Intern. J. Neuroscience, 2001. Vol. 108, pp. 21-30 Reprints available directly from the publisher Photocopying permitted by license only

2001 OPA (Overseas Publishers Association) N.V. Published by license under

the Gordon and Breach Science Publishers imprint.

CONTRALATERAL ROTATORY BIAS

UNILATERAL HEMISPHERECTOMY IN ADULT SWISS MICE*

IN THE FREE-SWIMMING TEST AFTER

THOMAS E. KRAHE', CLAUDIO c . FILGUEIRAS, EGAS M. CAPARELLI-DAQUER

and SERGIO L. SCHMIDT

Laboratbrio de NeuroJsiologia e Avaliqiio N~urocomportameiztal, Universidade do Estado do Rio de Janeiro (UERJ) ,

Avenida Manoel de Abreu 444, Yandar, Fisiologia - Vila Isabel, Rio de Janeiro, RJ, Brazil, 20 551 170

(Received 12 October 2000)

In the free-swimming rotatory test mice spend most of the time swimming close to the wall of the container attempting to escape from an aversive test situation. The attraction to the wall may suggest that turning behavior in the free-swimming test reflects the existence of intrinsic sensory asymmetries, which determine preferential attention adhesion to one side. In order to test this hypothesis, we investigated the rotatory swimming behavior of mice submitted to a unilateral hemispherectomy at adulthood, a condition of extreme sensory asymmetry. Fifteen days after surgery procedures, each mouse was tested for 5 min on 3 different days. We found that the hemispherectomized mice had a significant strong bias to turn in the direction contralateral to their lesion. These data could be explained considering that, in attempting to escape from the test situation, animals bring the recipient wall into their intact sensory field and, as a consequence, set the direction of locomotion. Thus, the free-swimming test may be useful to investigate sensory asymmetries during an aversive test situation.

Keywords: Side preference; Circling; Sensory asymmetry; Aversive situation

*This work was, in part, funded by grants given to SLS by Fundagao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), by Sr2 UERJ, and by P6s-GraduaFao em Biologia UERJ. The authors are thankful to Alexandre Medina, Fabiola Rocha and Yael Abreu-VillaFa for helpful comments, to Denise Oliveira and Marcel0 Mattos for technical assistance as well as to Edson Oliveira for animal care.

+Corresponding author. Tel.: (5521) 5876498, Fax: (5521) 5876295, e-mail: tkrdhe@ usanet

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_ _ -77 T. E. KRAHE (’t al.

Spontaneous and drug induced circling is frequently used as an experimental tool for the study of cerebral asymmetries (Glick and Shapiro. 1985; Gordon, Rehavi and Mintz, 1994; Noonan and Axelrod, 1989; Saji, Endo, Miyanishi, Volpe and Ohno, 1997; Wickens and Thornton, 1996). Circling behavior is usually measured on land with the aid of a spherical rotometer (Glick and Shapiro, 1985; Ungerstedt and Arbuthnott, 1970) and can also be measured in water through the free-swimming test (Collins, 1985; Denenberg, Talgo, Waters and Kenner, 1990; West, Reid and Schurr, 1986; Schmidt. Filgueiras and Krahe, 1999). In spite of similarities between the free- swimming test and the rotor test (West et d.. 1986), some conspicuous aspects of each test can be devised. Distinct from the rotor test, the frec-swimming test is recorded in a more stressful environment wherc the attraction to the wall has been associated with the animal’s attempts to escape from an aversive test situation (Schmidt ct 01.. 1999). In contrast, in a typical rotometer bowl the animals do not have the option of walking along a wall (Greenstein and Glick, 1975).

In another walled test setup ~ the open-field test ~ rodents also tend to locomote along the perimeter of the field: a phenomenon named “wall-facing” (Huston and Bures, 1983; Schiorring, 1979; Valle, 1970). In this setup, the time that the animal spends close to the wall is a behavioral measure of lateralized brain function (Steiner. Bonatz, Huston and Schwarting, 1988) and some investigators have shown dissociation between the laterality of wall-facing and the direction of turning in rotometers (Sullivan, Fraser and Szechtman, 1994; Sullivan, Parker and Szechtman. 1993). Due to the absence of walls in a traditional rotometer, animals will guide their movements in the direction of their motor asymmetries, a n asymmetry independent of latcralized external cues. However, in the presence of a wall, ;I

sensory bias can emerge during spontaneous investigation of environ- ment (wall-facing), which is cue-dependent and not predicted solcly by the direction of endogenous motor bias.

In summary, the data of attraction to the wall may suggest that turning behavior in the free-swimming test reflects the existence of 4ensory asymmetries, which determine the preferential attention adhesion to one side. Its well known that elimination of part of the brain, a widely used and reliable tool for the analysis of brain-behavior relations (r.g.. Huston, 1983), can lead to endurable contralateral

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ROTATORY BIAS AFTER HEMISPHERECTOMY 23

sensory neglect (Hicks and D’Amato, 1970). In this regard, in the present study we verified whether a unilateral hemispherectomy determines the side adhesion to the wall and consequently determines the direction of rotational behavior during the free-swimming test.

MATERIALS AND METHODS

Subjects

The animals used in this study were 39 Swiss mice that were bred and maintained in our laboratory on a 12: 12h light/dark cycle. Access to food and water was ad libitum. At adulthood (minimum = 3 months, maximum= 5 months), 25 animals (12 males and 13 females) were subjected to unilateral hemispherectomy and 14 animals (8 males and 6 females) were assigned to the control group.

Procedure for Unilateral Hemispherectomy and Sham Surgery

Under anesthesia with tribromoethanol (i.p.), the animals’ heads were fixed in a stereotaxic frame. After skin incision, the dorsal aspects of the frontal and parietal bones were unilaterally removed, and placed into saline solution (0.9%). The left or right hemisphere-side was chosen randomly - was then removed by aspiration and the surgical cavity filled with gel foam. The skull piece was then restored and the skin was sutured closed. The aim of the operation was to remove one lateral half of the brain rostra1 to the tentorium without crossing the midline or attempting to remove all of the olfactory bulb. In the control group the animals were anesthetized, heads fixed on the stereotaxic frame, the skin incised, and then sutured closed.

Test Procedure

Fifteen days after surgical intervention, each mouse was tested for 5 minutes on 3 different testing sessions of the free-swimming test with a 48 hours test-retest interval (testing sessions began at the same time of the day). The test procedure is described in details elsewhere (Schmidt et al., 1999). Briefly, each mouse was placed in a plastic container (diameter = 21 cm) filled with water in such a way that the animal

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24 T. E. KRAHE et ul

could not touch the bottom of the container. The swim rotations were continuously recorded by an overhead video camera.

Measurement of Turning Activity

A turn, either leftward (counterclockwise swimming) or rightward (clockwise swimming), was defined by using a 30" unit. For scoring the 30" turns, a transparent overlay with 30" axes was matched with the image of the circular apparatus on the screen of the video monitor. For each animal, the counting of any successive number of 30" turns in a particular direction was interrupted when the animal presented a shift in its initial direction, or when it ceased to move, or even when it floated passively.

Direction of Lateralization

For each testing session, a particular hemispherectomized mouse was assigned to a contraversive group if it turned to the contralateral side of the lesion more times than to the ipsiversive side. The opposite was true for the ipsiversive group. For the control group, the side preference classification was based on the number of turns to the right and to the left side, for each testing session.

Consistency of Laterality

Consistent turners were defined as mice that do not change their preferred side of rotation across the three testing sessions. Therefore, consistency of laterality is defined independent of the magnitude of the differences between contraversive and ipsiversive preference in the hemispherectomized group as well as between right and left preferences in the control group.

Turning Activity in Center and Periphery

Turning activity was also categorized according to the location where it occurred in the container. We counted the number of 30" turns in the periphery when the snout of the mouse was within a range of S.25cm from the wall. Accordingly, center activity was measured in a circle of 10.Scm in diameter. Both regions were

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ROTATORY BIAS AFTER HEMISPHERECTOMY 25

marked off using the same transparent overlay used to estimate the total number of 30" turns.

Histological Procedure

All hemispherectomized animals were perfused through the heart with saline followed by a solution of formaldehyde (4%). After post- fixation (always longer than 7 days) the brains were removed, embedded in albumin-gelatin, frozen, and cut into 40 pm coronal sec- tions. The first section in every three was stained with cresyl violet.

Statistical Analysis

The statistical analyses were conducted using Chi-square (x2) and Student's t-tests for two-sample comparisons of means. The two-tailed error probability ( p ) was set at 5%.

RESULTS

All hemispherectomized animals presented similar and extensive loss of the ablated hemisphere (Fig. 1). In all animals most of the cerebral hemisphere was eliminated, with a variable midline residuum of medial thalamic, hypothalamic, ventral forebrain structures, and medial remnants of the cortex.

Both hemispherectomized and control groups preferred to swim at the periphery of the recipient. For each testing session the amount of turning in the periphery is significantly greater than in the center (Tab. I). For both groups, the swimming behavior close to the wall was typically characterized by vigorous swimming during the first minute or so accompanied by frantic clawing at the side of the container. This initial response subsided with the progression of test- ing sessions. Defecation was also frequently noted in the beginning of each testing session.

Data on consistency of laterality indicated that 92% of the hemispherectomized animals (n = 23) were classified as side-consistent rotators. The difference between the percentage of side-consistent rotators and nonconsistent rotators reached significance (x2 = 15.4,

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T. E. K R A H E ('t cil

FIGURE I mized mouse brain at 6 rostro-caudal levels.

Camera lucida drawings of a coronally sectioned right heniispherecto-

TABLE I

Center ss. Prripher.y

Srssion

First StYOtId Third Control ( n - 14)

30" turns in periphcrq 483.6 * 73.5*** 331.9 t 48.9*** 295.4 + 54,2*** 30" turns in center 89.1 * 29.1 92.1 t 23.6 37.9 * I 0 . K

Hemispherectomized (n = 2 5 ) 30" turns in periphery 624.6 * 79.2*** 482.2 * 39.8*** 437.6 * 36.i*** 30" turns in center 130.4 5 56.7 67.3 I 18.8 60 2 + 12.9

Thc valucb repressnl means ( + SEM). T-test. *** [I <' 001

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ROTATORY BIAS AFTER HEMISPHERECTOMY 21

ttt

T ttt T

1* 2"d b Session

FIGURE 2 Mean number of 30" turns (contra and ipsiversive) of unilaterally hemispherectomized mice for each one of the three testing sessions. The values are means (+SEM) of 25 animals. T-test, * * * p < ,001.

df= I , p < .001). For the control group, 50% of the animals (n = 7) could be classified as side-consistent rotators. This percentage differed significantly from the hemispherectomized group ( x 2 = 7.2, df = 1, p < .01). Among the side-consistent hemispherectomized rotators, 91 YO of the animals (n = 21) turned preferentially to the contralateral side of the lesion. Accordingly, the mean number of contraversive 30" turns was significantly greater than the mean number of ipsiversive 30" turns for each testing session (Fig. 2). In addition, consistency of laterality and turning activity did not depend on sex or side of hemispherectomy. For the control group, no populational side- preference was found among the consistent rotators.

DISCUSSION

Our data show that hemispherectomized and control mice swim preferentially close to the wall of the container. When close to the wall,

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28 T. E. KRAHE et ul.

animals’ behavior is typically characterized by vigorous swimming accompanied by frantic clawing at the side of the recipient. These results are consistent with previous data of the free-swimming test in normal mice (Schmidt et ul., 1999) and rats (West et ul., 1986). suggesting that attraction to the wall reflects the animal’s attempt to escape from a frightening test situation.

Our results also clearly demonstrate that unilateral hemispherecto- mized animals turn preferentially to the side contralateral to the lesion. It is well known that, in rodents, unilateral hemispherectomy produccs severe sensory loss contralateral to the lesion without severe changes in motor function (e.g. , Hicks and D’Amato, 1970). Therefore, if an animal explores its environment, the only sensory input will be from the intact, largely ipsilateral, sensory hemifield. In this sense, the fact that most hemispherectomized animals turn to the side contralateral to the lesion could be explained considering that, in escape attempting to the test situation, animals bring the recipient wall into their intact sensory field and, as a consequence, set the direction of locomotion.

Besides the fact that severe sensory loss may guide the direction of turning behavior, we could also consider the effects of extensive lesions on the animal’s motor system. Kolb and Whishaw (1985) suggested that hemidecortication might produce a change in posture that causes the animal to display rotatory swimming bias ipsilateral to the lesion in the center of a large circular tank. However, the authors did not deny that these animals circle ipsilaterally in an attempt to bring the environment into its intact sensory field. Our data corroborates this second possibility, since if a posture imbalance is guiding the turning behavior a contralateral bias in the presence of a wall is not expected. Accordingly, unilaterally lesioned rats switch from ipsiversive movements in the center of an open field area to locomotion in the contraversive direction when exploring the edge (Fornaguera-Trias, Schwarting, Boix and Huston, 1993; Steiner et ul., 1988; Ziegler and Szerchtman, 198’8).

In conclusion, the present data support the hypothesis that during the free-swimming test, sensory asymmetries may guide the direction of turning behavior. Considering that in this test, rotatory behavior is recorded in an aversive condition, the free-swimming test may be useful to investigate cerebral asymmetries in sensory scanning of environment during a stressful test situation. This is particularly

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ROTATORY BIAS AFTER HEMISPHERECTOMY 29

interesting since it has been demonstrated that brain laterality is determinant of individual differences among rodents in their vulnerability to the deleterious effects of experiences involving lack of stressor control (Carlson and Glick, 1991).

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