Appl. Neurophysiol. 38: 291-301 (1975)

Quantitative Registration of Motor Disorders following Bilateral Lesions of Substantia nigra in the Rat W. W inkelmüller and F. M. N itsch Neurobiology Department of the Max-Pianck-Institut für Hirnforschung, Frankfurt/Main, and Neurochirurgische Klinik der Medizinischen Hochschule Hannover, Hannover

Key Words. Motor disorders • Substantia nigra • Rats • Behavioral studies • Con­ ditioned responses • Rigidity Abstract. Bilateral lesions in the substantia nigra were induced in rats trained in an instrumental learning program comparable with a fixed-interval schedule. Gener­ al observations and examinations of rough motor behavior revealed rigidity, a de­ crease in spontaneous activity and a loss of postural reflexes. The analysis of lever­ pressing rates results in a differentiation of rigidity and akinesia which is in accord­ ance with the overt motor disturbances, dependent on the size of the nigral lesions.

Introduction

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Since the causal relationship between cell loss in the substantia nigra and the Parkinson syndrome was established [5, 7, 9, 20], many attempts have been made to create an experimental model for the Parkinson syn­ drome by making lesions in the substantia nigra. These experimental re­ sults have been controversial. Discrete lesions in the nigra of the monkey have had no effect on motor behavior [1], Some investigators have pro­ duced Parkinson-like tremor by making lesions in the midbrain tegmen­ tum dorsomedial to the substantia nigra [6, 15, 16]. Other investigators have correlated experimentally induced tremor with the interruption of cerebellar efferents [2, 3, 19]. However, bilateral lesions in the nigra of monkeys consistently induce hypokinesia, which is characterized by a lack of spontaneous motor activity and decreased locomation [1, 6, 19]. In cats, we observed a comparable akinesia and ipsilateral circling move­

292

W inkei.muli. er/N itsch

ment after complete unilateral lesions in the area of the nigra [21]. Rigidi­ ty did not appear in these experiments. It is difficult to make a quantitative assessment of the motor disturb­ ances and of postoperative compensation after nigra lesions in experi­ mental animals. This study attempts to create a model for the Parkinson syndrome by bilateral elimination of the substantia nigra of rats.

Materials and Methods Coagulation of substantia nigra. 12 9-month-old Wistar rats were used in the in­ vestigation. The females weighed between 200 and 260 g and the males between 280 and 370 g. All rats had been in an instrumental training program for 6 months. Stereotactic coagulation by high-frequency current was made on both sides of the substantia nigra in 9 rats under pentobarbital anesthesia (30 mg/kg intraperitoneally). To control the lesion sites, the animals were killed after a survival time of 14 weeks. During this time, they served for further experiments. The entire brain was removed from each rat and fixed in 10% formalin. The frozen tissues were cut in frontal series and stained according to Woelke-Heidenhain and Nissl. Instrumental training procedures. Each rat was placed in one of three sound­ proof Skinner boxes. The presentation of light stimuli and rewards was regulated with programming equipment developed in our Institute [14]. The data from the three test boxes were registered by electromechanical counters, cumulative writers (Gerbrand) and a 24-digit printer. In addition, the entire experimental procedure was recorded on-line with a digital LINC 8 computer and stored on a magnetic tape. The data were later evaluated with the help of the LINC 8. The rats were to press a lever in response to a 2-sec light stimulus. The constant interstimulus time was 8 sec. Pressing the lever during the presentation of a light stimulus was reward­ ed with a food pellet. After the session, each rat received three pieces of pressed food. Water was always available. Checking motor behavior. The motor performances of the rats were studied in a fixed examination and test program apart from the registration of instrumental be­ havior. Spontaneous activity, especially locomotion, exploration and raising-rate, as well as rough power, skillfulness and correction of posture were checked on a verti­ cal balance wall made of Plexiglas. All findings were filmed and a protocol was re­ corded.

Results

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Histological Findings The lesions in the histological serial sections of each rat were trans­ ferred to the schematic drawing of the frontal section of the rostrocaudal nigra segments (fig. 1).

Motor Disorders following Substantia nigra Lesions

014

5

4

8

X

17

293

F ig.l. Extent of lesions in schematic drawings of the rostral and caudal division of the substantia nigra. Regions of damaged nerve cells in the pars compacta and needle tracks are denoted by the shaded areas. The foci of coagulation are indicated with black dots within a block boundary.

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Postoperative Motor Behavior The actions of the operated animals were calssified under the follow­ ing terms: spontaneous activity, corrections of posture, rigidity, and cir­ cling. Observations on the 4th and 32nd postoperative days are summa­ rized in table I. Spontaneous activity. Spontaneous activity refers to actions such as locomation, exploration and raising of the body. These actions are reduced in animals with larger lesions in the nigra. The rats remain immobile, and locomotion can be elicited only by employing a painful stimulus. Because they hold their heads low, they have difficulty reaching the drinking bot­ tle. The sniffing impulse is markedly reduced. While the spontaneous ac­ tivity of most animals returned to normal in 14-21 days, rats 5, l g, and 014 showed a slight reduction in exploratory behavior and raising rate up to the 32nd day of observation.

294

W inkelmuller/N itsch

Table I. Postoperative motor behavior on the 4th and 32nd days after treatment Group

A

B

C

Animal

014 5 4 18 3 4s X 8 17

Spontaneous activity loss 4lh 32nd

Posture cor­ rection loss 4th 32nd

Signs of rigidity 4th 32nd

+++ + + ++ + + +

+++ ++ +++ ++ ++ +

+ + + + + + +++ +

++ + + + ++ — —

++ ++ +

++ + + + + + -

+ +

+





+ + + —

Circling 4th

32nd

+ -

_ + ++ + + + + + -







The order of succession of the rats is based on the lesion size; group A contained the largest lesions and group C the smallest. The number of crosses (+ ) indicates the degree of deviation in respect to the symptoms observed on the 4th and 32nd days after coagulation. For description of symptoms see text.

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Postural corrections. The spontaneous correction of posture was eval­ uated by observing balance exercises at the climbing wall. Animals with an impairment of postural reflexes have difficulty in balancing skillfully. Although rough power is maintained, movements, especially of the hind legs, are heavy and sluggish. The animals are incapable of correcting im­ balance by quick movements or by shifting their weight (fig. 2a). Even when walking they tend to drag their hind legs. They bend forward and/or maintain awkward sitting positions for minutes. Rats X, 5, 3 and 014 still had slight movement correction difficulties at the end of the 32-day ob­ servation period. Rigidity. Rigidity is defined as hypertonus of the flexor muscles verte­ bral column, tail and hind legs. These animals have a characteristic hunchback so extreme that it cannot be lowered passively. Their tails stand out stiffly or stand straight up during all movements. The extensor movements seem to be more distinct than the flexor movements, so that walking looks strangely long-legged (fig. 2b). Although rough power and motility are preserved, rigidity through increased tonus can be noted when one passively moves the hind legs. The ability to contract the back

Motor Disorders following Substantia nigra Lesions

295

Fig. 2. a Before the nigra lesion animal 014 is able to balance skillfully (left), postoperatively there is a lack of automatic control of posture (right), b Typical signs of rigidity with hunched back and stiffness of the tail.

muscles was reduced in all animals during the first 10 days after the oper­ ation. On the 32nd day after the operation animals 5, 014, 18 and 4 showed a slight increase in the tonus of the tail and hind legs. Circling. The term circling refers to compulsive horizontal turning in one direction under the guidance of the head and foretrunk. Some rats ex­ hibited circling. The slightest sensory stimulus elicited this stereotyped turning. The direction of circling depends upon the size and the site of the lesion. Rats 5, 014, 4g, and 4 showed circling and turned toward the side with the larger lesion. While 3 animals compensated for the compulsive circling after 6-8 days, the tendency of animal 5 to turn to the left lasted until the end of the observation period.

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Data of the Instrumental Behavior Tests The lever-pressing rate indicates the activity of the rats in the instru­ mental behavior test. Lever pressing is a learned and therefore task-di­ rected activity [12]. A task-directed activity is not only influenced by Io-

296

W inkelmüller/N itsch

Fig. 3. Lever-pressing-rate (LPR) in the postoperative phase related to the norm. The norm (T=100) represents the mean value of the preoperative data. The postoperative lever-pressing-rate (each score is a mean value of 4 days) in plotted as T scores, a Data of the controls without lesions (animals 1, 11, 111), b-d Data of the animals with nigra lesions: X, 8, 17 (b), 18, 38, 48 (c), 4, 5, 014 (d).

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comotion, but also by components such as learning processes and motiva­ tion. Under constant conditions, each rat shows an individual distribution of lever-pressing rate after more training sessions. This distribution may be described by mean and standard deviation values. Therefore, we ex­ press the lever-pressing rate after treatment in test scores (T scores) (fig. 3). The rat’s postoperative lever-pressing rate scores (each score is the mean value of 4 days) show a decrease of 1-4 standard deviations in comparison with their previous mean values of norm distribution and in comparison to the scores of the control subjects. The tendency of the

Motor Disorders following Substantia nigra Lesions

297

Table II. Classification of the deviation in different instrumental parameters

X 8 17

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Mean deficit

X

X

X

X

X

X

X

X X X

X

X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X

-

X

X

X

X X X

X

Is 3 48

‘A’ times

HFLPR

X

014 5 4

LPR

X

Animal

X

X

X

X

X

X

X X

X

X

X

X

-

X

X



X

X

X

X

X

The order of succession of the rats is based on the lesion size; group A contained the largest lesions, i.e. cell decay, and group C the smallest. The number of X indicates the degree of deviation from preoperative data. LPR = Lever-pressing rate; HFLPR = highfrequency lever pressing rate.

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curves to increase during the 32 days of observation is remarkable. The point during the postoperative course at which the preoperative norm is reached is an important factor in estimating the extent of the lesion. The postoperative normalization refers to the time at which the lever-pressing rate reaches the norm, i.e., 0.5 standard deviation below the former mean value (norm line). The postoperative scores up to the beginning of nor­ malization were taken into consideration in order to classify the degree of disability (table II). Our training program can be regarded as a fixed-interval schedule. F ester and Skinner [4] describe the typical cumulative records of such fixed-interval ratios. After reinforcement, few or no responses may occur and then occur in correlation with the length of the fixed-interval and the amount of reinforcement. At a certain point the animal will respond by a sudden acceleration in the lever-pressing rate until the next reinforcement (fig. 4). Checking the interresponse time in respect to the time course of the program, we can discriminate between two types of interresponse time: (1) The interresponse time between the lever pressing which is reward­ ed and the new start of a task-directed activity, i.e., the first lever pressing after getting food, with the aim to get the next reward, designated as ‘A’

W inkelmuller/N itsch

298 l ig h t

c j

c h

lpr

_ ! _______i i ii

e h

c j

i m if ____________________ i

c j

u r n ___

X

REW ARD

A

'A'TIM E HFLPR

SEC

10

20

30

40

Fig. 4. Schematic drawing of the time course of lever-pressing rate (LPR) and the separation of interresponse time (IRT) in ‘A’ times and high-frequency lever pressing rate (HFLPR).

Fig. 5. Time course of high frequency lever pressing (HFLPR; interresponse times shorter than 1 sec) over 32 days after treatment. The norm value (100%) rep­ resents the mean value of the preoperative data.

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in figure 4. This ‘A’ time consists of the time it takes to eat the pellet and the consideration time. This time depends upon the motivation and train­ ing of the animal; (2) The interresponse time which follows the renewed task-directed ac­ tivity is shorter than the ‘A’ time. An analysis of the interresponse times

Motor Disorders following Substantia nigra Lesions

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of this second type revealed lever pressing at a rate faster than 1/sec. We call this phenomenon high-frequency lever pressing. It shows interindividual differences which are not due to differences in learning performance. The distribution of ‘A’ times is relatively constant in spite of differ­ ences in motivation. The distribution of ‘A’ times is characterized by the medians. Preoperatively, the medians shifted about 6.8 sec. Postoperatively, the medians increased. The shifting was the basis for classifying the lack of performance to react in the test program (table II). To check the lack of repetitive movements, we expressed the postoper­ ative high-frequency lever pressing rates as a percentage of the data be­ fore operation (fig. 5). The critical limit for normalization is 90% of the norm value. The classification of the total time course high-frequency lever pressing is shown in table II.

Discussion

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Many investigators have been able to produce a Parkinson-like syn­ drome in animals by placing lesions, i.e., causing a cell loss, in the pars compacta of the substantia nigra [6, 17], which is known to the source of the Parkinson syndrome in humans. In our experiments, the lesions were restricted to the substantia nigra. If they were located at an extranigral site (especially dorso-medial of the substantia nigra or rostral in the ventromedian tegmentum), they caused cell degeneration in the pars com­ pacta. Bilateral nigra lesions in the monkey have been associated with hy­ pokinesia in motor behavior [19]. However, none of the animals revealed any signs of rigidity. We found motor behavior dysfunctions showing all the criteria of rig­ idity after bilateral lesions in the rat. A reduction of spontaneous activity and disorders of postural reflexes was also apparent. The histological control showed a good correlation of motor dysfunction with the size of lesion in the substantia nigra. A classification of motor disorders after brain lesions made exclusively by behavior observation can only be approximate because the assessment of small changes in behavior is influenced by subjective criteria and can­ not be considered reliable. Instrumental behavior tests offer a possiblity to quantitavely register motor functions. One of our recorded parame­ ters, the lever-pressing rate, showed a decrease in most cases. As the lev­ er-pressing rate is a complex parameter consisting of components other

300

W inkelmüller/N itsch

than motor activity alone, the degree of decrease does not necessarily al­ ways correlate with the size of the lesion and the observed amount of mo­ tor dysfunction. The analysis of interresponse time offers the possibility to obtain parameters for akinesia or rigidity. An increased interval be­ tween receiving a pellet and starting a burst of activity (‘A’ times) is inter­ preted as a special sign for akinesia. All animals exhibiting a marked increase in ‘A’ times also showed a decrease in high-frequency lever pressing. In contrast, animals 3 and X showed marked signs of rigidity and had only very little or no increase in ‘A’ times. The reduction or loss of high-frequency lever pressing can be a special sign for rigidity. Akinesia and rigidity occur if large lesions are placed in the substantia nigra (animals 014, 5, 4 18). Discrete lesions in the medi­ al part of the substantia nigra (animals 3, X) only cause rigidity. The different parameters of the instrumental test cannot be correlated solely with the size of the lesion. However, when such correlations are compiled in a total score for each animal, the degree of instrumental defi­ cit corresponds with the histological finding and the observed motor dis­ orders. The symptoms of rigidity and akinesia are progressive in human Par­ kinson patients. However, when these symptoms are experimentally in­ duced in animals, they exhibit a compensatory tendency as the 32-day ob­ servation period demonstrates. Although rough motor disorders amelio­ rate, the data from the instrumental test still show a marked deviation from normal at the 32nd day, indicating disturbed motor performance.

References

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6 G oldstein , M.; A nagnoste, B.; O w en , W. S., and Battista, A. F.: The effects

of ventromedial tegmental lesions on the disposition of dopamine in the caudate nucleus of the monkey. Brain Res., Osaka 4: 298-300 (1967). 7 G reenfield , J. G. and Bosanquet, F. D.: The brainstem lesions in Parkinson­ ism. J. Neurol. N.S. 16: 213-226 (1953). 8 G uilford , J. P.: Fundamental statistics in psychology and education (McGrawHill, Maidenhead 1956). 9 H assler, R.: Zur Pathologie der Paralysis agitans und des postencephalitischcn Parkinsonismus. I. Psychol. Neurol., Lpz. 48: 387-476 (1938). 10 H assler, R.: Extrapyramidal-motorische Syndrome und Erkrankungen; in v. Bergmann, F rey und Schwiegk Handbuch für innere Medizin; 4. Auf!., vol. 5/3, pp. 676-904 (Springer, Heidelberg 1953). 11 H assler, R.: Physiopathology of rigidity; in S iegfried Parkinsons’s disease: rig­ idity, akinesia, behavior, vol. 1, pp. 20-45 (H. Huber, Bern 1972). 12 J arosch, E. und N itsch , F. M.: Experimente über paradoxe Wirkung von Tran­ quilizern im Tierversuch. Int. Pharmacopsychiat. 1: 168-183 (1968). 13 Jarosch, E.; N itsch, F. M. und L eber, W.: Schrittweise Synchronisation der aufgabenbezogenen Aktivität mit Lichtreizen als Lernprinzip bei Toten köpfäffchen (Saimirí sciureus) in optischen Diskriminierungsversuchen. Zool. Anz. 33: 284-289 (1969). 14 J arosch, E.; L eber, W. und N itsch , F. M.: Versuchssteuerungs- und Registrier­ system für instrumenteile Verhaltensuntersuchungen. Forma et functio 4: 271-282 (1971). 15 P eterson, E. W.; M agoun, H. W.; M c C ulloch, W. S., and L indsley, D. B.: Production of postural tremor. J. Neurophysiol. 12: 371-384 (1949). 16 P oirier , L. J.: Experimental and histological study of midbrain dyskinesias. J. Ncurophysiol. 23: 534-551 (1960). 17 P oirier, L. J. and Sourkes, T. L.: Influence of the substantia nigra on the ca­ techolamine content of the striatum. Brain 88: 181-192 (1965). 18 S impson, B. A. and I verson, S. D.: Effects of substantia nigra lesions on the lo­ comotor and stereotype responses to amphetamine. Nature new Biol. 230: 30-32 (1971). 19 Stern , G.: The effects of lesions in substantia nigra. Brain 89: 449-478 (1966). 20 T rétiakoff, C\: Contribution ä l’étude de l’anatomopathologie du locus niger de Soemmering; these (Jouve, Paris 1919). 21 W inkelmüller. W.: Wirkung von Reizeffekten und Ausschaltungen der Sub­ stantia nigra auf das motorische Verhalten der freibeweglichen Katze. Acta neurochir. 24: 269-303 (1971).

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Dr. W. W inkelmüller, Medizinische Hochschule Hannover, Neurochirurgische Kli­ nik, Postfach 61 01 80, D-3000 Hannover 61 (FRG)

Quantitative registration of motor disorders following bilateral lesions of substantia nigra in the rat.

Appl. Neurophysiol. 38: 291-301 (1975) Quantitative Registration of Motor Disorders following Bilateral Lesions of Substantia nigra in the Rat W. W i...
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