A measure of lateral paw preference in the mouse

Physiology& Behavior. Vol. 50, pp. 853-856. ©Pergamon Press pie, 1991. Printed in the U.S.A.

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A Measure of Lateral Paw Preference in the Mouse N I C H O L A S S. W A T E R S * A N D V I C T O R H. D E N E N B E R G # 1

*Biobehavioral Sciences Graduate Degree Program, University of Connecticut, Storrs, CT 06269-4154 "pBiobehavioral Sciences Graduate Degree Program and Department of Psychology University of Connecticut, Storrs, CT 06269-4154 R e c e i v e d 25 M a r c h 1991 WATERS, N. S. AND V. H. DENENBERG. A measure of lateral paw preference in the mouse. PHYSIOL BEHAV 50(4) 853-856, 1991.--A lateral paw preference testing unit is described. Mice are allowed access to preferred food with either their left or right forepaw, and the amount eaten with each paw is measured. The unit allows easy measurement and quantification of this behavior, without requiring food deprivation or continuous monitoring of the subjects, and may be performed in the subject's home cage. Its reliability under a number of conditions is reported. The results do not correlate with those obtained using the Collins paw preference test. Paw preference

Laterality

Asymmetry

(LPP) unit, because the activity, reaching for food with one paw or the other, is similar to the activity measured in the Collins paw preference test, except that the animal reaches to either side, rather than toward the center. The LPP unit consists of a chamber ( H x W × L : 4 . 4 x 2 . 8 x 4 . 3 cm) open at one end. In the closed end (Fig. 1A) are two oval holes ( 0 . 8 × 0 . 4 cm) located 1.2 cm above the floor, abutting the wall on each side. The mouse can enter the chamber and have access to food through either of the holes. The closed end extends 2.5 cm above the top of the chamber, and 0.6 cm wider on each side. This chamber is affixed to a base measuring 75 × 100 cm (Fig. 1B). Flush to the closed end and the base is a block measuring 4.6 x 3.3 cm and 0.9 cm high (Fig. 1C). Atop the block and flush to the chamber wall is a box 6.2 × 2.0 cm and covering the width of the closed chamber wall (Fig. 1D). The box is open at the top to allow the hoppers to slide in. The two hoppers are identical (Fig. 1E, F). They are 5.7 × 1.3 x 1.5 cm. The top, bottom, and three sides are completely blocked. One of the narrow sides has a 1.0-cm opening in the top into which food is poured, and a 1.5-cm opening at the bottom which is partially occluded by a 0.8 × 1.3-cm piece which slants inward from the top of the opening at a 55 ° angle. The piece has a slight indentation in the open end, which increases the size of the opening in the center. On the top of the hopper is a small handle for easy removal. The two hoppers fit snugly into the box with the openings facing the feeding holes. The LPP unit is constructed entirely of 2.5-mm clear acrylic, except the block and handles, which are solid acrylic. All measurements given are internal.

COLLINS (4,6) has developed an effective and reliable procedure for measuring paw preference in the mouse. The animal is placed into a small "telephone booth" unit containing a cylindrical feeding tube projecting at chest height when the subject stands on its hind legs. The mouse reaches for grains of sweetened rolled wheat inside the tube and is observed until 50 paw reaches have been made. The paw preference score is the number of right paw entries (RPE). Independent measures of direction and degree of asymmetry are obtained from the RPE score. Collins has shown that mice can be genetically selected for degree of asymmetry (6); the direction of asymmetry has been found to be associated with immune parameters (14,15), and with learning in autoimmune mice having cortical ectopias (9). There are two difficulties with the Collins procedure. First, it is labor intensive, since someone has to observe the 50 paw reaches made by each animal. Second, even though they have been food deprived for eighteen hours, some mice do not reach for food and thus cannot be used. The lateral paw preference test was designed to overcome these problems. It consists of a unit placed within the animal's cage from which food is available to either paw from two independent sources. The design of this apparatus was suggested, in part, by a conversation with Dr. Betty Zimmerberg. Results from this unit represent a reliable measure of "pawedness," but do not correlate with results from the Collins measure. APPARATUS

The apparatus (Fig. 1) is called a Lateral Paw Preference

tRequests for reprints should be addressed to Dr. V. H. Denenberg, Biobehavioral Sciences Graduate Degree Program, U-154, University of Connecticut, Storrs, CT 06269-4154. 853

854

WATERS AND DENENBER(i

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FIG. 1. Diagram of the LPP unit. (A) Closed end of chamber. (B) Base. (C) Block. (D) Box. (E, F) Hoppers. See text for measurements and further details.

METHOD

Ten BXSB-Yaa + mice born in our colony from progenitors received from The Jackson Laboratory were singly housed in standard cages and had access to food and water ad lib, except as indicated. These mice are congenic to the autoimmune BXSB strain, but do not have the Y chromosomal autoimmune accelerating factor (16). Table 1 gives the schedule of testing. At approximately six weeks of age, all subjects were food deprived for 24 hours and tested for paw preference using the Collins procedure (Day 1). In this test, the subject reached for grains of sweetened rolled oats from a centrally located tube (4,6). The number of reaches with each paw was counted until a total of 50 reaches was made. After 96 hours, a lateral paw preference testing unit was placed in each animal's cage (Day 5). The hoppers had been

TABLE 1 T E S T SCHEDULE FOR BXSB-Yaa ÷

Day 1

5 6 7 8 9 10 11 12 14

Test Collins Paw Preference Lateral Paw Preference (24-hour test) Lateral Paw Preference Lateral Paw Preference Lateral Paw Preference Lateral Paw Preference Food Deprivation and Lateral Paw Preference Food Deprivation, Novel Environment, and Lateral Paw Preference Reverse Lateral Paw Preference Collins Paw Preference

loaded with Maypo and weighed before the start ol the test. AI ter 24 hours, the unit was removed; each food hopper was weighed, reloaded, and weighed once again. The complete unil was replaced in the subject's cage lot 8-10 hours. ~r until one hopper was empty (or nearly so), then removed and reweighed (Day 6). This same procedure was followed for 3 additional days (Days 7-9t, giving a total of 5 consecutive days of LPP testing. Each day, the amount of food consumed from each hopper was recorded in grams as the difference from start to end of the tesl. All testing occurred during the light portion of a 12/12-h lighL/ dark cycle, except the 24-hour test on Day 5. Each animal was tested with the same unit each day. To test the effects of food deprivation and novelty stress on this behavior, nine of the original subjects were food deprived for 24 hours before the start of testing on Day 10, then allowed access to the LPP until one hopper was empty. The animals were allowed 2 hours free access to food, then food deprived for 20 hours again and placed in a novel cage for testing (Day 11 ~. After 24 hours' recovery, the subjects were tested with the LPP one more time (Day 12). with the hoppers in each unit reversed left to right, to test the possibility that one hopper was allowing easier access to food than the other. The subjects were allowed to rest for 24 hours, then food deprived again for a retest using the Collins technique (Day 14). Thirty-one additional mice were tested. Nine female NZB (age 5-6 weeks), 8 DBA/2 (2 male and 6 female, age 6 months), and 14 BXSB (5 male and 9 female, age 8 weeks) mice were tested twice. (The NZB and BXSB strains are autoimmune, whereas DBA mice have no immunity problems.) Each test consisted of placing an LPP unit in each subject's cage until the subject had consumed at least 1 gram of Maypo from at least one side of the unit. If the subject had not eaten enough at the end of the day, the unit was removed for the night and replaced the following morning without being weighed or reloaded. The two tests were always performed consecutively. Six of the DBA/2 mice were further tested for 2 days during the dark portion of the 24-hour day. RESULTS

Laterali~ Index A laterality index (LI) was calculated for each subject for each day as LI = [R - L]/[R + L]IA where R is the amount of food consumed from the right hopper and L is the amount from the left hopper. Pearson product-moment correlations were calculated on the Lls for each day, and are listed in Table 2. The reliability of the total test (rtt) was .899, and the reliability of a single test (rll) was .642 ( p < 0 . 0 1 ) (7). The scores of the five days were then pooled into a single LPP score and correlated against performance when testing was done under food deprivation, in a novel environment or with the hoppers reversed. These correlations, shown in Table 3, are positive, as expected, and repeated-measures ANOVAs revealed no significant differences in mean laterality index across the conditions. Note the correlation of .799 against the food deprivation and novel environment condition. These conditions are present in the Collins test. Test-retest reliability coefficients for the other three strains of mice are listed in Table 4. LIs from the six subjects tested in the dark were not different than those obtained under standard conditions ( r = .782, p < 0 . 0 5 ) . Performance on the Collins test was measured as the number of right paw entries (RPE) out of fifty entries. The test-retest reliability for the Collins procedure was .849, quite similar to

LATERAL PAW PREFERENCE

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TABLE 4

TABLE 2 INTERCORRELATIONSAMONG5 DAYSOF LPP TESTING LPP l LPP LPP LPP LPP

2 3 4 5

.791" .636* .507 .362

LPP 2

.801" .885* .630*

LPP 3

.848* .674*

PEARSON PRODUCT-MOMENTCORRELATIONSOF TEST-RETESTLPP IN THREE STRAINSOF MICE LPP 4

.706*

Strain

N

r

NZB DBA/2 BXSB

9 8 14

.571" .762t .778~:

*p<0.10 (one-tailed test); ?/)<0.05 (one-tailed test); ~:p<0.01 (onetailed test).

*p<0.05 (one-tailed test).

the values reported by Collins (6). The two Collins scores were pooled and correlated against the pooled LPP score, based upon the five days of successive testing. The correlation was .322, which is not significant. In addition, we tested 169 mice of the 15 NXSM recombinant inbred lines derived from crosses between NZB and SM mice (10) on both LPP and the Collins test, and found an insignificant correlation of .127 between the two tests.

Absolute Asymmetry Analyses were carried out on asymmetry indices, that is, degree of laterality regardless of direction. These were calculated for the LPP data as the absolute value of the LIs, and for the Collins test, as the absolute value of the number of right paw extensions minus 25. The reliability of the absolute LI was somewhat lower than LI, but still highly significant: rtt = .693 and rl~ = .311 (p<0.01). The Collins reliability coefficient was .70. The pooled Collins absolute asymmetry score did not positively correlate with any day of absolute LPP.

Left-Right Classification All 41 subjects were classified as either "right-pawed" (LI>0) or "left-pawed" (LI<0) for the first two days of LPP (no subject had an LI of 0.00). Direction of asymmetry was reliable, r = .810 (p<0.01), using the cosine-~r formula of the tetrachoric correlation (12). DISCUSSION The lateral paw preference test gives reliable results and is easily administered. Although one test yields reliable data, inspection of Table 2 suggests that two tests may be preferable, since the first LPP test was only significantly correlated with the second and third tests, while the second LPP test correlated with all the others. It is not apparent why the introduction of food deprivation on Day 10 should reduce the correlation against the mean of the prior five days (Table 3). This could represent sam-

TABLE 3 CORRELATIONSOF LPP SCORESPOOLEDOVER 5 DAYS AGAINSTOTHERTESTINGCONDITIONS Pooled LPP vs. Food Deprivation LPP Food Deprivation and Novel Environment LPP Reverse LPP *p<0.10 (one-tailed test); tp<0.01 (one-tailed test).

r .428 .799t .525*

piing error, since the combination of food deprivation and novelty exposure the following day correlated .799 with the LPP mean. This test measures a lateralized behavior that may be considered "paw preference" in a general sense, but the lack of significant correlations establishes that is not related to paw preference as measured by the Collins technique. The difference between the techniques does not seem to be related to factors such as food deprivation or novelty stress, though these have been shown to have effects on other lateralized behaviors (1-3), nor does the lateralization seem to be an artifact of the individual testing units, as reversing the food hoppers in units had no effects upon the mean laterality index, and results from different groups of animals tested in our lab with the same units did not show intraunit correlations. The difference between this test and the Collins test is difficult to explain. It is unlikely that the animals are reaching across with the contralateral paw to receive food when it was always available ipsilaterally. None was observed to do this, and in tests when food is only available unilaterally, less than 15% of the animals have been observed to reach contralaterally (5). We believe that the difference between the two procedures may be related to the different reaching requirements. In the Collins procedure, the animal makes a medial and proximal reach with either paw, whereas in our procedure, the animal makes a lateral and more distal reach. It was suggested (A. Galaburda, 1991, personal communication) that the difference may be associated with a bias in visual fields. In the Collins test, the reward occupies a central, binocular location, whereas in LPP, stimuli are located in distal, monocular fields. However, the high correlation between results from testing in the light and from testing in the dark does not support this hypothesis. Results from other mice tested in our lab show no correlation between LPP and performance on a swim rotation or a free-choice T-maze procedure, indicating that LPP is not related to locomotor laterality (unpublished results). The different paw preference on different tasks is not unusual, as different "handednesses" in different situations has been reported in humans (13) and other primates (11). Though the results from the two paw preference tests were uncorrelated, each test seems to be related to learning. Performance on the Collins test is related to performance on a simple water escape task (8,9), while preliminary data indicate that performance on LPP is related to performance on the Morris spatial maze (17). The lateral paw preference test is useful for the continued study of lateralized behavior in rodents. We are currently designing an LPP for use with rats, and in continuing studies with inbred strains of mice, it will provide another tool to reveal the genetic and environmental factors that contribute to laterality in behavior.

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ACKNOWLEDGEMENTS The authors wish to thank Nancy Talgo for aid in the data collection. This research was supported, in part, by Grant No. HD20806 from the National Institute of Child Health and Human Development, NIH.

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