Nocturnal rotation in normal rats: Correlation with amphetamine-induced rotation and effects of nigro-striatal lesions

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).

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Correlation of nocturnal and amphetamine-induced rotation In every instance, the direction of rotation at night was the same as the direction of rotation elicited by D-amphetamine. Moreover, as shown in Table I, nocturnal rotation was quantitatively correlated with amphetamine-induced rotation during the day. This was true for both parameters of rotation.

Early effects of lesions Within the first 2n. 48 h after surgery, all lesions produced transient contralateral rotation (Fig. 3). The time-course of this effect varied with the rostral-caudal level of the lesion within the nigrostriatal system whereas the intensity of this effect varied with the size and side of the lesion. Thus contralateral rotation after unilateral caudate lesions began about four hours after surgery, peaked at 6-10 h and disappeared by 16-20 TABLE I

Correlation o f nocturnal rotation (12 h) with D-amphetamine-induced rotation (1 h) Results for 66 rats.

Nocturnal D-Amphetamine Correlation (r)

Mean net rotations q- S.D.

Mean per cent dominance -4- S.D.

47.1 ~ 10.8 55.2 ~ 11.1 0.85*

71.4 -4- 4.0 87.3 -4- 4.8 0.89*

* Significant at P < 0.001, t-test.

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Fig. 3. h whereas the effect of unilateral nigral lesions began at 10-12 h after surgery, peaked at 20-30 h and disappeared by 44--48 h. The time-course of the effect of unilateral nigrostriatal bundle lesions was intermediate between that of nigral and caudate lesions. At each rostral-caudal level, the magnitude of this transient 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. The intensity of effect was also proportional to the size of the lesion, at least in the caudate nucleus. By 3 days after surgery, all rats returned to a mostly normal diurnal cycle - - that is, as in normal rats, approximately 90 ~o of all net rotations occurred at night, though in each case, the direction of rotation was now ipsilateral to the lesion and the magnitude of net rotation was greater than before surgery (Fig. 3). When administered D-amphetamine on the seventh postoperative day, all rats rotated toward the side of the lesion in direct relation to how much they rotated the previous night (Table II). Although, in all rats, the magnitude of druginduced rotation was greater postoperatively than preoperatively, rats with lesions in the same side of the brain as the preoperative direction of rotation had larger drug responses than rats with lesions in the opposite side of the brain (Table II).

Long-term effects of lesions With the exception of rats having large caudate lesions, the direction of spontaneous rotation was the same as the preoperative direction by one month after surgery. If the side of the lesion and the direction of rotation were the same, the magnitude of rotation was greater than that occurring preoperatively, though less than that occurring at one week after surgery. If the side of the lesion was opposite to the direction of rota-

TABLE 11

Effects o['lesious on nocturnal (NOC) and D-Amphetamine-imluced ~p-A ) rotation ( 12 h am/1 h, respectively Net = mean net rotations + S.D. ; i~,]D =: mean per cent dominance

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N

Preoperative SN-S 3 SN-0 3 NSB-S 3 NSB-0 3 CN (S)-S 3 CN (S)-0 3 CN (M)-S 3 CN (M)-0 3 CN (L)-S 3 CN (L)-0 3 Sham 4 Postoperative (6-7 SN-S SN-0 NSB-S NSB-0 CN (S)-S CN (S)-0 CN (M)-S CN (M)-0 CN (L)-S CN (L)-0 Sham

Noe: Net, %D

45.3 44.2 43.2 44.8 47.1 47.3 46.2 45.5 45.6 48.3 47.8 days) 194.1 101.0 198.3 98.7 97.4 69.2 200.2 101.9 287.3 170.1 48.3

S.D.)

D-A: Net, %D

9.1, 8.2, ± 7.6, ± 8.5, ±8.1, 8.0, ± 7.3, ± 7.9, ±9.1, i 9.4, ± 8.7,

70.5 ± 4.4 71.0 ± 4.3 69.8 ± 4.0 70.2 i 4.1 71.2i3.6 70.6 ± 3.8 71.3 -j 4.0 71.4 ± 3.9 71.8 ± 4 . 3 70.6 ± 4.5 70.9 ~: 4.2

± 15.1, ±10.2, ± 12.1, i8.3, ± 7.2, ± 3.8, i 14.3, ± 11.2, t 15.5, _L 12.3, ± 7.9"*,

95.4 ± 2.5 83.6±5.1 94.2 ± 1.8 80.1 t 4.5 90.7 ± 3.1 82.1 ± 3.1 94.9 i 2.8 84.1 i 2.8 98.6 J: 1.5 90.5 i 2.6 69.8 i: 4.2**

Postoperative (35 36 days)*** SN-S 97.3 ± 6.1, 84.6 £ 3.1 SN-0 26.4 ± 3.8, 66.4 _£ 2.8 NSB-S 101.5 ±: 5.3, 83.9 £ 2.9 NSB-0 25.4 :t: 3.6, 66.8 ~ 2.6 CN (S)-S 74.8 ± 4.1, 80.3 i 2.6 CN (S)-0 31.5 ± 5.6, 67.2 ± 3.1'** CN (M)-S 101.8 ± 5.9, 84.0 t 2.4 CN (M)-0 21.6 ± 3.9, 63.7 ~: 1.2 CN (L)-S 172.1 ± 12.1, 90.1 ± 3.6 CN (L)-0 90.6 :L 8.6, 81.2 ± 2.1 Sham 50.1 ± 8.6"**,71.5 ± 4.4***

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* S and 0 refer to lesions made in the same or opposite sides of the brain as the preoperative direction of rotation, respectively. SN, substantia nigra; NSB, nigrostriatal bundle, CN, caudate nucleus with small (S), medium (M) or large (L) lesion. ** In all cases except these, postoperative values were significantly (P <: 0.05-0.001, paired t-tests) greater than preoperative values. The effects of S lesions were always significantly greater (P < 0.05 0.01, t-tests) than the effects of 0 lesions. Regression correlations (across all lesioned groups) between Noc and D-A rotation were 0.92 (net) and 0.95 ( ~ D ) (P < 0.001). *** In all cases except these, postoperative values were significantly (P < 0.05-01, paired t-tests) different from preoperative values. The effects of S lesions were always significantly greater (P 0.05-0.001, t-tests) than the effects of 0 lesions. Except for group CN (L)-0, the direction of rotation was always the same as the preoperative direction whereas the direction of D-A rotation was always ipsilateral to the lesion. When the net direction was also ipsilateral to the lesion, the regression correlations (across all such groups) between Noc and D-A rotation were positive (r - 0.91 and 0.90 for Net and ~ D , respectively; P < 0.001 ). When the net direction was eontralateral to the lesion, the regression correlations were negative (r - ~ . 8 6 and ---0.91 for Net and ~oD, respectively; P < 0.001).

159 tion, the magnitude was less than that occurring preoperatively (Table II). All rats with large caudate lesions still rotated toward the side of the lesion, though less so than at one week after surgery; rats with same-sided lesions (i.e. the side of the lesion and the preoperative direction of rotation were the same) rotated more than rats with opposite-sided lesions. In all rats with lesions, the diurnal patterns of rotation remained normal, as they had been at one week after surgery. When administered D-amphetamine at the end of the experiment, the direction of rotation, in all cases, was toward the side of the lesion, though the magnitude was always less than that observed at one week after surgery. In each instance, rats with same-sided lesions had larger drug responses than rats with opposite-sided lesions (Table 11). In all rats with large caudate lesions and in all other rats with same-sided lesions, the magnitude of drug-induced rotation was directly correlated with nocturnal rotation; in the remaining rats with opposite-sided lesions, there was an inverse relationship (Table 1I). DISCUSSION Aside from transient effects during the first couple of days after surgery, most investigators have assumed that 'rats with unilateral nigro-neostriatal lesions do not turn unless treated with drugs'L The present study leaves no doubt that this assumption is not correct. Moreover, it is now clear that normal rats also rotate without drugs. Both observations were obviously missed in previous studies because rats were only tested during the inactive part of their diurnal cycles. Contralateral rotation occurring early after nigrostriatal lesions has been noted by other investigators14,15. Although other interpretations are possible (e.g. destruction ofnon-dopaminergic neurons functioning in opposition to dopaminergic neurons), it is commonly attributed to a degeneration release of dopamine 15 which is known to accumulate in nigrostriatal terminals upon cessation of impulse activity16. The results of the present investigation suggest that the duration of this release phenomenon is related to the length of the degenerating axon, since contralateral rotation lasted longer after nigral lesions than after nigrostriatal bundle lesions and least after caudate lesions. A similar relationship between length of degenerating axon and temporal release of transmitter has been established at peripheral junctions 8. The time-course of contralateral rotation did not appear to be affected by the size of the lesion. Larger caudate lesions produced more intense rotation than smaller lesions but the time-courses were comparable. Lesion sidedness was a significant variable in all phases of this study. When the side of the lesion was the same as the preoperative direction of rotation, the early contralateral rotation was of lesser magnitude than when a similar lesion was in the opposite side of the brain. Conversely, the subsequent ipsilateral rotation was of greater magnitude with same-sided than with opposite-sided lesions. The same relation applied when ipsilateral rotation was augmented following the administration of D-amphetamine. A lesion sidedness interaction was also apparent in the degree and kind of recovery a month after surgery. Except in the case of large caudate lesions, all lesioned rats then rotated in their preoperative directions. In all cases, the magnitude of rotation

160 was greater than the preoperative level if the lesioned side was the same as the preopeJative direction and lower than the preoperative level if the lesioned side was opposite to the preoperative direction. These results confirm and extend previous findings (e.g. 10) of laterality effects on lesion sequelae. In normal animals, D-amphetamine simply appeared to potentiate the endogenous asymmetry 6,11 - - the more a rat rotated at night, the greater its sensitivity to Damphetamine during the day. In lesioned animals, this was also true except at one month after surgery, when some lesioned rats rotated away from the side of their lesions at night - - that is, when the lesions were opposite to the preoperative direction of rotation and recovery had occurred. In such animals, nocturnal rotation was inversely correlated with D-amphetamine sensitivity during the day. In other words, the more a rat rotated contralateral to a lesion at night, the less it rotated ipsilateral to a lesion after o-amphetamine. The effect of D-amphetamine in lesioned animals therefore depended on the nature of the interaction between the side of the lesion and the preoperative asymmetry at the time the drug was administered. One can only speculate on the mechanism of the recovery phenomena. It is particularly curious how rats with opposite-sided lesions could revert back to their preoperative directions of rotation. Since the two nigrostriatal pathways of the normal rat appear to function at different metabolic rates 1', it is conceivable that following a lesion, presynaptic compensatory changes 1 would occur to a greater extent in the normally more active side. Although such changes might be sufficient to mediate partial recovery, one might also expect there to be less functional reserve in terms of the total amounts of dopamine available for release. Hence D-amphetamine might deplete the lesioned side more quickly than normal 11 and induce ipsilateral rotation regardless of the nocturnal direction. After the first couple of postoperative days, none of the lesions altered the normal cyclical pattern of diurnal rotation. This observation, together with all of the lesion sidedness interactions, indicate that unilateral lesions of the nigrostriatal system do not produce qualitative changes in behavior. Contrary to the usual assumption, implicit in a great deal of the recent literature, rotation (spontaneous as well as drug-induced) is not an effect per se of unilateral lesions. Rather, such lesions may alter the set-point of a normally operative mechanism. An accurate assessment of the lesion phenomenon requires an understanding of the baseline upon which the lesion is superimposed. ACKNOWLEDGEMENT S.D.G. is supported by N I D A Research Scientist Development Award DA 70082.

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