Pharmacological characteristics of tremor, rigidity and hipokinesia induced by resperrine ni rat

7/18/2019 Pharmacological characteristics of tremor, rigidity and hipokinesia induced by resperrine ni rat

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europharmacology Vol.

26 No. 9 pp. 1431-1440 1987

0028-3908/87 3.00 + 0.00

Printed in Great Britain. All rights reserved

Copyright 0 1987

Pergamon Journals Ltd

PHARMACOLOGICAL CHARACTERISTICS OF

TREMOR RIGIDITY AND HYPOKINESIA

INDUCED BY RESERPINE IN RAT

F. C. COLPAERT*

Department of Psychopharmacology, Janssen Pharmaceutics, B-2340 Beerse, Belgium

(Accept ed 23 Februa ry 1987)

Summary-The experiments characterized the dose- and time-dependence of parkinsonian motor signs

induced by reserpine in rats and a standardized system of manipulation of animals, evaluation of

symptoms and analysis of data was devised. The assay procedure yielded no more than 0.5, 4.5 and 0.0%

false positives with the evaluation of tremor, rigidity and hypokinesia, respectively. A dose-dependent and

often complete blockade of all three signs was obtained with L-DOPA plus carbidopa (10: 1) as well as

with other classes of pharmacological agents that are used in the treatment of Parkinsons disease, i.e.

direct or indirect dopamine (DA) agonists (amantadine, pergolide, lisuride) and inhibitors of monoamine

oxidase (MAO) (clorgyline, pargyline, deprenyl, tranylcypromine). The inhibitor of the uptake of DA,

nomifensine, and anticholinergics, 5-hydroxytryptamine (5HT) antagonists, histamine antagonists and

tricyclic antidepressants exerted little or no effect. The effects of putative agonists and antagonists at q-

and a,-adrenoceptors were also examined. Yohimbine blocked tremor and rigidity, but not hypokinesia,

at 0.66 and 0.28 mg/kg, respectively. It is suggested that alpha-adrenergic mechanisms and, in particular,

a,-adrenoceptors, may be involved in reserpine-induced tremor and rigidity. Noradrenergic and dopami-

nergic systems can conceivably interact to progressively generate these different motor signs.

Key words: reserpine, Parkinsons disease, motor signs, rat, tremor, rigidity, hypokinesia.

Reserpine induces symptoms resembling those of

Parkinsons disease in humans (Flach, 1955; Kline

and Stanley, 1955) and signs of similar motor dis-

turbance in laboratory animals (Glow, 1959; Windle

and Cammermayer, 1958). In the rat, reserpine in-

duces rigidity of skeletal muscles (Morrison and

Webster, 1973a, b) as well as tremor, postural flexion,

hypokinesia and several other signs of motor

disturbance (Jurna and Lanzer, 1969; Wagner and

Anderson, 1982).

Pharmacological studies of reserpine-induced

motor signs have revealed antagonist activity of

reserpine with L-DOPA and some putative dopamine

(DA) agonists (Goldstein, Barnett and Malick 1975;

Johnels, Steg and Ungerstedt 1978; Jurna and

Lanzer, 1969), with anticholinergics (Jurna, 1976;

Morrison and Webster, 1973a) and with putative

a-adrenoceptor blockers (Morrison and Webster,

1973b; Wagner and Anderson, 1982). However, the

available pharmacological analyses of reserpine-

induced motor signs in rats and other species of

laboratory animals have at least two limitations. One

is that most studies relate only to muscle rigidity (e.g.

Goldstein et al., 1975) or to indirect measurements

thereof (e.g. the stretch response; Johnels and Steg,

1982), while other symptoms have been left largely

unexamined. A second limitation is that the effects

*Present address: Fondax-Groupe de Recherche Servier,

7 rue Ampere, 92800 Puteaux, France.

of DA agonists, anticholinergics, GI-adrenoceptor

blockers and other drugs have been examined in

varied experimental conditions, and a data base

comparing the effects of these different pharma-

cological treatments in a standardized set of condi-

tions, is not available. A further problem is that

several of the motor disturbances induced by re-

serpine are highly variable in time as well as within

and between subjects (e.g. Goldstein et

al.,

1975),

thus making it difficult to define and quantify possible

effects of drugs on the symptoms.

The purpose of the studies reported here was

three-fold. First, the dose- and time-relationship of

some of the motor signs induced by reserpine in the

rat were characterized. Second, a standardized system

of manipulation of animals, evaluation of symptoms

and analysis of data was developed which permitted

that effects of drugs on tremor, rigidity and hypo-

kinesia induced by reserpine be determined and com-

pared in a reliable manner. Third, a series of experi-

ments was conducted in which the effects of L-DOPA,

DA agonists, anticholinergics, a-adrenoceptor block-

ers and various other pharmacological treatments on

these symptoms were examined.

Animals

METHODS

Female Wistar strain rats, weighing 22&250 g,

were used throughout. Observations were carried out


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1432

F. C. COLP ERT

between 1 and 5 p.m. and took place in an air-

conditioned laboratory (21 f 1C; R.H.: 65 f 5C).

Animals were used only once.

Scoring system

The following scoring system was used to evaluate

symptoms induced by reserpine. All evaluations were

carried out by a single examiner who was unaware of

treatment conditions.

Tremor.

The animal was grasped by the trunk and

held in the supine position; possible tremolous move-

ments of the limbs were scored 2 when they were

visible immediately and clearly; score 1 was assigned

when tremolous movements were intermittent and/or

of modest amplitude. Score 0 was assigned when no

tremolous movements could be observed by this

visual method. The animal was then placed back on

the table and the examiner now gently palpated the

four limbs. Tremor was scored 2 if it was detectable

immediately and clearly by this method of palpation.

A score 1 was assigned when the tremor was intermit-

tent and/or of modest amplitude; score 0 indicated

that no tremor could be detected. These manoeuvres

were repeated 3 times in all, yielding 3 visual and 3

palpation scores, The median of the 3 visual scores

and that of the 3 palpation scores were found and a

final single score for tremor was obtained as the

median of these two.

Rigidity.

The animal was grasped by the hind-

quarter and held vertically, head up. Trunk rigidity

was evaluated from the degree to which the trunk

resisted gentle movements in the horizontal plane.

Rigidity of the limbs was evaluated from the degree

to which the limbs resisted gentle bending and

stretching. Score 2: any of these manoeuvres revealed

severe (lead pipe) rigidity, compared to normal

rats. Score 1: any manoeuvre revealed muscle tone to

be increased but less than severe. Score 0: no clear

evidence of increased muscle tone.

Zfypokinesia. The animal was grasped by the trunk,

manipulated for a few seconds and then placed back

on the table. A score 0 was assigned if, in response

to this manipulation, the animal moved some dis-

tance over the table. Score 1: some movements of

head and limbs occurred, but there was no loco-

motion. Score 2: the animal failed to move the head

or the limbs.

Postural jl exion. Flexion of the back was scored

from observing the animal while it rested on the

table. Flexion of limbs and digits was scored after the

animal had been lifted off the table and turned on its

back. Score 2: severe flexion, score 1: flexion was

marked but less than maximal, score 0: no marked

flexion occurred; not markedly different from normal

animals.

Postural immobil i t y.

The animal was lifted off the

table and held in the supine position in the examiners

hand. Righting movements were scored 2 if they were

absent or did not result in actual righting. Score 1:

righting movements were clearly slowed and/or im-

paired, but righting eventually occurred. Score 0:

righting was not markedly impaired.

Reserpine: time-response relationship

Rats were given a subcutaneous injection of either

40mg/kg of reserpine (n = 9) or saline (n = 9).

Tremor, rigidity, hypokinesia, postural flexion of

digits, limbs and back, and postural immobility were

scored at the following intervals after injection, i.e.

20, 40, 60, 80, 100 and 120 min and then every hour

for up to 8 hr after injection of reserpine.

Reversal of symptoms

Ten rats were injected with 40 mg/kg of reserpine,

whereas 10 other animals received a subcutaneous

injection of saline. Sixty minutes after injection, pairs

of rats, consisting of one reserpine- and one saline-

treated animal, were put in a tank which measured

30 x 30 cm and contained water, 30 cm deep at 35C.

The animals stayed in the water for 90 set during

which they were observed.

Reserpine: dose-response relationship

In this and subsequent experiments, evaluations

were limited to the classic triad of symptoms of

Parkinsons disease (Selby, 1968), i.e. tremor, rigidity

and hypokinesia.

Animals were given either saline

(n =

9) or 0.63,

2.5, 10 and 40 mg/kg of reserpine

(n =

9 per dose)

and were confined to individual cages until 80 min

after the injection, which was when evaluations were

carried out.

Ant agonism of reserpi ne-i nduced sympt oms

The animals used in these experiments were fasted

overnight because several of the experiments involved

oral pretreatments; experiments not involving oral

pretreatments also utilized fasted rats for the sake of

comparability.

Pretreatments consisted of the oral or sub-

cutaneous administration of either saline, drug or

drug vehicle. Sixty minutes after this pretreatment, all

animals were injected subcutaneously with 40 mg/kg

of reserpine. Evaluations of tremor, rigidity and

hypokinesia were carried out in the manner described

above 40, 60 and 80min after the injection of

reserpine.

From the time of pretreatment on, the rats were

housed individually. Any single experiment involved

15 animals of which at least one received pre-

treatment with saline; one or several rats received a

vehicle as appropriate, while other rats were pre-

treated with one of several drugs. Drugs were tested

on 7 rats per dose. Vehicles were also tested on 5 or

7 rats; none of the vehicles produced significant

activity and the data for drug vehicle are not re-

ported. A total number of 220 (control) rats received

pretreatment with saline by either the oral or the

subcutaneous route. The drugs and doses tested are

specified below (Results section).


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Reserpine-induced parkinsonian si ns

1433

Int eractions w it h haloperidol . Rats (n = 7 per treat-

ment condition) were treated orally with L-DOPA or

one of the DA agonists specified below, and, at the

same time, were given a subcutaneous injection of

either saline or 2.5 mg/kg of haloperidol. L-DOPA

was given at 640 mg/kg; the other treatments consis-

ted of 0.16 mg/kg lisuride, 160 mg/kg amantadine,

160 mg/kg bromocryptine or 10 mg/kg pergolide.

Sixty minutes after this double pretreatment, all

animals were given a subcutaneous injection of

4Omg/kg of reserpine, and tremor, rigidity and

hypokinesia were evaluated as indicated above.

Drugs

The compounds used were reserpine, L-DOPA

(3,4-dihydroxyphenylalanine), amantadine HCl,

phenylephrine HCl (Janssen Chimica, Belgium),

dexetimide HCl, pirenperone, ritanserin, astemizole,

aceperone, haloperidol (Janssen Pharmaceutics,

Belgium), carbidopa, amitryptiline HCl (Merck,

Sharp & Dohme, U.S.A.), bromocryptine mesylate,

apomorphine HCl, ketotifen fumarate (Sandoz,

Switzerland), pergolide mesylate, lergotrile mesylate

(Eli Lilly, U.S.A.), lisuride maleate (Schering A. G.,

West Germany), yohimbine HCl (Sigma, U.S.A.),

piperoxan HCl (Rhone-Poulenc, France), idazoxan

(Reckitt and Colman, U.K.), tranylcypromine sul-

phate (Smith Kline and French, U.S.A.), clorgyline

HCl (May and Baker, U.K.), pargyline HCl (Abbott,

U.S.A.), methoxamine HCl (Burroughs Wellcome,

U.S.A.), N-(2-chloro-5-(trifluoromethyl) phenyl)-4,5-

dihydro-1H imidazole (St-587) nitrate and clonidine

HCl (Boehringer, West Germany), trihexyphenidil

HCl (Lederle, U.S.A.), atropine sulphate (Mann,

Belgium), biperiden HCl (Knoll, West Germany),

nomifensine (Hoechst A. G., West Germany),

imipramine HCl, desipramine HCl (Ciba-Geigy,

Switzerland), prazosin HCl (Pfizer, U.S.A.), xylazine

HCI (Bayer, West Germany) and (-)-deprenyl

(Semmelweis University, Hungary).

Doses of amantadine, bromocryptine, lergotrile,

lisuride, apomorphine, yohimbine, tranylcypromine,

pargyline,

trihexyphenidil, atropine, dexetimide,

biperiden, ketotifen, imipramine, amitryptiline and

desipramine refer to the salt; doses of other com-

pounds refer to the base. L-DOPA, carbidopa and

bromocryptine were given as suspensions, while

the other compounds were dissolved in water. One

equivalent of tartaric acid was added to solutions of

lisuride, St-587, nomifensine, aceperone and halo-

peridol. Two equivalents of tartaric acid were used

with pirenperone and astemizole and three with

prazosin. Two equivalents of tartaric acid were added

to concentrations of ritanserin of up to 1 mg/ml; to

larger concentrations were added two equivalents of

lactic acid in 20% polypropyleneglycol. Ascorbic acid

was added to solutions of reserpine in an amount

(w/w) which was four times that of reserpine.

Because of limitations in solubility, the 40 mg/kg

dose of reserpine was administered in a volume of

4 ml/100 g body wt; injections of similar volumes of

saline were ineffective in control experiments.

Other doses of reserpine and all other drugs and

vehicles were administered in a volume of 1 mg/lOO g

body wt.

RESULTS

Reserpine: time-response relationship

A dose of 40mg/kg of reserpine induced tremor,

rigidity, hypokinesia, postural flexion and postural

immobility; all 9 rats showed each of these symptoms

to at least some extent at one or several intervals of

time after the injection. Tremor reached its maximal

amplitude within 4Omin, while other symptoms

reached a peak at 60-100 min after injection (Fig. 1).

Tremor, rigidity and flexion of the limbs and digits

decayed from about 3 hr onward and had essentially

disappeared by 8 hrs after the injection. In contrast,

severe hypokinesia and postural immobility persisted

throughout the 8 hr observation period. This latter

resemblance in time course may suggest that both

hypokinesia and postural immobility reflect one de-

fect, i.e. a difficulty in initiating movement. Neither

symptom would seem to be the result of rigidity, since

hypokinesia and postural immobility persisted at

times (i.e. 68 hr) at which rigidity had essentially

disappeared (Fig. 1). After this observation period of

8 hr, the 9 animals injected with reserpine were put in

living cages where they had food and water available

ad li bit um.

Regular checks on subsequent days indi-

cated that the animals remained essentially immobile

and ate or drank little, if anything. One animal died

7 days after injection and all 9 rats were dead after

12 days. In view of the fact that the animals did not

eat or drink and then died after this interval of time,

it is likely that the cause of death was dehydration

and malnutrition.

Reversal of sympt oms

Immediately upon being thrown into the water,

reserpine-treated rats began to swim, much like

normal animals. The tremor, postural flexion and

hypokinesia that were apparent prior to the session

in the water tan%, disappeared abruptly and seem-

ingly completely. After a period of time ranging from

about 10-40 set, some signs became apparent again.

That is, episodes of swimming alternated with epi-

sodes of passive floating during which some flexion of

the limbs and some degree of hypokinesia were

apparent. In none of the rats tested was tremor ever

observed in the course of the 90 set episode during

which the animals were in the water.

Reserpine: dose-response relationship

A dose of

2.5

mg/kg of reserpine induced tremor

and hypokinesia, while rigidity became apparent at

10 mg/kg (Fig. 2). All three symptoms were maximal

or near-maxima1 at 40mg/kg.


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1434

F. c. COLP ERT

,~~.-________---------.

POSTURAL FLEXION BAC,

HYPOKINESIA

Jd1. _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ - -

Ill

0

11

POSTURAL IMMOBILITY

__.____-__ _.______ ___..______

206OlOOl 1

I

I I

I I

min

234567 8 hr

time after injection

Fig. 1. Time-course of motor signs induced by 40 mgjkg of

reserpine in rats. Data points represent the mean (+ I SEM)

of n = 9. Asterisks indicate one-tailed P c .05 (Mann-

Whitney U-test; Siegel, 1956) for differences with a saline

control group (n = 9). Control data are not shown; with

none of the symptoms did the control mean exceed 0.1 at

any time after the injection. Note that symptoms were

measured along an ordinal scale and to compute averages

is a less than perfectly admissible operation when the data

are ordinal (Stevens, 1946). The mean was nonetheless

used here because other measures of central tendency (e.g.

median, modal value) appeared to reflect very poorly the

graded changes in response that occurred as a function of

time after injection.

&-_____.____

HYPOKlNESlA

I II I

0

0.63 25

lb Lo

dose of reserp~ne mgtkg J

Fig. 2. Dose-response curves for reserpine in producing

tremor, rigidity, and hypokinesia in rats. Rats were given a

dose of reserpine (n = 9 per dose) or saline (dose 0; n = 9)

and evaluations were carried out 60 min after the injection.

See also

legend to Figure 1.

Antagoni sm of reserpine-in duced tremor, rigidity and

hypokinesia: control data

In the course of experiments which examined the

effects of various drugs on the symptoms induced by

reserpine, control data were accumulated with 178

rats that received saline by gavage and with

42

animals that were injected subcutaneously with saline

1 hr prior to receiving reserpine. There were no

apparent differences between control data obtained

with these two routes and the data were pooled

(n = 220) to yield the frequency distributions shown

in Figure 3.

Forty minutes after reserpine, 6% of control rats

had a median tremor score 0 and another 7% had a

score of only 0.5; other animals had scores ranging

from 1 to 2. Similar distributions of tremor scores

were obtained at 60 and 80 min after reserpine.

However, the animals that scored 0 at one interval of

time were most often given a score greater than 0 at

another interval and only 1 out of 220 rats (0.5%)

was assigned a score 0 at each of the three intervals.

Rigidity scores appeared to be even more variable

and up to 35% of the animals were assigned a score

0. However, no more than 10 of 220 rats (4.5%) were

assigned a score 0 at each of the three intervals.

Hypokinesia became prominent 60 and 80 min

after reserpine and none of the 220 control animals

was assigned a score 0 at each time interval.

To account for variability within and between

subjects and in order to define criteria of activity of

antagonist drugs, protection from tremor induced by

reserpine was considered to have occurred in animals

pretreated with drug if the median tremor score was

0 at 40, 60 and at 80min after injection reset-pine.


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Reset-pine-induced parkinsonian signs

1435

80-

VI

m

E

70-

; 60-

; 50-

b LO-

2

z

30-

y 20-

% lo-

o-

score

time min 1: LO

TREMOR

RIGIDITY

60 80

LO

60

80

HYPOKINESIA

lj

___.________

0

7

2 0

7

2 0;1

LO 60 80

Fig. 3. Frequency distribution of tremor, rigidity and hypokinesia induced by 40 mg/kg of reserpine in

(control) rats (n = 220) that were pretreated with saline. Observations were made 40, 60 and 80 min after

injection of reserpine, and the distribution of scores is given for each of these three time intervals. Scores

assumed values of 0, 1 or 2. Rigidity and hypokinesia were scored only once at each interval. At each

interval, tremor

was scored 3 times by a visual method, and 3 times by a method involving palpation.

Scores given here represent the median of the median visual score and the median palpation score (see:

Methods).

Protection from rigidity and hypokinesia induced by

reserpine was similarly defined by the animal having

been assigned a score 0 at each of the three

time intervals after injection of reserpine at which

observations were made.

L-DOPA

Carbidopa had little or no effect on tremor and

hypokinesia, but protected up to 43% of the animals

from rigidity. L-DOPA produced dose-dependent

effects on each symptom, but with none of the

symptoms did up to 640mg/kg of the compound

produce a complete effect (Fig. 4).

Combinations of L-DOPA plus carbidopa blocked

all three symptoms. With tremor and hypokinesia,

dose-response curves were orderly and relatively

steep. However, increasing the dose from 0.01 to

40 mg/kg failed to increase the effect on rigidity much

beyond 40%, which was the level of effect that doses

of 4-64 mg/kg of carbidopa alone produced.

Doses producing protection in 50% of the animals

tested were computed according to the method of

Finney (1971) and are reported in Table 1. The results

indicate that the addition of carbidopa increased the

effectiveness of doses of L-DOPA; for example, the

ED, dose for L-DOPA protecting against tremor,

was about 8 times less in the presence of carbidopa

than it was in its absence (Table 1).

Table 1. The ED, values for compounds protecting rats from tremor, rigidity and hypokinesia induced by reserpine; ED, values

(and 95% CL) were computed according to the method of Finney (1971) and are expressed in mg/kg

Comoound (route)

TWll0r Rigidity Hypokinesia

Carbidopa

L-DOPA

L-DOPA plus carbidopa

Amantadine

Bromocryptine

Pergolide

Lergotrile

Lisuride

Apomorphine

Tranylcypromine

Clorgyline

Pargyline

Deprenyl

Phenylephrine

Methoxamine

St-587

Piperoxan

Idazoxan

Yohimbine

>64

>64

>640 606 (35z46). 2-640

(4w38)

0.84 (0.49-I .5) 265 (154458)

I;::; ;:

(p.:.)

(1442) 37

(22-w

110 (64-191)

29

(17-50) 7.2 (4.2-12) >I60

;;:z:; 1.3 (0.75-2.2)

0.46 (0.274.79) 2.4 (1.4-4.1)

7.6 (4.4-l 3)

1.5 (0.8s2.5) >I60

0.080 (0.04ti.14)

0.11 (0.062XI. 18) 1.0 (0.60-1.8)

I:;:;

>40 3.2

(1.9-5.6) >40

(S.C.) 0.60 (0.35-1.0)

0.54 (0.32-0.94) 0.73 (0.42-1.3)

(KC.) 0.31 (0.18-0.54)

0.17 (0.0974.29) 0.081 (0.0474I.14)

(S.C.) 7.8 (4.5-13)

5.0 (2.9-8.6) 5.0 (2.W3.6)

(S.C.) 14 (S&24)

3.2 (1.9-5.6) 14 (8.0-24)

(S.C.) 5.0 (2.9-8.6)

8.7 (5.0-15) >I0

(S.C.) 9.5 (5.5-16)

5.0 (2.9-8.6) >I0

(S.C.) 7.8 (4.5-13)

9.5 (5.5-16) >lO

(S.C.) 12 (6.7-20)

9.5 (5.W6) 240

(S.C.) 1.8 (l&3.1)

2.7 (1.S4.6) >I0

(S.C.) 0.66 (0.38-1.1)

0.28 (O.lWl.48) >I0


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1436

F.

c. COLPAERT

car btdopa L- DOPA

__ __ _ . _ _ . _ .

L- DOPA . carbfdopa

-- .__ ._ _________

E

go-

;: 60-

1003

90.

EO-

70

60-

SO-

LO-

30-

20-

10.

O-

4 16 SL

LO 160640 00100401606325 10 LO160 640

dose mgfkgJ

Fig. 4. Effects of earbidopa, I+-DOPA, and carbidopa plus

L-DOPA on the tremor, rigidity and hypokinesia induced by

resetpine in rats. Rats were given any one of the pre-

treatments indicated by the oral route and, 1hr later, were

injected with 40 mg/kg of reserpine. N = 7 for each dose.

Observations were made 40, 60 and 80

min after reserpine.

Ordinate: percentage of animals that were protected against

reserpine&duced iremor (upper graph),.rigidity (middle

graph) and hypokinesia (lower graph). Combinations of

L-DOPA and carbidopa were used at a dose ratio of IO: 1.

Putative DA agonists

The putative DA agonists amantadine, bromo-

cryptine, pergolide, lergotrile and lisuride antago-

nized tremor in a dose-dependent manner {Fig. S),

but up to 40mg/kg of apomorphine was effective in

at most 2 of 7 animals tested. All the putative DA

agonists also antagonized rigidity. With some com-

pounds (i.e. bromocryptine and apomorphine), the

dose-effect curve was particularly orderly; the effects

of lisuride, however, failed to reach the 100% level.

Pergolide, lisuride and amantadine antagonized

hypokinesia in a dose-depentent manner, while

bromocryptine, lergotrile and apomorphine exerted

little effect.

Computation of ED,-values (Table 1) indicated

that there was no apparent relationship between the

potency of L-DOPA and the putative DA agonists in

antagonizing the three symptoms that were studied.

With L-DOPA, bromo~ryptine, pergolide and lergo-

trile, the (ED,) dose which antagonized rigidity was

from 3 to 95 times greater than the dose which

antagonized tremor, while these two doses were

similar with amantadine and lisuride. With all com-

pounds the dose that antagonized hypokinesia was

greater than that at which tremor and rigidity were

blocked, but the magnitude of the difference in

dose varied greatly between the different compounds

(Table 1).

Monoamine oxidase inhibitors

Subcutaneous injections of tranylcypromine (0.16,

0.63 and 2.5 mg/kg), clorgyline (0.04, 0.16 and

063mg/kg), pargyline (2.5 and lOmg/kg) and de-

prenyl (2.5, 10 and 40mg/kg) antagonized tremor,

rigidity and hypokinesia in a dose-dependent manner

(not shown). The ED,,-values at which each

compound produced the three effects are shown in

Table I.

Miscellaneous compounds

Large subcutaneous doses (i.e. up to 10 and

40 mg/kg) of the anticholinergics trihexyphenidil,

atropine, dexetimide and biperiden consistently failed

to exert any apparent effect on either tremor or

hypokinesia (not shown). All four compounds pro-

duced a partial effect on rigidity, but the effect did not

relate in an orderly way to dose with fhose two

compounds (i.e. dexetimide and biperiden), that were

examined at three or more doses.

The putative 5-HT antagonists pirenperone

(Colpaert and Janssen, 1983; 0.04 and 0.16 mg/kg)

and ritanserin (Colpaert, Meert, Niemegeers and

Janssen, 1985; 0.63, 10 and 160 mg/kg) exerted a

partial effect on tremor; 2 or 7 animals were also

protected against rigidity at 10 and 160 mg/kg doses

of ritanserin. Doses of 10 mg/kg of the putative

histamine antagonists (Niemegeers, Awouters and

Janssen, 1982) astemizole and ketotifen, of the in-

hibitor of the uptake of DA, nomifensine and also of

the tricyclic antidepressants imipramine, amitryp-

tiline and desipramine produced essentially no effect

(data not shown).

Putative adrenoceptor antagonists and agonists

The putative a,-adrenoceptor agonists phenyl-

ephrine (2.5 and lOmg/kg, s.c.), methoxamine (2.5

and lOmg/kg, s.c.) and St-587 (2.5 and 10 mg/kg,

s.c.) prevented the tremor and rigidity, but not the

h~okinesia, at ED, doses ranging from 5.0 to

9.5 mg/kg (Table 1). The q-antagonists prazosin

(0.16, 0.63, 2.5 and 10 mg/kg, p.o.) and aceperone

(2.5 and 10 mg/kg, p.o.) exerted less and/or some-

what more variable effects. The a-agonist xylazine

(s.c.) had little effect at 2.5 mg/kg; 10 mg/kg protected

617 rats from tremor but in addition produced se-


7/10

Reserpine-induced parkinsonian signs

Am ntr dl nc

Bromocrypl l ne Pcrgol l de

1437

Apomorphl ne

,oo_ _ _ _ _ - . _ _ _

go-

60-

70-

60-

Lergotr l l e Ll surt dc

-- _______

I

1

1

0

rvuul l r , , ,

2510 LO160 OOL 0160632. 5 10 LO 160 0010.0L016063i 5 2' 50

dose ( rnglkgl

i

E

z

R

R

G

b

:

Y

I i

;

0

K

I

N

E

S

I

A

Fig. 5. Effects of putative dopamine agonists on the tremor, rigidity and hypokinesia induced by reserpine

in rats. Apomorphine was injected subcutaneously; other drugs were administered by gavage. N = 7 for

each dose. See also legend to Figure 4.

dation and relaxation of skeletal muscles. Clonidine

(0.01, 0.04, 0.16 and 0.63 mg/kg, s.c.) protected from

tremor and rigidity in a dose-related manner, but

sedation and muscle relaxation were apparent at

0.16 and 0.63mg/kg, respectively. Like putative

a,-agonists, the putative a,-antagonists protected

from tremor and rigidity, but not hypokinesia, in a

manner which related in an orderly way to the dose

(Fig. 6). The EDSo values were 12 and 9.5 mg/kg,

respectively, with piperoxan, but only 0.66 and

0.28 mg/kg, respectively, with yohimbine (Table 2).

Int eracti ons w it h haloperidol

Doses of 2.5 mg/kg of haloperidol had little or

no influence (Fisher exact probability; one-tailed

P >

0.05; Siegel, 1956) on the effects of L-DOPA or

the DA agonists on tremor, rigidity and hypokinesia

induced by 4Omg/kg of reserpine (Table 2).

DISCUSSION

The present data extend earlier reports (e.g.

Wagner and Anderson, 1982) that reserpine pro-

duces, in a dose-dependent manner (Fig. 2), motor

signs in rats which morphologically resemble the

symptoms of Parkinsons disease.

Specifically,

40 mg/kg reserpine produced tremor, skeletal muscle

rigidity, postural immobility, hypokinesia and

postural flexion involving the digits, limbs and the

back. Some stimulus conditions can set off a period

of time during which the Parkinsonian patient is

relatively free from symptoms (kinesia paradoxa:


8/10

1438

F. C. COLP ERT

Vohtmbtne

80-

70

60-

50-

LO-

30.

20-

10-

OJ -

0

0;

P1peroxan

l daroxan

25 10 LO OOLO.1606325 10

dose f mgl kgl

Fig. 6. Effects of putative cc,-adrenoceptor antagonists on

the tremor, rigidity and hypokinesia induced by reserpine in

rats. All drugs were given subcutaneously in n = 7 rats per

dose. See also legend to Figure 4.

Babinski, Jarkowski and Plichet,

1921). An abrupt

reversal of symptoms also occurred in the reser-

pinized rats that were put in a water tank. The time

course of the different signs which reserpine produced

in rats were also examined (Fig. 1).

A standardized system of manipulation of animals,

evaluation of symptoms and analysis of data was

devised to obtain a reliable procedure to assay effects

of drugs on the classic triad of symptoms (Selby,

1968) of Parkinsons disease, i.e. tremor, rigidity and

hypokinesia. False positive rates were only 0.5, 4.5

and O.O%, respectively (Fig. 3). L-DUPA

plus car-

bidopa, the treatment of choice in the idiopathic

disease (Quinn, 1984), blocked rigidity, tremor and

hypokinesia with this order of potency (Fig. 4; Table

1). Other direct or indirect DA agonists, that are in

use in treatment (Burton and Caine, 1984), were also

effective, but bromocryptine and lergotrile essentially

failed to effect hypokinesia, and apomo~hine only

prevented rigidity (Fig. 5). The (ED,) doses at which

L-DOPA and the putative DA agonists blocked any

one sign failed to correlate with those at which the

other two signs were prevented (Table I). These

differences may in part relate to the different tnech-

anisms by which these compounds interfere with

dopaminergic neurotransmission.

Anticholinergics had no effect on tremor and hypo-

kinesia but did affect, as in an earlier study (Goldstein

et al. 1975), rigidity, The anticholinergic effect on

rigidity related poorly to the dose and failed to reach

the 100% level. While anticholinergics antagonize the

extrapyramidal symptoms induced by neuroleptics

(Klawans, 1968; Simpson and Sramek, 1981), the

present data are consistent with the fact (Quinn,

1984) that these compounds are of limited value in

the idiopathic disease. Putative S-HT and histamine

antagonists and also tricyclic antidepressants, exerted

little or no effect.

Previous reports (Morrison and Webster, 1973b;

Wagner and Anderson, 1982) suggested that adre-

nergic mechanisms contribute to the rigidity induced

by reserpine and several compounds were examined

which act on alpha-adrenoceptors. At lOmg/kg

doses, the putative a,-agonists phenylephrine, meth-

oxamine and St-587 protected partially against

tremor and rigidity, but also produced overt behav-

ioural depression. A similar picture emerged which

the @,-agonists, clonidine and xylazine which, at

effective doses, also depressed behaviour and pro-

duced muscle relaxation (Colpaert, 1987a). The

q-antagonists, prazosin and aceperone, exerted only

limited effects. A dose-dependent and complete

protection against tremor and rigidity (Fig. 6) was

produced by the cr,-antagonists, yohimbine and piper-

oxan, at doses which had no apparent effects on overt

behaviour. Idazoxan was less effective, however, and

none of the a~-antagonists affected hypokinesia.

That relatively small doses (Colpaert, 1987b) of

the qantagonists yohimbine and piperoxan blocked

Table 2.

Effects of L-DOPA and

putative

DA agonists on the tremor,

rigidityand hypokinesia induced by reserpine in the presence or

absence of haloperidot. Rats were given any one of the pretreatments

indicated by the oral route and also a subcutaneous injection of

either saline (S) or 2.5 mg/kg haloperidol (H);

I

hr later, all animals

were injected subcutaneously with 4Omgjkg of reserpine. Per-

centages of effect against tremor, rigidity and hypokinesia induced

by reserpine are based on n = 7 rats per treatment condition.

Differences between S and H conditions were evaluated by means of

the Fisher test (Siegel, 1956); all (one-tailed) P-values were >O.OS

Percentage of effect

Compound (dose in m&g) Tremor Rigidity

Hypokinesia

L-DOPA (640) +s 57 71

0

+H 29 57 29

Lisuride

(0.16) +s

86 100 I4

+H 71 loci

0

Amantadine (160) +s 100 86 29

+H 86 100 14

Bromocryptine (160) +S 100 100

43

+H 71 86 0

Pergolide (IO) +s 57 86 57

+H 100 43

14


9/10

Reserpine-induced parkinsonian signs

1439

tremor and rigidity induced by reserpine may be

surprising in view of the widely held theory of

DA-deficiency (Schultz, 1984) in Parkinsons disease.

However, extensive behavioural (Antelman and

Caggiula, 1977) and neurophysiological (Woodward,

Moises, Waterhouse, Hoffer and Freedman, 1979)

evidence indicates that noradrenaline (NA) and DA

systems in the brain may interact. Of particular

relevance to the present data are findings that yohim-

bine increases the synthesis and utilization of DA

(Anden and Grabowska, 1976), that piperoxan

increases the turnover of DA in the striatum (Geyer

and Lee, 1984) that presynaptic a,-receptors on DA

nerve terminals in the hypothalamus mediate release

of DA (Ueda, Goshima and Misu, 1983) and that NA

nerve terminals, originating in the locus coeruleus,

regulate the turnover of DA in the striatum (Ponzio,

Hollman and Jonsson, 1981). There also is evidence

that the synthesis (Persson and Waldeck, 1970a,b)

and release of NA (Jackisch, Moll, Feuerstein and

Hertting, 1985) is modulated by DA neurones and

that NA activity modulates the locomotor stimulant

effects of activation of DA receptors in reserpinized

rats (Anden, Stromborn and Svensson, 1973; Dolphin,

Jenner and Marsden, 1976). It is thus conceivable

that a,-antagonists increase DA activity through an

interaction with a,-receptors that are located on NA,

as well as on DA neurones. This is in addition to

evidence (Jackisch et al., 1985; Colpaert, 1987b) that

several of the DA agonists, and also DA itself, may

act directly on a,-receptors. In the present experi-

ments, a massive dose of the DA antagonist halo-

peridol (Janssen, 1967) did not markedly prevent

the blockade of the symptoms induced by reserpine

by L-DOPA and putative DA agonists. It is thus.

suggested that alpha-adrenergic mechanisms, in par-

ticular a,-adrenoceptors, may play an important role

in tremor and rigidity induced by the reset-pine.

There is little evidence at present that NA is

involved in Parkinsons disease (Cools, 1984; Schultz,

1984). The disease is associated with a loss of pig-

mented DA neurons in the substantia nigra (SN; pars

compacta) and a subsequent decrease of DA in the

striatum (Ehringer and Hornykiewicz, 1960); also,

the compensation of the deficit in endogenous DA by

administration of its precursor L-DOPA powerfully

reverses the symptoms (Quinn, 1984). However, the

symptoms of Parkinsons disease develop as a result

of an underlying progression (Selby, 1968) and this

progression does not seem to be governed by a

dopaminergic feedback loop. This is because (i) com-

pensating the DA deficit by means of L-DOPA

reverses already established symptoms but fails to

slow

the

progression of the disease (Horstink, 1984;

Klawans, 1984; Quinn, 1984) and (ii) simulating the

DA deficit by means of DA antagonists elicits symp-

toms (Ayd, 1961; Simpson and Sramek, 1981) but

fails to install progression.

There are possibilities, then, to consider non-

dopaminergic mechanisms in the progression. For

example, NA pathways have a powerful influence on

the collateral sprouting of DA neurones (Tassin,

Lavielle, Herve, Blanc, Thierry, Alvarez, Berger and

Glowinski, 1979). Repair mechanisms, such as

sprouting, could conceivably account for the fact that

extensive lesions of the substantia nigra are on occa-

sions found with humans that never presented par-

kinsonian symptoms (Meyer, 1958). Repair may of

course be deficient in patients that do develop the

disease and who, invariably, have a loss of neurones

in the locus coeruleus (Escouralle, De Recondo and

Gray, 1970; Hassler, 1938; Hornykiewicz, 1982) the

principal source of NA in the brain (Levitt and

Moore, 1979). Noradrenergic mechanisms can thus

conceivably be involved in the repair which compen-

sates (see also: Zigmond and Stricker, 1984) for the

neuronal death (Wright and Whalley, 1984) in DA

systems, which is associated with the normal ageing

process; a deficient control of repair may result in

functional expressions of the deficit in DA (i.e. symp-

toms) that are reversible by L-DOPA but progressive.

These considerations suggest that further animal

research should perhaps be concerned with (i) differ-

entiating effects of drugs on the progressive devel-

opment of symptoms compared to the acute reversal

of established symptoms, with (ii) determining the

neurophysiological and biochemical correlates of the

effects of various pharmcological manipulations on

symptoms induced by reserpine and with (iii) the

neuropathological changes which may perhaps occur

late after injection of reserpine in rats.

Acknowledgements-Mr Patrick De Haes gave expert tech-

nical assistance. This work was supported in part by a grant

from the Instituut voor Wetenschappelijk Onderzoek in

Nijverheid en Landbouw.

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Pharmacological characteristics of tremor, rigidity and hipokinesia induced by resperrine ni rat