) = 2.5, p < 0.01. In the coat-hanger test, lurcher mutants showed inferior performance on all measures of equilibrium. The mutants had higher values for latencies 1-3: l, Rl(10,30) = 72.5, p < 0.001; 2, R1(7,23) = 36.5, p < 0.001; 3, Rl(10,30) = 46, p < 0.001; and lower values for latency 4, Rl(10,30) = 99.5, p < 0.01. Moreover, normal mice had more successful climbs than lurcher mutants, t(28) = 3.38, p < 0.005. There were missing data for the first 3 normal mice and for 7 mutants for the latency 2 and successful climb measures because we did not think of measuring these behaviors until later shipments. In the stem of the T-maze, lurcher mutants had less wall hole-pokes, t(19) = 1.73, p < 0.05 and floor hole-pokes, t(19) = 2.64, p < 0.01. There was no group difference in terms of cork hole-pokes, t(19) = 1.68, 0.1 > p > 0.05. Moreover, the groups did not differ in the number of rears on the corks, t(19) = 0.7, p > 0.1. One of the iurcher mice froze in the apparatus and therefore was not included in the results. In the steel cage, both normal mice and lurcher mutants had less hole-pokes during the second half of the sessions than the first: normal mice, t(10) = 2.78, p < 0.01; lurcher mutants, t( l 0) = 1.87, p < 0.05. The lurchers had more hole-pokes during the first 3 min, t(20) = 3.19, p < 0.005 and the last 3 min, t(20) = 4.24, p < 0.001, than normal mice. DISCUSSION
Lurcher mutants were impaired in all aspects of the coathanger test. In comparison to normal mice, mutant mice took
TABLE 1 EQUILIBRIUM BEHAVIOR AND EXPLORATORY ACI'IVITY IN LURCHER MUTANTS AND NORMAL MICE
Tests
Coat hanger (latencies in s) I 2 3 4 Successful climbs Wooden beam (latency in s until fall) Grid (number of falls) Stem of T-maze Wall hole-pokes Cork hole-pokes Floor hole-pokes Rears Steel cage hole-pokes First 3 min Last 3 min
Normal Mice
Lurcher Mutants
17.2 ± 6.5 22.0 _+ 8.4 25.0 _+ 9.2 59.5 _+ 1.4 0.7 _+ 0.5 56.1 _+ 7.6 0 +_0
46.0 51.2 57.3 41.0 0.2 53.2 0.5
+_ 16.9" _+ 13.8* ± 6.4* ± 19.0t _+ 0At _+ 14.0 ± 0.7+
0.9 + 1.0 0.7 + 1.0 5.8 _+ 3.0 12.5 _+ 6.3
0.2 ± 0.2 +_ 2.8 _+ 10.8 ±
20.9 ± 6.3 16.4 ± 7.9
31.2 +__ 6.0127.0 _+ 5.4*
0.6~ 0.3 1.9~" 7.6
Mean _+ SD. 0.001. t p
