Brain Research, 150 (1978) 149-161 © Elsevier/North-Holland Biomedical Press
149
N O C T U R N A L R O T A T I O N IN N O R M A L RATS: C O R R E L A T I O N W I T H A M P H E T A M I N E - I N D U C E D R O T A T I O N A N D EFFECTS OF N I G R O S T R I A T A L LESIONS
STANLEY D. GLICK and RUSSELL D. COX Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York, N. Y. 10029 (U.S.A.)
(Accepted November 3rd, 1977)
SUMMARY Normal unoperated rats were observed to rotate (turn in circles) at night. For most (91.7 ~ ) rats, the preferred direction of rotation was consistent across hours and days and was the same as the direction of rotation elicited by D-amphetamine (1.0 mg/ kg) during the day. The magnitudes of nocturnal and D-amphetamine-induced rotation were also highly correlated. After rats showed stable diurnal patterns of rotation, unilateral lesions were made in either the substantia nigra, the nigrostriatal bundle or the caudate nucleus. All lesions produced transient contralateral rotation within the first 24 48 h after surgery. The time-course of this contralateral rotation was more prolonged after nigral lesions than after nigrostriatal bundle lesions and least after caudate lesions, suggesting that the duration of a degeneration release of dopamine is proportional to the length of the degenerating axon. Lesion size was correlated with the intensity of contralateral rotation but not with the time-course. At each rostralcaudal level, the magnitude of contralateral rotation was greater if the lesion was in the side of the brain opposite to the preoperative direction of rotation than if in the same side. By three days after surgery, all rats returned to a mostly normal diurnal cycle with the direction of rotation now being ipsilateral to the lesion. D-Amphetamine potentiated the ipsilateral rotation, though rats with lesions in the same side of the brain as the preoperative direction of rotation had larger drug responses than rats with similar lesions in the opposite side of the brain. By one month after surgery, the direction of spontaneous rotation of most rats had returned to the preoperative direction. As at all other times, the magnitude of rotation was, in part, dependent on the side of the lesion with respect to the preoperative bias. It is suggested that, following a unilateral lesion, compensatory processes occur to a greater extent if the lesion is in the normally more active side of the brain.
150 INTRODUCTION Previous studies in this laboratory have established that rats have a normal asymmetry in nigrostriatal function. The existence of such an asymmetry was initially suggested by the observations that o-amphetamine induces side preferences 4 and circling behavior or rotation 9 in normal rats. Because rotation had previously been reported only in rats with unilateral lesions of the nigrostriatal system '~, it was postulated that rotation in normal rats reflects an intrinsic nigrostriatal asymmetry. Subsequently, it was found that normal rats have asymmetries in striatal dopamine content 6 as well as in dopamine metabolism and dopamine-stimulated adenylate cyclase activitylL The intrinsic asymmetry in nigrostriatal activity has been related to spontaneous side preferences determined in a T maze 17. Amphetamine potentiates the dopaminergic asymmetry as it induces rotation 11 - - rotation appears to be the ultimate consequence of a consistent and persistent side preference 7. If laterality is a significant component of the rat's behavioral repertoire, it is reasonable to expect this to be more evident during the active part of its day-night cycle. For obviously pragmatic reasons, all previous studies of rotation, in lesioned as well as in normal rats, were conducted during the light non-active part of the day. We now report that normal rats rotate spontaneously at night, that nocturnal rotation is correlated with amphetamine-induced rotation during the day and that unilateral nigrostriatal lesions produce time-dependent changes in nocturnal rotation which vary with the size and side of the lesion. MATERIALS AND METHODS
Subjects The subjects were 72 naive female Sprague-Dawley rats approximately 3 months old at the beginning of the experiment. Animals were provided with food and water ad libitum during all phases of testing.
Apparatus All testing was conducted in 4 cylindrical (30.5 cm in diameter) Plexig?as enclosures having grid floors. An attached graduated drinking tube provided water, and food pellets were scattered on the floor. The apparatuses were situated in an environmentally controlled room (temperature -- 22 °C; humidity = 60 %) having a normal 12-12 h day-night cycle (lights on/off at 7:00 AM/PM). In each apparatus, a flexible wire, which harnessed the animal, was connected to a shaft which activated a photoelectric position sensing device that differentiated between incomplete and full (360 °) rotations 8.
Surgery and histology All surgery was conducted under sodium methohexital anesthesia. Unilateral lesions were made in either the caudate nucleus (CN), the substantia nigra (SN) or the nigrostriatal bundle (NSB) by a direct anodal current of 2 mA for 5-30 sec. Stereotaxic
151 coordinates ~3 for the caudate nucleus were 2.0 mm anterior to bregma, 3.0 mm lateral to the midline and 5.5 mm from the dura; for the substantia nigra, coordinates were 3.2 posterior, 2.5 mm lateral and 7.5 mm depth; for the nigrostriatal bundle, coordinates were 1.6 mm posterior, 2.2 mm lateral and 8.5 mm depth. Following the experiment, all rats were killed and perfused first with 0.85 ~ saline solution and followed by perfusion with 10 ~ formalin. The brains were removed and immersed in formalin for at least a week before sections (40/~m stained with Luxol blue and cresyl violet) were made and histological examination was conducted.
Procedure All 72 rats were placed individually in one of the cylindrical 'rotometers' and tested for at least 7 days. Baseline non-drug data were collected during the first 6 days. On the morning (9.30 a.m.) of the seventh day, each rat was injected i.p. with o-amphetamine sulfate (1.0 mg/kg). Three days later, 30 rats received unilateral lesions in either the caudate nucleus, substantia nigra or nigrostriatal bundle; these rats, as well as 4 sham-operated rats, were replaced in the rotometers and tested for another seven days, with D-amphetamine being administered again on the seventh day. At one month after surgery, the same 34 rats were again tested for another seven day sequence. Of the 30 lesions, 15 were made ipsilateral and 15 were made contralateral to the preoperative direction of rotation. Full rotations to the left and right were always recorded automatically by a printout counter. Left and right rotations were separately totalled each hour and the net rotational difference (i.e. rotations in the dominant direction minus rotations in the opposite direction) was determined for each rat. An index (i.e. per cent ( ~ ) dominance) of the consistency of each rat's directional preference independent of variations in the total number of rotations was also determined by the following formula: rotations in the dominant direction x 100/total rotations. RESULTS
Histology All lesions were located in the intended structures. The extent of each kind of lesion is shown in Fig. 1. In other rats, similar lesions of the substantia nigra (N = 5) and nigrostriatal bundle (N -- 6) decreased striatal dopamine levels to an average of 56.2 ~o and 53.1 ~ of control, respectively.
Diurnal pattern of normal rotation A consistent rotational bias across days was significant (Chi square test, P < 0.01) for the vast majority of rats (66 out of 72). Some rats consistently rotated to the left (N --= 36) and others to the right (N = 30). Approximately 90 ~ of all net rotations were made at night. Fig. 2 shows the daily pattern of rotation averaged across 66 rats for the first 6 days in the test apparatus. Since ~ dominance is incalculable when rats are motionless (e.g. sleep), it was not meaningful to determine ~ dominance on an hour-to-hour basis. However, when summated across the 12 h light and 12 h dark periods separately, ~ dominance appeared to have little or no diurnal variation (mean dominance during the light and dark was 68.8 ~ and 70.2 ~o, respectively, P > 0.1).
152
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149
N O C T U R N A L R O T A T I O N IN N O R M A L RATS: C O R R E L A T I O N W I T H A M P H E T A M I N E - I N D U C E D R O T A T I O N A N D EFFECTS OF N I G R O S T R I A T A L LESIONS
STANLEY D. GLICK and RUSSELL D. COX Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York, N. Y. 10029 (U.S.A.)
(Accepted November 3rd, 1977)
SUMMARY Normal unoperated rats were observed to rotate (turn in circles) at night. For most (91.7 ~ ) rats, the preferred direction of rotation was consistent across hours and days and was the same as the direction of rotation elicited by D-amphetamine (1.0 mg/ kg) during the day. The magnitudes of nocturnal and D-amphetamine-induced rotation were also highly correlated. After rats showed stable diurnal patterns of rotation, unilateral lesions were made in either the substantia nigra, the nigrostriatal bundle or the caudate nucleus. All lesions produced transient contralateral rotation within the first 24 48 h after surgery. The time-course of this contralateral rotation was more prolonged after nigral lesions than after nigrostriatal bundle lesions and least after caudate lesions, suggesting that the duration of a degeneration release of dopamine is proportional to the length of the degenerating axon. Lesion size was correlated with the intensity of contralateral rotation but not with the time-course. At each rostralcaudal level, the magnitude of contralateral rotation was greater if the lesion was in the side of the brain opposite to the preoperative direction of rotation than if in the same side. By three days after surgery, all rats returned to a mostly normal diurnal cycle with the direction of rotation now being ipsilateral to the lesion. D-Amphetamine potentiated the ipsilateral rotation, though rats with lesions in the same side of the brain as the preoperative direction of rotation had larger drug responses than rats with similar lesions in the opposite side of the brain. By one month after surgery, the direction of spontaneous rotation of most rats had returned to the preoperative direction. As at all other times, the magnitude of rotation was, in part, dependent on the side of the lesion with respect to the preoperative bias. It is suggested that, following a unilateral lesion, compensatory processes occur to a greater extent if the lesion is in the normally more active side of the brain.
150 INTRODUCTION Previous studies in this laboratory have established that rats have a normal asymmetry in nigrostriatal function. The existence of such an asymmetry was initially suggested by the observations that o-amphetamine induces side preferences 4 and circling behavior or rotation 9 in normal rats. Because rotation had previously been reported only in rats with unilateral lesions of the nigrostriatal system '~, it was postulated that rotation in normal rats reflects an intrinsic nigrostriatal asymmetry. Subsequently, it was found that normal rats have asymmetries in striatal dopamine content 6 as well as in dopamine metabolism and dopamine-stimulated adenylate cyclase activitylL The intrinsic asymmetry in nigrostriatal activity has been related to spontaneous side preferences determined in a T maze 17. Amphetamine potentiates the dopaminergic asymmetry as it induces rotation 11 - - rotation appears to be the ultimate consequence of a consistent and persistent side preference 7. If laterality is a significant component of the rat's behavioral repertoire, it is reasonable to expect this to be more evident during the active part of its day-night cycle. For obviously pragmatic reasons, all previous studies of rotation, in lesioned as well as in normal rats, were conducted during the light non-active part of the day. We now report that normal rats rotate spontaneously at night, that nocturnal rotation is correlated with amphetamine-induced rotation during the day and that unilateral nigrostriatal lesions produce time-dependent changes in nocturnal rotation which vary with the size and side of the lesion. MATERIALS AND METHODS
Subjects The subjects were 72 naive female Sprague-Dawley rats approximately 3 months old at the beginning of the experiment. Animals were provided with food and water ad libitum during all phases of testing.
Apparatus All testing was conducted in 4 cylindrical (30.5 cm in diameter) Plexig?as enclosures having grid floors. An attached graduated drinking tube provided water, and food pellets were scattered on the floor. The apparatuses were situated in an environmentally controlled room (temperature -- 22 °C; humidity = 60 %) having a normal 12-12 h day-night cycle (lights on/off at 7:00 AM/PM). In each apparatus, a flexible wire, which harnessed the animal, was connected to a shaft which activated a photoelectric position sensing device that differentiated between incomplete and full (360 °) rotations 8.
Surgery and histology All surgery was conducted under sodium methohexital anesthesia. Unilateral lesions were made in either the caudate nucleus (CN), the substantia nigra (SN) or the nigrostriatal bundle (NSB) by a direct anodal current of 2 mA for 5-30 sec. Stereotaxic
151 coordinates ~3 for the caudate nucleus were 2.0 mm anterior to bregma, 3.0 mm lateral to the midline and 5.5 mm from the dura; for the substantia nigra, coordinates were 3.2 posterior, 2.5 mm lateral and 7.5 mm depth; for the nigrostriatal bundle, coordinates were 1.6 mm posterior, 2.2 mm lateral and 8.5 mm depth. Following the experiment, all rats were killed and perfused first with 0.85 ~ saline solution and followed by perfusion with 10 ~ formalin. The brains were removed and immersed in formalin for at least a week before sections (40/~m stained with Luxol blue and cresyl violet) were made and histological examination was conducted.
Procedure All 72 rats were placed individually in one of the cylindrical 'rotometers' and tested for at least 7 days. Baseline non-drug data were collected during the first 6 days. On the morning (9.30 a.m.) of the seventh day, each rat was injected i.p. with o-amphetamine sulfate (1.0 mg/kg). Three days later, 30 rats received unilateral lesions in either the caudate nucleus, substantia nigra or nigrostriatal bundle; these rats, as well as 4 sham-operated rats, were replaced in the rotometers and tested for another seven days, with D-amphetamine being administered again on the seventh day. At one month after surgery, the same 34 rats were again tested for another seven day sequence. Of the 30 lesions, 15 were made ipsilateral and 15 were made contralateral to the preoperative direction of rotation. Full rotations to the left and right were always recorded automatically by a printout counter. Left and right rotations were separately totalled each hour and the net rotational difference (i.e. rotations in the dominant direction minus rotations in the opposite direction) was determined for each rat. An index (i.e. per cent ( ~ ) dominance) of the consistency of each rat's directional preference independent of variations in the total number of rotations was also determined by the following formula: rotations in the dominant direction x 100/total rotations. RESULTS
Histology All lesions were located in the intended structures. The extent of each kind of lesion is shown in Fig. 1. In other rats, similar lesions of the substantia nigra (N = 5) and nigrostriatal bundle (N -- 6) decreased striatal dopamine levels to an average of 56.2 ~o and 53.1 ~ of control, respectively.
Diurnal pattern of normal rotation A consistent rotational bias across days was significant (Chi square test, P < 0.01) for the vast majority of rats (66 out of 72). Some rats consistently rotated to the left (N --= 36) and others to the right (N = 30). Approximately 90 ~ of all net rotations were made at night. Fig. 2 shows the daily pattern of rotation averaged across 66 rats for the first 6 days in the test apparatus. Since ~ dominance is incalculable when rats are motionless (e.g. sleep), it was not meaningful to determine ~ dominance on an hour-to-hour basis. However, when summated across the 12 h light and 12 h dark periods separately, ~ dominance appeared to have little or no diurnal variation (mean dominance during the light and dark was 68.8 ~ and 70.2 ~o, respectively, P > 0.1).
152
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