The effect of α-methyl-p-tyrosine and substantia nigra lesions on spinal motor activity in the rat

EUROPEAN JOURNAL OF PHARMACOLOGY 20 (1972) 341- 350. NORTH-HOLLAND PUBLISHING COMPANY

THE EFFECT OF a-METHYL-p-TYROSINE

AND SUBSTANTIA NIGRA LESIONS

ON SPINAL MOTOR ACTIVITY IN THE RAT * I. JURNA, N. RUZDIC **, T. NELL and W. GROSSMANN *** Institut ffir Pharmakologie und Toxikologie der Unversitiit des Saarlandes, D-665 Homburg/Saar, W..Germany

Accepted 18 August 1972

Received 26 May1972

I. JURNA, N. RU~DIC, T. NELL and W. GROSSMANN, The effect ofa.methyl-p-tyrosine and substantia nigra lesions on spinal motor activity in the rat, European J. Pharmacol. 20 (1972) 341-350. The effects of a-methyl-p-tyrosine (aMT) and of bilateral lesioning the substantia nigra on spinal motor activity were studied in rats and compared with reserpine rigidity, aMT, as well as the lesions produced by electrocoagulation or microinjection of 6-hydroxydopamine (6-OH-DA) produced rigidity signalled by tonic activity in the electromyogram during sustained muscle stretch, high a- and low "t-reflex activity, and a short latency of a-reflex discharges. The effect on spinal motor activity of aMT and of bilateral electrocoagulation of the substantia nigra was antagonized by DOPA. Motor disturbances following microinjection of 6-OH-DA into both substantiae nigrae were not influenced by DOPA, metamphetamine or amantadine. Atropine abolished the effect of substantia nigra lesioning by electrocoagulation or by microinjection of 6-OH-DA. On the basis of the results it is concluded that the rigidity produced by reserpine is due to dopamine depletion in the striatum. Spinal motor activity Substantia nigra lesions

a-Motoneurones -r-Motoneurones

1. INTRODUCTION An i.v. injection of a high dose of reserpine ( 5 - 1 0 mg/kg) produces rigidity, tremor, and akinesia in rats (Steg, 1964). Reserpine rigidity is due to c~motor hyperactivity since a-motor discharge is increased, 7-motor discharge is decreased, and the rigidity persists after deafferentation (Steg, 1964, 1966). It most probably results from a depletion of monoamines in the brain because removal of the striatum or lesioning the dorsolateral funiculi of the spinal cord, abolishes the rigidity (Arvidsson et al., 1967). It is assumed that this type of rigidity is not caused by monoamine depletion alone but by a *

Supported by a grant of the Deutsche Forschungsgemeinschaft. ** Supported by a grant of the Eilebrecht-Kemenah-Stiftung. *** From the Institute of Pharmacology and Toxicology of the Ruhr-University, Bochum.

c~Methyl-p-tyrosine DOPA

Atropine Amantadine

change in central monoaminergic and cholinergic interaction in favour of cholinergic dominance. The rigidity is depressed by drugs stimulating monoaminergic transmission (1-DOPA and metamphetamine) and by cholinolytic agents (atropine, biperiden and trihexyphenidyl (Arvidsson et al., 1966; Jurna et al., 1969). Moreover, physostigmine produces a rigidity which resembles reserpine rigidity in all details yet investigated, and this rigidity is also abolished by cholinolytic agents and by 1-DOPA or metamphetamine (Arvidsson et al., 1966; Jurna et al., 1969). For further support o f the view that reserpine rigidity is mainly due to a diminished content of dopamine or noradrenaline in the brain, the effect of a-methyl-p-tyrosine (ctMT) on spinal motor activity was studied, c~MT reduces the level of dopamine and noradrenaline, but not of 5-hydroxytryptamine in the brain (And~n et al., 1966a). This effect o f a M T is due to its inhibitory action of the enzyme tyrosine hydroxylase (Udenfriend et al., 1965), which is respon-

342

I. Jurna et aL, Substantia nigra lesions and spinal motor activity

sible for the formation of DOPA from tyrosine (Nagatsu et al., 1964), and not to interference with the uptake of catecholamines into the stores (Corrodi et al., 1966). To establish that reserpine produces rigidity by depleting dopamine in dopaminergic neurones, which are located in the substantia nigra and send their axons into the striatum, lesions of both substantiae nigrae were produced either by electrocoagulation or by microinjections of 6-hydroxydopamine (6-OHDA). Injections of 6-OH-DA into the substantia nigra of rats have been shown by Ungerstedt et al. (1969) to induce selective anterograde degeneration of the whole nigrostriatal dopamine neurone system.

2. MATERIALS AND METHODS 2.1. General procedure The experiments were performed on 44 Wistar rats of 2 5 0 - 3 0 0 g body weight. All surgical procedures were carried out under halothane anesthesia. Recording of spinal motor activity was performed under light halothane anesthesia. The level of anesthesia employed did not interfere with reflex activity, as tested in some experiments in which the surgical wounds were instilled with procaine and the animal allowed to recover for a short period from anesthesia. The rectal temperature was kept between 36.5 and 38.0°C. 2.2. Lesioning o f substantia nigra The substantia nigra on both sides was located according to the coordinates of the stereotaxic atlas of Fifkova and Marsala (1967). The coordinates used were as follows: anterior +4.0 mm; lateral 2.0 mm; horizontal 9.0 mm (7.8 mm from dorsal surface of the brain). For electrocoagulation, bipolar electrodes (David Kopf NEX-200) were used. The distance between the electrode tips was 1 mm, and the plane between the tips was arranged in parallel with the sagittal plane. Coagulation was made with a current of approximately 15 mA applied for 25 sec (Grass Lesion Maker). The lesions were checked microscopically. Microin]ections of 6-OH-DA hydrobromide were made with a cannula tightly fitting into another cannula which was stereotaxicaUy inserted into the substantia nigra. 6-OH-DA was dissolved in saline with

ascorbic acid added (0.2 mg/ml). On each side, 4/al of the solution containing 8 !lg of 6-OH-DA (calculated as the base) were injected at a rate of 1/al/min. 28 days after electrocoagulation, or 5 - 8 days after the injection of 6-OH-DA were allowed to elapse before the experiments on spinal motor activity were carried out. 2.3. a- and 7-motor activity a- and 3,-motor activity was measured according to the method described by Steg and coworkers (Steg, 1964; Roos and Steg, 1966; Arvidsson et al., 1966). The procedure is illustrated by fig. 1. After laminectomy and opening the dura, the animals were fixed rigidly. A thin filament containing a- and 3'-motor fibres was isolated from the ventral roots supplying the calf muscles (L 6 or S1). The greater part of the central roots remained intact to allow the recording of reflex activity in the electromyogram during muscle stretch. Activity of a- and 3'-motor units was recorded with two pairs of electrodes (RI and R2). The distance between both pairs of electrodes was about 5 mm. After conventional amplification, the potentials obtained with the electrode pair proximal to the spinal cord (R1) were displayed with the upper beam of a dual beam oscilloscope, and the potentials recorded with the electrode pair (R2) placed distally to the proximal pair of electrodes were displayed with the lower beam. Reflex" activity was elicited by electrical stimulation of the corresponding dorsal root (S) with single rectangular pulses of 0.05 msec duration. Stimulation strength was supramaximal for a-reflex discharges, i.e. an increase of the stimulation strength above that chosen did not increase the number of a-reflex discharges. In general, it ranged between 3 - 5 V. Stimulation frequency was 3 pulses per 10 sec. a- and 3'-discharges were distinguished by differences in amplitude, shape and conduction velocity of the potentials, a-potentials are sharp and of relatively high amplitude, whereas 7-potentials are round and of low amplitude. However, since the amplitude of the potentials recorded depends not only on the diameter of the fibres in the filament but also on the resistance of the tissue between active fibres and the recording electrodes, differences in conduction velocity provide a more reliable means of distinguishing between aand 3'-discharges. Potentials in a-axons are conducted

1. Jurna et aL, Substantia nigra lesions and spinal motor activity

343

S

y

c~

a

y

¥

Y

y

[;>_. ~

I 1 I I I ¼ ~..,

R2 2

msec

Fig. 1. Schematic representation of the procedure for recording c~- and 3,reflex activity. A thin f'flament was isolated from the ventral root L 6 or S 1 and placed on two pairs of recording electrodes (R 1 and R2). Reflex activity was elicited by electrical stimulation with the electrode pair S attached to the corresponding dorsal root. The potentials obtained with the electrode pair proximal to the spinal cord (Rl) were displayed on the upper beam, and the potentials with the distal pair of electrodes (R2), on the lower beam of a dual beam oscilloscope. The recording was taken from an experiment performed on a preparation without substantia nigra lesion and drug application, c~- and 3"-discharges are indicated by arrows. Identical potentials on the upper and lower beam are connected by broken (3"-discharges) or unbroken lines (a-discharges). Note that the bias of the broken lines is more pronounced than that of the unbroken lines. This indicates that the time lag between identical potentials on the upper and lower beam is greater for 3'- than for a-potentials. The first downward deflection on both beams is the stimulus artifact. at higher speed than potentials in 7-axons; therefore, the time lag between identical potentials on the upper and the lower beam of the oscilloscope is smaller in the case of a-unit discharge than in the case of "/-unit discharge. This is indicated in the recording of fig. 1 by the different bias of the lines drawn between identical potentials on the upper (R1) and the lower (R2) beam. The recording was taken from an experiment in which no drug had been administered or lesion made. It shows that in the unpretreated rat the number of a-reflex discharges is lower than 7activity (Arvidsson et al., 1966). This method of distinguishing between a- and 7-motor discharges does not allow to state how many individual m o t o r units are involved in the reflex discharge or whether a single unit discharges repeatedly. The drugs, tested for their action against the effect o f aMT or substantia nigra lesions, were applied only once in each preparation. 5 - 6 preparations were used to determine the action of a drug. Before the application o f the drugs, and at constant intervals (1-5, 30 and 45 min) after their injection, 15 to 20 recordings (sweeps) o f reflex activity were made each time, and

a- and 7-discharges per sweep counted. The total duration of each sweep was 10 msec. The number of aand 7-discharges per sweep obtained before and at a given time after drug injection in each preparation of a series of experiments was pooled for statistical evaluation. The latency between the stimulus artifact and the first a-discharge was also measured in each recording. 2.4. Electromyogram The electromyogram was recorded with concentric needle electrodes from the calf muscles at rest and during stretch produced by dorsiflexion o f the foot to determine whether tonic or phasic activity could be elicited by muscle stretch. Tonic activity signalled the presence o f rigidity. No quantitative measurements were performed.

2.5. Drugs The drugs used were a-methyl-p-tyrosine (ethyl ester o f a-methyl-p-tyrosine hydrochloride, Merck, Sharp and Dohme, Rahway, New Jersey); 6-hydroxydopamine (6-hydroxydopamine hydrobromide, Hoff-

344

L Jurna et al., Substantia nigra lesions and spinal motor activity CtMT 500mg/kg

ct

r~fl~x

5' ~

DOPA

lO0rng/kg

12h

L~/AI~,.~,___.

30min

.~9"~t'~'..,.-~,_.

45min

..j.~k'~,-..-_..~ 2 rnsec

electro- ~

~

~] _

myogrom

&~_ "-,

-. ~ t ~ , _ *-,

"

-

20rnsec

Fig. 2. Effect of ~MT and DOPA on spinal motor activity. Upper row of recordings: a- and "),-reflex discharges from ventral root f'flament Sl. Upper beam: potentials recorded with electrode pair proximal to spinal cord. Lower beam: potentials recorded with distal electrode pair. An a- and -r-discharge are indicated by arrows. Lower row of recordings: electromyogram recorded from right (upper beam) and left (lower beam) calf muscle. First column of recordings: recordings made 12 hr after i.p. injection of aMT, 500 mg/kg. Second and third columns: recordings made 30 and 45 min after i.v. injection of DOPA, 100 mg/kg. Note that before DOPA the electromyogxam shows tonic activity during sustained muscle stretch, whereas after DOPA there is only phasic activity elicited by quick stretch. m a n n - L a Roche Grenzach/Baden); reserpine (Sedaraupin ®, C.F. Boehringer & S6hne, Manheim); a 1% solution of DOPA (1-dihydroxyphenyl-alanine) in saline was obtained by heating to 75°C under constant stirring; atropine (atropine sulfate); metamphetamine (d-metamphetamine hydrochloride) and amantadine (amantadine hydrochloride, Merz & Co, Frankfurt, Main). aMT was injected i.p. either in a dose of 500 mg/kg 12 hr before the beginning of the experiment, or in a dose of 50 mg/kg every 4 hr over a period of 20 hr before the beginning of the experiment (total dose: 300 mg/kg). With the exception of 6-OH-DA, all drugs were injected through a cannular inserted into a tail vein.

3. RESULTS 3.1. E f f e c t o f o_MT on spinal m o t o r activity

aMT, 500 mg]kg, injected i.p. 12 hr before the beginning of the experiment produced marked rigidity in the 6 rats tested, which manifested itself by the presence of tonic activity in the electromyogram (fig. 2). a-Reflex activity was found to be higher than 3'reflex activity. The number of a-reflex discharges per sweep was 10.1 -+ 0.2 (mean -+ standard deviation; number o f sweeps counted ( n ) : 112), wheras the number of 3'-discharges was 7.6 +- 0.4. This result cor-

responds with that obtained after an application of reserpine (Arvidsson et al., 1966; Roos and Steg, 1966; Jurna and Lanzer, 1969) or tetrabenazine (Jurna et al., 1969). l.v. injection of DOPA (100 mg/kg) reduced a- and increased 3'-reflex activity. Fig. 2 shows that tonic activity disappeared in the electromyogram as areflex activity decreased. 45 min after the injection of DOPA, the number of a-refllex discharges per sweep had decreased to 5.3 +- 0.2 (n = 112), and the number of 7-reflex discharges had increased to 8.7 -+ 1.4. The change produced in a-reflex activity is statistically significant ( p < 0.001). The state of spinal motor activity after DOPA corresponds to the one observed in untreated rats (Arvidsson et al., 1966; Jurna and Lanzer, 1969; Jurna et al., 1969). It has been reported by Moore et al. (1967) that aMT may cause renal damage leading to polyufia, glucosuria and proteinuria when the drug is injected in a single, high dose ( 2 0 0 - 3 0 0 mg/kg). The 6 rats treated with aMT 500 mg/kg excreted twice as much urine within 12 hr as untreated rats. The glucose concentration in the urine was determined enzymatically and found to amount to about 110 rag%. Thus it is probable that the animals suffered from renal injury. Therefore, experiments were performed on 6 rats in which repeated doses of aMT (50 mg/kg) were administered every 4 hr over a period of 20 hr. The total amount

L Jurna et al., Substantia nigra lesions and spinal motor activi O, %

u)

12-

12-

, ,coogu,o,ioo ~73after 30

3o- V////,t I

O 108-

8-

}

6-

60--

I

t,.~

E r-

O-

I~i 115 30 0 15 f RESERPINE DOPA

I

30

I

/.5

0

l

401 30

2-

2-

I 1

I 115 3~0 1,5 min ATROPINE

~)or@: p <0.01

Fig. 3. Effect of reserpine, DOPA and atropine on a- and -r-reflex discharges after bilateral electrocoagulation of the substantia nigra. Ordinates: number of a- or 3,-reflex discharges. Abscissae: time after the injection of reserpine, 10 mg/kg, DOPA, 100 mg/kg, and atropine, 5 mg/kg. The moment of injection is indicated by arrows and vertical bars. Each point represents the mean calculated on 90-120 sweeps recorded in 6 preparations for each drug. The thin vertical lines in these points give the standard deviation. Filled circles and unbroken lines: a-discharges. Open circles and broken lines: ")'-discharges. The experiments were performed 24-28 days after electrocoagulation. Note that before the injection of DOPA or atropine a-reflex discharge is higher than ~/-reflex discharge.

of aMT administered was 300 mg/kg. In accordance with the finding of Moore et al. (1967), no polyuria and glucosuria was observed after this treatment. As in the experiments performed with the single, high dose of aMT, however, the number of a-reflex discharges was higher than the number of 3'-reflex discharges. The number of a-discharges per sweep was 7.3 -+1.8, and the number of "),-discharges per sweep was 6.2 -+ 1.3 (n = 120). 45 min after the injection of DOPA (100 mg/kg), the number of a-reflex discharges was reduced to 2.7 + 2.1, and the number of 7-discharges had increased to 9.6 -+ 1.1 (n = 118). It is, therefore, unlikely that the changes in a- and3,reflex activity produced by an injection of a high dose of aMT are due to renal damage and its consequences. Blood pressure was recorded under light halothane anesthesia from carotid arteries of 6 rats which had received aMT 500 mg/kg 12 hr before starting the

2010 ~

I

I

I

I 2

I~"~

I

I

I

I

I

rTTJ

I

I

l 2

I

trr~after ~ reserpine

T

I

I

I

3

Ir-'-II coagulation ofter r77~a f t e r

I I o

, '°oogo,o,,on

I I

1

I~ I

r

0

ater

II I I

reserpine

10-

•--

%

oln ater

11.-

345

p

m

e

0 I T I I I f I I I I I I I I 2 3 msec

Fig. 4. Effect of reserpine, DOPA and atropine after bilateral electrocoagulation of the substantia nigra on the distribution of the interval between stimulus artifact and the first a-reflex discharge. The interval is plotted in msec against the frequency of distribution in percent of the total number of determinations (n). All values obtained in the experiments, performed 24-28 days after electrocoagulation of both substantiae nigrae (i.e. before the application of the drugs), are presented in the 3 diagrams by the heavy lined area (n = 237). The values obtained 30 min after the i.v. injection of reserpine, 10 mg/kg (n = 127), 75 min after reserpine and 45 min after the i.v. injection of DOPA, 100 mg/kg (n = 118), and 45 min after the i.v. injection of atropine, 5 mg/kg (n = 116), axe presented by the faint lined - shaded areas in the respective diagrams.

recording. In all animals the mean arterial pressure was above 110 mm Hg. Therefore, the changes in spinal motor activity observed after aMT do not derive from hypotension. 3.2. E f f e c t o f substantia nigra lesions by electrocoagulation In 12 rats, lesions were produced by electrocoagulation in the substantia nigra of both sides. Spinal motor activity was tested 2 4 - 2 8 days after the operation. No changes in behaviour or gross motor activity were observed at that time. However, the electromyogram always showed tonic activity during sustained stretch of the calf muscles. a-Reflex activity was found to be higher than 3'activity (fig. 3), as is the case after an application of reserpine or tetrabenazine (Arvidsson et al., 1966; Roos and Steg, 1966; J u m a and Lanzer, 1969; Jurna

I

346

L Jurna et al., Substantia nigra lesions and spinal motor activity

et al., 1969). The frequency of distribution of the interval between stimulus artifact and the first areflex discharge shows a maximum at 1.4 msec (fig. 4). Similar latencies have been observed after reserpine and tetrabenazine, whereas in the untreated rats reflex latency was 2.0 msec or even longer (Jurna and Lanzer, 1 9 6 9 ; J u m a et al., 1969). Reserpine, 10 mg/kg, was given to 6 operated rats to test whether a- and 3'-reflex activity could be further changed. The result is summarized in fig. 3. Reserpine increased the number of a-reflex discharges, whereas 3'-reflex activity remained unchanged. The effect on a-reflex discharge was not significant. The maximum of the distribution of latencies was shifted from 1.4 msec to 1.0-1.2 msec (fig. 4). DOPA, 100 mg/kg, injected after reserpine reduced significantly the number of a-reflex discharges below the value obtained before the application of reserpine and increased the number of -/-reflex discharges (fig. 3). Furthermore, DOPA increased the interval between stimulus artifact and the first a-reflex discharge (fig. 4). After DOPA, the frequency of distribution of the latencies shows a maximum at 2.0 msec, which is the value that has been obtained in non-pretreated rats (Jurna and Lanzer, 1969; J u m a et al., 1969). Atropine, 5 mg/kg, was given to 6 rats without previous administration of reserpine. It reduced the number of a-reflex discharges and increased the number of 3'-reflex discharges (fig. 3); both effects were significant. The interval between stimulus artifact and the first a-reflex discharge was increased by atropine (fig. 4). After the end of the experiments on spinal motor activity the brains were removed, fixed in neutral 10% formaline, cut and checked microscopically. The lesions were found to comprise the larger part of the substantia nigra, but were not complete. Partial destruction of both substantiae nigrae by electrocoagulation thus produced a disturbance of a- and 3'-motor activity which is similar to that observed after an injection of reserpine (Steg, 1964) or tetrabenazine (Jurna and Lanzer, 1969) and, accordingly, depressed by either DOPA or atropine. In 3 rats the electrodes for coagulation were inserted into the substantia nigra o f both sides without applying current. These sham-operated rats showed low a- and high 3,-reflex activity, as is the case in normal untreated rats (Steg, 1964; Jurna and Lanzer, 1 9 6 9 ; J u m a et al., 1969).

12-

12-

10-

10-

Ct

86-

--~-0---_~___ 0

o ---<)---

-,b - - ' ° -y

"5 r,

2-

2-

E

:3 C

0-0

0-

I

I

L

15

30

/.5

I

I

I

15

30

45

METAMPHETAMINE

DOPA 12-

,0T

10-

8

:5

7

__-6---~---o

~-~- -~ Y

"6 ~'

2-

c=

0-

I

I

I

15

30

/-.5

AMANTADINE

0

I

I

I

0

15

30

I /.5 rain

ATROPINE

~ ) o r (~ : p <0.02

Fig. 5. Effect of DOPA, m e t a m p h e t a m i n e , a m a n t a d i n e and atropine o n a- and 7-reflex discharges after bilateral micro-

injection of 6-OH-DA into the substantia nigra. Ordinates: number of a- or ~,-reflex discharges. Abscissae: time after the injection of the drugs. The moment of i.v. injection of DOPA, 100 mg/kg, metamphetamine, 2 mg/kg, amantadine, 50 mg]kg, and atropine, 5 mg/kg, is indicated by arrows and vertical bars. Each point of the curves represents the mean calculated on 75-100 sweeps recorded in 5 preparations used for testing each drug. The thin vertical lines in these points give the standard deviation. Filled circles and unbroken lines: a-discharges. Open circles and broken lines: 7-discharges. The experiments were performed 5-7 days after microinjection of 6-OH-DA (8 ug in 4 ul). 3.3. Effect of microin]ections into the substantia

nigra of 6-OH-DA Bilateral microinjections of 6-OH-DA into the substantia nigra were performed in 20 rats. a- and 3'reflex activity was determined 5 - 7 days after the operation, when manipulation of the limbs revealed rigidity. In all experiments a-reflex activity was found to be higher than 3'-activity (fig. 5), as is the case after

I. Jurna et al., Substantia nigra lesions and spinal motor activity

347

%

%

d&e, 50 mg/kg, failed to influence a- and 7-motor

50-

50-

t.0-

r~]after {*0I 16-OH-DA

30-

g T ~ a f t er V_/_~DOPA

activity. Both drugs, like DOPA, antagonized the effect of reserpine on a- and 7-motor discharges (Jurna and Lanzer, 1969;Jurna et al., 1970). A t r o p i n e , 5 mg/kg, markedly depressed a-hyperactivity (fig. 2B) and increased the number of 7-reflex discharges, so that 7-activity was higher than a-activity (fig. 5). Tonic activity in the electromyogram was abolished and no rigidity was palpable on dorsiflexion of the feet. The distribution of the latencies was shifted by atropine towards a maximum at 1.8 msec (fig. 6). After atropine, the spinal motor activity was similar to that in untreated rats (Steg, 1964; Arvidsson et al., 1966; Juma and Lanzer, 1969; Juma et al., 1969). The brains of the operated rats were not assessed histologically. Experiments performed on 4 rats which had been treated with bilateral microinjections of saline and ascorbic acid into the substantia nigra yielded results resembling those obtained in experiments on non-pretreated animals, i.e. a-activity was low and 7-activity high, a-reflex latency amounted to about 2.0 msec, and no tonic activity could be elicited in the electromyogram by sustained muscle stretch.

20-

10-

10-

I

I 1

I

1

I

I 2

~ " A aft er metamphetamine

0-

,I

I

I

I

I

..... I 2

I

~'~

I

I

d/1//I] I I 3

I

I

60-

60v/J

~,,

50-

so-

/,0-

r ~ l a f t er /,0I 16-OH-DA

30-

FT)Toffer 30rZ/.d ainu ntadine

20-

20-

10-

10-

0-

I'~lafter I 16-OH-DA

30-

20-

0-

~~[~

11

I

i

I

I

I 2

0-

' ~ l a f t er I 16-OH-DA

I

I

I

I

I

I

I 2

I

I

I

I

I 3

I

I rnsec

Fig. 6. Effect of DOPA, metamphetamine, amantadine and atropine after bilateral microinjection of 6-OH-DA into the substantia nigra on the distribution of the interval between stimulus artifact and the first a-reflex discharge. The interval is plotted in msec against the frequency of distribution in percent of the total number of determinations (n). All values obtained in the experiments performed 5-7 days after microinjection of 6-OH-DA (8 pg in 4 pl) into both substantiae nigrae are presented in the 4 diagrams by the heavy lined area (n = 368). The values obtained 45 min after i.v. injection of DOPA, 100 mg/kg (n = 94), metamphetamine, 2 mg/kg (n = 89), amantadine, 50 mg/kg (n = 81), and atropine, 5 mg/kg (n = 75) are presented by the faint lined-shaded areas in the respective diagrams. administration of a high dose of reserpine (Steg, 1964; Arvidsson et al., 1966). The interval between stimulus artifact and the first a-reflex discharge was short, the frequency of distribution of the latencies showing a maximum at 1.4 msec (fig. 6) which corresponds with the value obtained after reserpine (Juma and Lanzer, 1969). Tonic activity was always present in the electromyogram during sustained stretch of the calf muscles. DOPA, 100 mg/kg, did not reduce a- or increase 3,-reflex activity (fig. 5), nor did it increase the interval between the stimulus artifact and the first a-reflex discharge (fig. 6). Rigidity remained unchanged throughout the experiments performed with DOPA. Similarly, m e t a m p h e t a m i n e , 2 mg]kg, and amanta-

I

4. DISCUSSION a-MT produced a disturbance of spinal motor activity similar to that observed after the application of reserpine or tetrabenazine. The number of a-reflex discharges was higher than the number of 3'-discharges. The relatively high a-reflex activity may be due to an increase either in the number of active motoneurones or in the impulse discharge of single motoneurones. aMT is an inhibitor of tyrosine hydroxylase (Udenfriend et al., 1965) the enzyme which mediates the formation o f DOPA from tyrosine (Nagatsu et al., 1964). If the rigidity and the changes in a- and 7motor activity after aMT were due to a reduced synthesis of dopamine or noradrenaline from DOPA in the brain, exogenous DOPA should abolish the motor disturbance. This, in fact, was the case. Since aMT diminishes the concentration of dopamine and noradrenaline in the brain but not of 5-hydroxytryptamine (And6n et al., 1966a), reserpine rigidity in the

348

L Jurna et al., Substantia nigra lesions and spinal motor activity

rat must be assumed to follow rather from a lack of dopamine or noradrenaline than from a depletion of central monoamines in general. Reserpine rigidity had been shown to be depressed by drugs either facilitating monoaminergic or inhibiting cholinergic transmission (Arvidsson et al., 1966; Jurna and Lanzer, 1969). It has, therefore, been suggested that it results from a disturbed balance between monoaminergic (dopaminergic) and cholinergic mechanisms in the brain. Much evidence has accumulated in favour of the view that the striatal system serves as a site of dopaminergic and cholinergic interaction. For instance, microelectrophoretic application of dopamine to caudate neurones inhibits neuronal activity, whereas similar application of acetylcholine activates the neurones (Bloom et al., 1965; Herz and Zieglg~insberger, 1966; McLennan and York, 1966; 1967; Herz and Zieglg~/nsberger, 1968). Furthermore, systemic application of reserpine or physostigmine increases the activity of striatal neurones activated by glutamate, and the facilitatory effect of both drugs is inhibited by DOPA or atropine (Steg, 1969). Finally, when the striatum, the site of cholinergic dominance after depletion by reserpine of dopamine, is removed rigidity is abolished (Arvidsson et al., 1967). Dopaminergic neurones sending their axons into the striatum are situated in the substantia nigra (And~n et al., 1964; Fuxe et al., 1964; Fuxe, 1965), and interference with this dopaminergic nigro-striatal system has been reported to result in marked motor disturbance (And~n et al., 1966b; Poirier et al., 1966; Goldstein et al., 1967; Ungerstedt, 1968; Ungerstedt et al., 1969). The results presented in this paper show that bilateral lesioning of the substantia nigra either by electrocoagulation or by microinjection of 6-OHDA exerted an effect similar to that observed after the application of reserpine or aMT: a-reflex activity is higher than 3'-reflex activity, and a-reflex latency is very short. This is in contrast to spinal motor activity in intact rats where a-reflex activity is low and 3'activity is high, and the latency of a-reflex discharge is much longer (Steg, 1964; Arvidsson et al., 1966; Roos and Steg, 1966; Jurna and Lanzer, 1969; Juma et al., 1969). The rigidity present in the operated rats may be ascribed to a-hyperactivity. When assessing a- and 3'-reflex activity with the method employed in this study, isolation of filaments containing only few active units is attempted for better discrimination. This includes that a process of

fibre selection is applied so that, in non-pretreated preparations, among the a-motor axons isolated those predominate which respond to reflex stimulation with a relatively long latency, i.e. axons from motoneurones activated polysynaptically. After pretreatment with aMT or bilateral lesioning of the substantia nigra, however, the latency of the first a-reflex discharge was found to be reduced to values which are compatible with monosynaptic activation. To explain the reduction in reflex latency it may be assumed that predominance of cholinergic activity facilitates monosynaptic transmission in the spinal cord. Chlorpromazine has been reported to increase monosynaptic reflex amplitude in curarized cats with an intact neuraxis (Stern and Ward, 1962). Chlorpromazine produces rigidity and a-reflex hyperactivity (Roos and Steg, 1966), which most probably are due to inhibition of noradrenergic (or dopaminergic) transmission in the brain. Although electrocoagulation and microinjection of 6-OH-DA induced the same type of rigidity, DOPA abolished the disturbance of spinal motor activity only after electrocoagulation. The reason for this may be that coagulation destroyed only part of the dopamine neurones in the substantia nigra. The remaining neurones may still have been able to compensate for the deficit when dopamine release was activated by injection of its precursor DOPA. After microinjection of 6-OH-DA, however, virtually all dopamine neurones must have degenerated (Ungerstedt, 1968). Together with the cell bodies, axons and terminals, also the substrates for the formation, storage and release of dopamine disappeared so that DOPA was ineffective. However, this is speculative since the effects of the lesions produced by electrocoagulation or by microinjection of 6-OH-DA were not checked by chemical determinations. A complete break-down of the dopaminergic nigro-striatal system after microinjection of 6-OH-DA into the substantia nigra might also explain the failure of metamphetamine and amantadine to 'normalize' spinal motor activity. Metamphetamine abolishes reserpine rigidity (Juma and Lanzer, 1969), possibly by releasing dopamine. Amantadine stimulates the synthesis and release of dopamine in the striatum (Scatton et al., 1970), which may account for the reserpine antagonism of the drug (Juma et al., 1972). The ineffectiveness of metamphetamine and amantadine in 6-OH-DA-treated preparations indicates an indirect mode of action in reserpine rigidity.

L Jurna et al., Substantia nigra lesions and spinal motor activity

349

Table 1 Conclusions Problem Reserpine rigidity results from central depletion of

j . . D o p a m i n e or noradrenaline or

"'~ Reserpine rigidity results from central depletion of

Answer

5-Hydroxytryptamine This alternative is favoured by experiments with lesioning of nigrostriatal dopamine neurones

.../~Dopamine or Noradrenaline

f

Reserpine rigidity results from central depletion

This alternative is favoured by experiments with txMT

in a specific structure (the striatal system)

or.,...~ in general

Metamphetamine and amantadine antagonize reserpine rigidity by

an indirect action (via dopamine release) or

~

This alternative is favoured by experiments with microinjection of 6-OH-DA

a direct action

A t r o p i n e abolished m o t o r disturbance in b o t h kinds o f lesioned preparations. This is apparently due to the fact t h a t the lesions did n o t affect the site o f action o f atropine, i.e. the cholinergic r e c e p t o r sites. We c o n c l u d e that the p r o b l e m s studied and the answers o b t a i n e d are summarized in table 1. On the basis o f the results it is p o s t u l a t e d that reserpine rigidity in the rat is mainly due to the lack o f d o p a m i n e as a transmitter in the nigro-striatal system which gives rise to cholinergic d o m i n a n c e in striatal cholinergic and dopaminergic interaction.

ACKNOWLEDGEMENTS The authors axe indebted to Dr. Rebholz of HoffmannLa Roche, Mr. Gall of Merck, Sharp & Dohme and to Dr. Scherm of Merz & Co for the kind supply of test substances.

REFERENCES And6n, N.-E., A. Caxlsson, A., Dahlstrtim, K. Fuxe, N.-A. Hillarp and K. Larsson, 1964, Demonstration and mapping out of nigro-neostriataldopamine neurones, Life Sci. 3, 523.

And6n, N.-E., H. Corrodi, A. Dahlstr~im and T. H61~felt, 1966a, Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons, Life Sci. 5, 561. Anti,n, N.-E., A. Dahlstr~m, K. Fuxe and K. Laxsson, 1966b, Functional role of nigro-striatal dopamine neurons, Acta Phaxmacol. (Kbh.) 24, 263. Arvidsson, J., B.-E. Roos and G. Steg, 1966, Reciprocal effects on ~- and ~,-motoneurones of drugs influencing monoaminergic and cholinergic transmission, Acta Physiol. Scand., 67,398. Arvidsson, J., I. Jurna and G. Steg, 1967, Striatal and spinal lesions eliminating reserpine and physostigmine rigidity, Life Sci. 6, 2017. Bloom, F.E., E. Costa and G.C. Salmoiraghi, 1965, Anesthesia and the responsiveness of individual neurons of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine administered by microelectrophoresis, J. Pharmacol. Exptl. Therap. 150, 244. Corrodi, H., K. Fuxe and T. HiSkfelt, 1966, Refillment of the catecholamine stores with 3,4-dihydroxyphenylalanine after depletion induced by inhibition of tyrosine hydroxylase, Life Sci. 5,605. Fifkov~, E. and J. MarSala, 1967, Stereotaxic atlases for the cat, rabbit and rat, in: Electrophysiological Methods in Biological Research, eds. J. Bureg, M. Petran and J. Zachar (Academic Press, New York and London) p. 65 3. Fuxe, K., 1965, Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine terminals in the central nervous system, Acta Physiol. Scand. 64, Suppl. 247. Fuxe, K., T. H6kfelt and O. Nilsson, 1964, Observations on the cellular localization of dopamine in the caudate nucleus of the rat, Z. Zellforsch., 63,701.

350

L Jurna et aL, Substantia nigra lesions and spinal motor activity

Goldstein, M., B. Anagnoste, W.S. Owen and A.F. Battista, 1967, The effects of ventromedial segmental lesions on the disposition of dopamine in the caudate nucleus of the monkey, Brain. Res. 4,298. Herz, A. and W. Zieglg~insberger, 1966, Synaptic excitation in the corpus striatum inhibited by microelectrophoretically administered dopamine, Experientia (Basel) 22,839. Herz, A. and W, Zieglg~insberger, 1968, The influence of microelectrophoretically applied biogenic amines, cholinomimetics and procaine on synaptic excitation in the corpus striatum, Intern. J. Neuropharmacol. 7,221. Jurna, I. and G. Lanzer, 1969, Inhibition of the effect of reserpine on motor control by drugs which influence reserpine rigidity, Naunyn-Schmiedeb. Arch. Pharmakol. 262,309. Jurna, I., C. Theres and T. Bachmann, 1969, The effect of physostigmine and tetrabenazine on spinal motor control and its inhibition by drugs which influence reserpine rigidity, Naunyn-Schmiedeb. Arch. Pharmakol. 263,427. Jurna, I., W. Grossmann and T. Nell, 1972, Depression by amantadine of drug-induced rigidity in the rat, J. Neuropharmacol. 11,559. McLennan, H. and D.H. York, 1966, Cholinergic mechanisms in the caudate nucleus, J. Physiol. (London) 187,163. McLennan, H. and D.H. York, 1967, The action of dopamine on neurones of the caudate nucleus, J. Physiol. (London) 189,393. Moore, K.E., P.F. Wright and J.K. Bert, 1967, Toxicologic studies with a-methyltyrosine, an inhibitor of tyrosine hydroxylase, J. Pharmacol. ExptL Therap. 155,506. Nagatsu, T., M. Levitt and S. Udenfriend, 1964, Conversion of l-tyrosine to 3,4-dihydroxyphenylalanine by cell-free preparations of brain and sympathomimetically innervated tissue, Biochem. Biophys. Res. Commun. 14, 543.

Poirier, L.J., T.L. Sourkes, G. Bouvier, R. Boucher and S. Carabin, 1966, Striatal amines, experimental tremor and the effect of harmaline in the monkey, Brain 89, 37. Roos, B.-E. and G. Steg, 1966, The effect of l-3,4-dihydroxyphenylalanine and d,l-5-hydroxytryptophan on rigidity and tremor induced by reserpine, chlorpromazine and phenoxybenzamine, Life Sci. 3, 351. Scatton, B., A. Cheramy, M.J. Besson and J. Glowinski, 1970, Increased synthesis and release of dopamine in the striatum of the rat after amantadine treatment, European J. Pharmacol. 13, 131. Steg, G., 1964, Efferent muscle innervation and rigidity, Acta Physiol. Scand. 61, Suppl. 225. Steg, G., Efferent muscle control and rigidity, in: Nobel Symposium I, Muscular Afferents and Motor Control, ed. R. Granit (Almqvist and Wiksell, Stockholm) p. 437. Steg, G., 1969, Striatal cell activity during systemic administration of monoaminergic and cholinergic drugs, in: Third Symposium on Parkinson's Disease, eds. F.J. Gillingham and I.M.L. Donaldson (Livingstone, Edinburgh) pp. 26-29. Stern, J. and A.A. Ward, 1962, Supraspinal and drug modulation of the a-motor system, A.M.A. Arch. Neurol. 6,404. Udenfriend, S., T. Nagatsu and P. Zaltzman-Nierenberg, 1965, Inhibition of purified beef adrenal tyrosine hydroxylase, Biochem. Pharmacol. 14,837. Ungerstedt, U., 1968, 6-Hydroxydopamineqnduced degeneration of central monamine neurons, European J. Pharmacol. 5,107. Ungerstedt, U., L.L. Butcher, S.G. Butcher, N.-E. And6n and K. Fuxe, 1969, Direct chemical stimulation of dopaminergic mechanisms in the neostriatum of the rat, Brain Res. 14,461.