Effects of pharmacological manipulation of dopaminergic and cholinergic neurotransmission in genetically dystonic hamsters

European Journal of Pharmacology, 213 (1992) 31-39 ~3 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

31

EJP 52338

Effects of pharmacological manipulation of dopaminergic and cholinergic neurotransmission in genetically dystonic hamsters Wolfgang L6schcr and Gabrielc Frcdow Department of Pharmacology, Toxwology and Pharmacy, School of Veterinary Medicine, Hannot'er, F R. G. Received 2 September 1991, accepted 17 December 1991

In an inbrcd line of Syrian hamsters, attacks of sustained dystonic postures of the limbs and trunk can be initiated by handling or mild environmental stimuli (e.g. new cage). The severity of the dystonic syndrome in these mutant hamsters (gcne symbol dt sz) is age-dependent, with a peak at about 30-40 days of age. A scoring system for grading the type and severity of the dystonic attacks can be used to study the activity of drugs against dystonic movements with individual pre- and post-drug vehicle trials as control. The effects of drugs which alter dopaminergic or cholinergic functions in the brain were studied in selectively bred dystonic hamsters and age-matched non-dystonic controls. The dopamine precursor Icvodopa (injected together with carbidopa) and the dopamine receptor agonist apomorphinc increased the severity of dystonia in hamsters when administered prior to the age of maximum severity of dystonia. A very similar effect was observed with the cholinomimetic pilocarpine. In contrast, the dopamine receptor antagonist haloperidol caused a marked overall reduction in dystonic movements. Anticholinergic drugs, i.e. trihexyphcnidyl and biperiden, increased the latency to onset of the dystonic attack, but did not reduce its severity. No differences were observed between dystonic and non-dystonic hamsters with respect to extent and duration of stereotypies induced by dopaminergic and cholinergic drugs or hypolocomotion and catalepsy produced by haloperidol. The data suggest that dopaminergic hyperactivity might be involved in the pathophysiology of dystonia in dt sz mutant hamstcrs.

Dystonia; Dopamine; Acetylcholinc; Anticholincrgic drugs; Haloperidol; Lcvodopa; Apomorphinc

I. Introduction

The clinical term 'dystonia' is used for relatively common movement disorders characterized by inappropriate prolonged muscle contractions which forcefully distort the body into abnormal postures or repetitive movements (Fahn et al., 1987; Marsden and Quinn, 1990). Both pharmacological and pathological data suggest that the dystonias may be a group of disorders with multiple aetiologies (McGeer and McGeer, 1988). Dystonias are classified in three ways: by age of onset (childhood versus adult), by aetiology (idiopathic versus symptomatic) and by body distribution (focal or segmental versus generalized) (Fahn et al., 1987). Most dystonias are idiopathic, i.e. without any identified pathological cause for the illness (Marsden and Quinn, 1990). Pharmacological treatment of dystonias is unpredictable and often disappointing (McGeer and McGeer, 1988). About 40-50% of children and adults

Correspondence to: W. L/Jscher, Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Biinteweg 17, D-3000 Hannover 71, F.R.G.

with idiopathic dystonia obtain moderate to dramatic benefit from treatment with anticholinergic drugs, such as trihexyphenidyl or biperiden (Fahn and Marsden, 1987). Furthermore, a small population of patients with dystonia responds favourably to the dopamine precursor, lcvodopa (plus carbidopa), or to dopamine agonists, such as apomorphine, while another subset of approximately twice that magnitude responds favourably to oppositely acting dopamine antagonistic drugs, such as dopamine receptor blockers (e.g. haloperidol) and dopamine storage deplctors (Fahn and Marsden, 1987). The efficacy of other drugs in the treatment of dystonia is less well documented and there is a need for development of new drugs for dystonic patients not helped by currently available drugs. One of the major obstacles to the development of rational pharmacological protocols in idiopathic dystonia is the lack of reliable animal models (Lang, 1988). The only model which has been characterized pharmacologically to a sufficient extent is the mutant dystonic (dt) rat (Lorden et al., 1988), probably the most useful model of idiopathic torsion dystonia available to date (McGeer and McGeer, 1988). However, in contrast to

32 torsion dystonia in patients, cholinergic or dopaminergic blockers fail to produce a significant overall reduction in dystonic movements in dt mutant rats (Lorden el al., 1988). Furthermore, the dystonic rat shows a reduced sensitivity to the akinetic effects of dopamine antagonists, such as halopcridol, which is in contrast to clinical experience because drug-induced parkinsonism is one of the most limiting factors in the use of dopamine antagonists in human dystonia (Lang, 1988). Thus, the validity of this model for human dystonia is uncertain (Lang, 19881. L6scher et al. (1989) recently described a mutation of Syrian hamsters with the clinical and electrophysiological characteristics of paroxysmal dystonia, i.e. a typc of dystonia characterized by episodes of sustained gcneralized dystonic contractions of muscles which may bc precipitated by strcss or centrally stimulating compounds, such as coffec (Brcssmann et al., 19881. The mutant hamsters, for which wc proposed the genc symbol dt sz (for detailed description of genetics sec Fredow and lJischer, 1991), display sustained gcneralized dystonic movements and posturcs during stress-inducing situations, e.g. weaning, physical stimulation or change of environmcnt (L6schcr et al., 1989; Fredow and l.,6scher, 19911. Consistent with paroxysmal dystonia in humans (Fahn ct al., 19871, dystonic attacks in the mutant hamsters are followcd by a return to normal with no neurological deficit between attacks (L6scher et al., 1989). Similar to idiopathic dystonia in humans, the dystonic movements in hamsters occur in the absencc of morphological alterations in the brain or spinal cord (Wahnschaffc et al., 1990). In the first pharmacological experiments in dystonic hamsters, drugs, such as benzodiazepines, which facilitate GABAergic neurotransmission were shown to exert potent antidystonic effects (l_,6scher et al., 1989; Fredow and L6scher, 1991), which is consistent with clinical experience in patients with paroxysmal dystonia (Bressmann et al., 1988). For further pharmacological characterization of the hamster model, we examined the susceptibility of the mutant animals to drugs which exert effects on cholinergic and dopaminergic systems in the brain.

2. Materials and methods

2.1. Animals The mutant dt sz hamsters and non-dystonic control hamsters used in the present experiments were obtained by selective breeding as described in detail elsewhere (Fredow and L6scher, 19911. The hamsters were housed in groups of three to five animals in Makrolon plastic cages at an ambient temperature of 23-25°C with a light cycle of 12 h (light on at 7:(10

a.m.), and were fed on Altromin 1320 standard diet (Altromin, Lage, F.R.G.). All experiments were carried out in the forenoon at controlled temperature (2325°C). In all experiments, animal groups consisted of male and female hamsters, since there was no indication for sex-related differences in dystonia or for the effect of drugs on dystonia.

2.2. Drug testing Prior to drug experiments, groups of 9-14 dystonic hamsters were repeatedly challenged by using a triple stimulation technique consisting of (1) taking the animal from its home cage and placing it on a balance; (2) i.p. injection of vehicle (usually saline); and (3) placement of the animals in a new (clean and empty) plastic cage (one animal per cage). Latency to dystonic movemerits was defined as the time from placing the animal in the new cage to the onset of the abnormal movement or posture (see below). The triple stimulation technique resulted in shorter and more reproducible latencies to onset of the dystonic attack than each of the three stimulation components alone, indicating a 'stimulus summation' phenomenon. In view of this stimulus summation and the fact that weighing and injection are necessary components of drug experiments, it was essential to use these components also during control trials. In order to obtain reproducible latencies with the triple stimulation technique and to avoid onset of dystonia during weighing or injection, it was important to keep the time from taking the animals out of their home cage to placing them in the new cage as short and constant as possible. Hamsters that exhibited dystonic movements before placement in the new cage were omitted from the evaluation. Pre-drug testing was started after weaning of the hamsters at the age of 21 days, and was repeated at interstimulation intervals of 2-3 days until the latency and severity of dystonic movements were reproducible. The maximum severity of dystonia was usually reached at an age of 30 days, after which reproducible latency and severity were recorded for about 10 days. After 40 days of age, the severity of the dystonic movements slowly declined, and the latency to onset of dystonic movements markedly increased. Except for some experiments with levodopa, apomorphine and pilocarpine, all drug experiments were performed when the animals were 30-40 days of age. Once the maximum severity of the dystonic attack was reached, the triple stimulation technique used for control and drug experiments induced a typical sequence of dystonic movements that was subdivided into six stages as described by L6scher et al. (1989). For grading of the dystonic movements, the hamsters were observed in the empty cage for 3 h and the severity of the dystonic attack was rated as follows: stage 1, flat-

33 tened ears and flattened posture while walking, preceded by wet-dog shakes, grooming, and rapidly twitching vibrissae; stage 2, facial contortions, rearings with forelimbs crossing, disturbed gait with retarded setting of forepaws, stage 3, stiffened hindlimbs so that the animal appears to walk on tiptoes in a dysmetric hypcrgait; stage 4, loss of balance; stage 5, hindlimbs hyperextended caudally, animal continues to pull itself with the functional forelimbs; stage 6, animal immobilized in a twisted, hunched posture with both hindlimbs and forelimbs tonically extended forward, Straub tail, opisthotonus, alternating unilateral forelimb elevation, swaying movements of the head, and copious red eye mucus and salivation. This final stage persisted for 2-5 h, but rapid recovery occurred thereafter. Not all mutant hamsters progressed through the entire sequence described; the individual maximum stage was usually reached within 45-170 min. Electromyographical (EMG) and electroencephalographic ( E E G ) recordings before, during and after dystonic attacks showed that the onset of the attack coincided with continuous tonic muscle activity and phasic bursts, which were present even when the animals did not move, whereas the EEG showed no abnormalities (l_dischcr et al., 1989). For drug testing, a control trial was undertaken with the triple stimulation techniquc, injecting the vehicle used for drug administration (see below), and latencies to the different stages of the dystonic attack were noted after the animals had been placed in the new cage. Two days later, the drug was administered to the same group of animals and latencies were noted again. Two days later, a control trial with the triple stimulation technique was again undertaken. Hamsters in which the maximum severity of dystonic movements in the pre-drug and post-drug control trial differed by more than two stages were omitted from the drug evaluation. For illustration of drug experiments, individual control and drug data arc shown in order to prevent differences in individual drug responses from being masked by calculation of average values. Indeed, patients with idiopathic dystonia show qualitative and quantitative differences in individual drug responses, the reasons of which are still not understood (Fahn and Marsden, 1987) so that the occurrence of such differences in the hamster model would be interesting in terms of the predictive value of the model. The following doses of drugs were administered to groups of 9-14 mutants hamsters: levodopa, 100 m g / k g i.p. (administered immediately after i.p. injection of 1(1 m g / k g of carbidopa, an inhibitor of peripheral aromatic L-amino acid decarboxylase); apomorphine, 1 m g / k g i.p.; haloperidol, 0.25 and 0.5 m g / k g i.p.; pilocarpine, 50 m g / k g i.p. (administered immediately after s.c. injection of 1 m g / k g of the peripheral anticholinergic N-methyl-scopolamine); trihexyphenidyl, 10 and 20 m g / k g i.p.; and biperiden, 10, 2(1 or 40 m g / k g i.p.

Thesc doses were chosen on the basis of previous studies with these drugs using rats or gerbils (Skirboll et al., 1979; Sanberg, 1980; Turski ct al., 1983; L6scher, 1985; Carvcy et al., 1986). All drugs were also injected in agc-matchcd non-dystonic control hamsters (four to five animals per dose) in order to study if any differcnces in type, extent and duration of behavioural effects produced by manipulation of dopamincrgic and cholinergic neurones existed between dystonic and non-dystonic hamsters.

2.3. Drugs Carbidopa was provided by MSD Sharp & Dohme (Munich, F.R.G.). Haloperidol, pilocarpine (used as hydrochloridc), N-methyl-scopolamine (used as nitrate), trihcxyphenidyl (used as hydrochloridc) and Icvodopa were obtained from Sigma (Munich, F.R.G.). These drugs were freshly dissolved in distilled water (haloperidol, levodopa and carbidopa by means of dilute HCI) before each experiment. Apomorphine (as hydrochloride) and biperiden (as lactate) were used as commercial aqueous solutions and were diluted with water prior to use. The injection volume for all drugs was 5 ml/kg. All doses given in this study refer to the free acid or base forms of the drugs.

2.4. Statistics The significance of differences between control trials and drug trials was calculated with the Wilcoxon signed rank test for paired replicates. A drug effect was only considcred significant when a significant difference was present to both the pre- and post-drug control trial. Data from only one control trial are in the figures.

3. Results

3.1. Lecodopa Administration of the dopamine precursor levodopa (in combination with carbidopa) to mutant hamsters at the age of maximal severity of dystonia exerted no effects on the severity but significantly increased the latency to onset of the dystonic attack (fig. 1). In contrast, when lew)dopa was administered to dystonic hamsters prior to the age of maximum severity of dystonia, the severity of the attack was markedly increased in most animals (fig. I). Again, the onset of the attack was retarded. Furthermore, the latency to the individual maximum stage of dystonia was significantly increased, because higher severity grades were reached in the animals (fig. 1).

34 Levodopo 100 rng/kg. C ~ 10rng/~ in homsters with d ~ = n t se~arity of dysto~o Seventy Isc~e]

l.~tencylSt Irr=nl

5 4

20

///~ o

3

,0oio.

ol.

60' !

1

0

I.~lency mox [n'ml 180 l 160 1 I lt.O 1 ok ns 120 I

i!/

2

Control Drug

0 o

Control

Drug

the extent and duration of these effects between dystonic and non-dystonic animals.

~"'~t~°

401 o" _!o 2OJo' ! o ~ontr~ O----~ug

3.2.

Apornorphine

The results obtained with the dopamine receptor agonist apomorphine were similar to those obtained with levodopa (fig. 2). Thus, no effect on the severity of dystonia was observed in mutant hamsters at the age of maximum severity of the syndrome, although animals reached the maximal stage more rapidly with apomor-

~phine l l m g l k g ] in hemsters w,th different severity of dystonbo n=13

n=9 Loteno/1st Ira,n]

Seventy [scorel 6 5 4 3 2 I 0

z"

J

Lotencyn~x In".n]

r ol o

Sever~y

Latency1st

(score]

[turn]

Lotency mox Irr~nl

20

180 • 160 140 120 100 8O 6O

180'

~6o', 5

120 ~

100

?01~ " 80 I:X0.0

3 2

!p(0.001

Control Drug

20

0

Control

Drug

0

10

]

Control Drug

n:13 Fig. 1. Effect of lew)dopa, administered in combination with the peripheral Dopa decarboxylase inhibitor carbidopa, on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment on (1) the severity of the dystonic movements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two groups of 9 and 13 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with levodopa were 32-35 days old at the time of drug injection in the experiments shown in the upper graph, but 25-27 days old (i.e. prior to the age of maximum severity of dystonia) in the experiments shown in the lower graph. Seizure severity during treatment with levodopa in the experiments shown below was significantly increased both with respect to pre-drug values (illustrated in the figure) and post-drug values (not illustrated).

In addition to effects on dystonia, levodopa induced ataxia, which was observed after 3 - 5 min following injection and lasted up to about 40 min. After about 30 min, all animals exhibited stereotyped sniffing and biting, which lasted for about 60 min. Furthermore, in all animals vomiting occurred after about 60-70 min. The same behavioural effects of dopamine receptor stimulation were also observed in age-matched nondystonic hamsters without there being differences in

0

rls

Controt Drug

Seventy lscorel 6

~

O.

Control

4

Latency mox I~nl

30

180 160 lZ,O 1201 1001 801

3 2

10"

0

;p~O.O01 Control Drug

ns

~201 .

1

Control Drug

Loter~y1st lm,nl

20

0

Control

Drug

::p~&O01

z,0 2O

Drug

5

i

60 401

o.

Control Drug

Fig. 2. Effect of apomorphine on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment on (1) the severity of the dystonic m~wements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two groups of 13 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with levodopa were 32-36 days old at the time of drug injection in the experiments shown in the upper graph, but 25-28 days old (i.e. prior to the age of maximum severity of dystonia) in the experiments shown in the lower graph. Seizure severity during treatment with apomorphine in the experiments shown below was significantly increased both with respect to pre-drug values (illustrated in the figure) and post-drug values (not illustrated).

35

phine than during control sessions. In mutant hamsters prior to the age of maximum severity of the syndrome, apomorphine significantly increased the severity of the attack so that most animals exhibited stage 6. Only one animal showed no increase in severity compared with control trials (fig. 2). In all dystonic animals and all non-dystonic controls, stereotyped licking and biting and hyperactivity were observed 2-5 min after injection. These behavioural alterations lasted for about 20-30 min without there being differences between mutant hamsters and controis.

Pitocorpme 50 mg/kcj • N-Methyl-Scop~aminelrng/kg in hamsters with different severityof dystonio Severity lscoreI

I ntencylst Im~l

61~.~i"---,:.~ 3o 1

i

4

20

'

?

]

:.os

, o

Severity lscorel

Lol:ency1st Ira,hi

Severity

Lotencymax Irnin]

[score]

°-

Controt Drug Severity Iscore]

0 Control Drug

::

Control. Drug Hatoperid~ 0.25mg/kg, n=11 L~encylst Ira,hi

j

:11

5

2010 /

Pl0'05

Control Drug

Latencymox [~n] 180' • 160"

,,/D

\/

,

120 /~t,~ .~' 100

3 2 1

::p(005

0 Control Drug

~x~~.m m

/ i / %

Control

Drug

,got

Control Drug n=14

LotencylSt ~nl

z.Oi 20: n s i';x~ 01 : ~, Control Drug

Hot00endO. 0,5 rng/kg, n: 10 Fig. 3. Effect of haloperidol on dystonic movements in dt sz mutant hamsters. The figure show the effects of drug treatment on (1) the severity of the dystonic movements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two group of 11 and l0 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with haloperidol were 35 days old at the time of drug injection in the experiments shown in the upper graph, and 31 days old in the experiments shown in the lower graph.

oI

:

Control Drug

Latencymox [~n] 180] 1601 1401 p(0.01

1 °i

lO0

80 2 040

!

120t o,e\ !

i

o 'i~r&~m~

~-

.~/i?,, /~// ' ;

10

1 1

18o]

56j ~ 3 0 t. 20

I001q,,N~,p! px(O i 0'5

::

Control Drug

]

Latencymax /min)

ns

0

Control Drug n=10

20t ........ ::o

oj'~Control 4 ! Drug

Fig. 4. Effect of pilocarpine, administered in combination with the peripheral anticholinergic drug N-methyl-scopolamine, on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment on (l) the severity of the dystonic movements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two groups of 14 and 10 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with pilocarpine were 32 days old at the time of drug injection in the experiments shown in the upper graph, and 26 days old (i.e prior to the age of maximum severity of dystonia) in the experiments shown in the lower graph. Seizure severity during treatment with levodopa in the experiments shown below was significantly increased both with respect to pre-drug values (illustrated in the figure) and post-drug values (not illustrated).

3.3. Haloperidol The dopamine receptor antagonist haloperidol at both doses tested (0.25 and 0.5 mg/kg) significantly decreased the severity of dystonia in mutant hamsters at the age of maximum severity of the syndrome (fig. 3). At the higher dose, haloperidol also increased the latency to onset of the attack. It should be noted that 30% of the hamsters did not exhibit a reduced severity at the higher dose of haloperidol, thus pointing to

36 d i f f e r e n c e s in i n d i v i d u a l s u s c e p t i b i l i t y o f d y s t o n i a , s i n c e all a n i m a l s s h o w e d t h c s a m e b c h a v i o u r a l r c s p o n s c to h a l o p c r i d o l ( s c c b c l o w ) . B c c a u s c o f t h c m o r c consist e n t r e d u c t i o n in t h e s e v e r i t y o f d y s t o n i a o b t a i n e d at 0.25 m g / k g h a l o p e r i d o l , t h c l a t c n c y to t h c i n d i v i d u a l m a x i m u m s t a g e was s i g n i f i c a n t l y r e d u c e d (fig. 3). A t 0.25 m g / k g h a l o p e r i d o l , s p o n t a n e o u s l o c o m o t i o n in t h c c a g e w a s r e d u c e d for a b o u t 30 m i n a f t e r injcction in b o t h d y s t o n i c h a m s t e r s a n d c o n t r o l s . A t 0.5 m g / k g , l o c o m o t o r activity was s u p p r e s s e d for a b o u t 60 m i n a n d all d y s t o n i c a n d n o n - d y s t o n i c h a m s t c r s c x h i b ited c a t e l c p s y d u r i n g this t i m e w i t h o u t any e v i d e n c c for d i f f e r e n c c s in t h e e f f e c t i v e n e s s o f h a l o p c r i d o l b e t w e e n normal and affected hamsters.

Seventy {scorel 6 O*O--.O0 5

I--.

4 E.tlI I

:/

~ IO- --:O 3

O~-

"l

Lotencyl st ~n~n]

Latency max [~n] 1801 160

1<°1\ 120 I

20

.:O~, : p(0.05

'

2

ns

1 0

1°i

il

Seventy Iscorel

Control Drug Bipenden 10mg/l
L~tencyl st ban

6

30

iN,,m~

Irr~l 180 160

S / 2

*

ns

10

/.:'-5;. i

1 0

p(0,001

.Severity [se0rel

2 I

~o,A '@

~0 Control Drug

Control Drug Control Drug Tr,hexyphenidyl 10 mg/kg , n :12

Latency max Im,nl

LotencylSt [mml

180]

6

1601

5

I

A

100{ ~. :: 801 q _ ~ : :

p,O.O01

3

2O p¢0.01

3

100 8O 6O

0

0

5 L

@

2

10

]

0~, ~':"-0 li

0

.-',,\ aS~'~..! • '"~

Control Drug

180" 160 Z %01

,max

140 120

I

Lotency mo x iq~,n]

Latency 1st Im nl

6 ~ - - ~ ' - I ° 4 ~ I~ 30 Severity Iscore]

iOOi i \ !as 80 o-\-5,1o ',

20

Control Drug

!

.! :i

I

::

,: / i/I

1201 100 •

ns .....!' . ' • / • \

:,'

?

.

,

.X / / . /

.

c

80: 601 -,, .~..!\;

-V', ~o •

/ s j ' , ">

201 :" 0 ooP~-'-JiPU', Control Drug Control Drug Control Drug Bipenden 20mg/kg, n=9 Fig. 6. Effect of biperidcn on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment on (I) the severity of the dystonic mcwements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two groups of eight and nine hamsters arc shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with biperiden were 38-39 days old at the time of drug injection in the experiments shown in the upper graph, and 32-36 days old in the experiments shown in the lower graph. 0

Z)o/o. , , 1

0

0

oJ

b,,

Control Drug Control Drug Trihexyphenidyt 20mg/kg, n =IL Fig. 5. Effect of trihexyphenidyl on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment on (1) the severity of the dystonic movements and postures, (2) the latency to the onset of dystonic movements ('latency Isr), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from two groups of 12 and 14 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal state recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with trihexyphenidyl were 38 days old at the time of drug injection in the experiments shown in the upper graph, and 30 days old in the experiments shown in the lower graph. Control Drug

3.4. Pilocarpine T h e c e n t r a l l y a c t i n g c h o l i n o m i m e t i c d r u g pilocarpine (injected together with N-methyl-scopolamine to a n t a g o n i z e p e r i p h e r a l c h o l i n o m i m e t i c e f f e c t s ) was i n j e c t e d at a s u b c o n v u l s i v e d o s e o f 50 m g / k g . T h e e f f e c t s o n d y s t o n i a w e r e s i m i l a r to t h o s e o b s e r v e d w i t h l e v o d o p a o r a p o m o r p h i n e . T h u s , t h e s e v e r i t y o f dystonia in a n i m a l s at t h e a g e o f m a x i m u m s e v e r i t y o f t h e s y n d r o m e was not a l t e r e d by p i l o c a r p i n e , b u t m a x i m u m s t a g e s w e r e r e a c h e d m o r e rapidly, i n d i c a t i n g a p r o d y s t o n i c e f f e c t (fig. 4). W h e n p i l o c a r p i n e was i n j e c t e d

37

prior to the age of maximum severity, it significantly increased the severity of the attack in all hamsters. Because of this increase in severity, the time to reach the individual maximum stage was increased. In addition to prodystonic effects, pilocarpinc induced stereotypies, i.e. purposeless chewing and stereotyped sniffing, grooming and yawning, in all animals about 4-7 rain after injection; the stereotypies lasted for about 10-20 rain. About 50% of the animals also showed tremor of the head. After these effects, spontaneous locomotor activity was reduced for about 60-90 min. No differences in the behavioural alterations were noted between dystonic animals and controis.

3.5. Anticholinergic drugs Trihexyphenidyl, injected at doses of 10 or 20 mg/kg, exerted no significant effects on the severity of dystonia in mutant hamsters, but significantly increased the latency to onset of the attack (fig. 5). However, at the higher dosage, the maximum individual stage was reached more rapidly than in control trials. The effects of biperiden were similar. Thus, while the severity of dystonia was not altered, biperiden significantly increased the latency to onset of the attack at 10 or 20 mg/kg (fig. 6). However, at 40 mg/kg this antidystonic effect was lost and animals instead progressed more rapidly to the individual maximal stages (fig. 7). Neither drug induced any adverse effects at the doses administered in dystonic animals or non-dystonic controls.

Seventy '.sc~re~

L~eocy 1st ira,hi

L~ency mox I;'~1 180

30

5 41"

i.

23

i

160

!

140

i

120 100

3I

=:~s

2

~

10

60 ! ns

o J ~

Cor~rot Drug

\,, \ t\\ ~ IP \~

Co~r~ Drug Bipenden 40 mg/kg , n :11

40

Cor~ro~ Drug

Fig. 7. Effect of biperiden on dystonic movements in dt sz mutant hamsters. The figure shows the effects of drug treatment in (1) the severity of the dystonic movements and postures, (2) the latency to the onset of dystonic movements ('latency lst'), and (3) the latency to the maximum stage reached in the individual hamsters ('latency max'). Individual data from a group of 11 hamsters are shown for both control and drug trials. The severity of dystonia is indicated as the maximal stage recorded during the 3-h observation period. Significant differences between control and drug trial are indicated in the figure; n.s. signifies that the differences were not significant. The animals used for the experiments with biperiden were 33-36 days old at the time of drug injection.

4. Discussion

The present data obtained with mutant dt sz hamsters, a new genetic animal model of idiopathic generalized dystonia, show that drugs which increase dopaminergic and cholinergic transmission in the brain exert marked prodystonic effects in these hamsters, while antagonism of dopaminergic transmission produces a significant overall reduction in dystonic movements. In contrast to the prodystonic effect of the cholinomimetic pilocarpine, anticholinergic drugs did not attenuate the severity of the motor syndrome of the mutant hamsters, but retarded the onset of the dystonic attacks. Furthermore, at very high doses both biperiden and trihexyphenidyl exerted prodystonic rather than antidystonic effects. With respect to the behavioural response to manipulation of dopaminergic and cholinergic neurones, there was no indication that dystonic hamsters had an altered susceptibility compared to non-dystonic controls. For instance, in contrast to the dt rat (Lorden et al., 1988), dt sz mutant hamsters did not show an attenuated response to the akinetic and cataleptic effects of haioperidol. There are several lines of evidence which strongly implicate abnormalities in dopaminergic neurotransmission as one pathophysiological factor involved in dystonia. Evidence in favour of a dopamine deficit in dystonia derives from (a) the dramatic therapeutic response to levodopa in cases of idiopathic (childhoodonset) dystonia with diurnal fluctuations (Lang, 1988); (b) studies showing lowered levels of homovaniIlic acid (HVA), the main dopamine metabolite, a n d / o r tetrahydrobiopterin, the cofactor for tyrosine hydroxylase, in cerebrospinal fluid (CSF) of some dystonic patients (De Yebenes et al., 1988); (c) findings of subnormal dopamine levels in the nucleus accumbens and striaturn of brain of patients with idiopathic torsion dystonia (Hornykiewicz et al., 1988); and (d) positron emission tomography (PET) studies showing a significant reduction in L-[nSF]fluorodopa uptake into be caudate and putamen of levodopa-rcsponsive dystonic patients (Sawle et al., 1991). However, the majority of patients with dystonia do not improve after administration of levodopa or dopamine receptor agonists, suggesting that a dopamine deficiency is only involved in a minority of patients with this syndrome (De Yebenes et al., 1988). In dt sz mutant hamsters, levodopa and apomorphine exerted no antidystonic effects, thus demonstrating that these animals are not suited as a model of levodopa-responsive dystonia. Indeed, both levodopa and apomorphine exerted marked prodystonic effects in the mutant hamsters, while haloperidol proved to be a potent antidystonic agent in this model. These data are consistent with clinical reports showing that levodopa or dopamine agonists worsen the symptoms of many patients with dystonia (Lang, 1988). In this re-

38

spect, it should be noted that lcvodopa is the most common cause of drug-induced dystonia (Lang, 1988). Indeed, in a recently developed primate model of dystonia, levodopa or dopamine agonists are used to produce dystonic movements which are virtually identical to those of the human syndrome (Mitchell et al., 1990). In idiopathic dystonia in humans, antidopaminergic agents, such as the neuroleptic haloperidol, are among the most effective therapeutic agents (Fahn and Marsden, 1987). Based on the present data, the dt sz mutant hamsters would be a model of antidopaminergic-responsive dystonia. The prodystonic effects of levodopa and apomorphine and the antidystonic effect of haloperidol would seem to indicate that a dopamine excess rather than a dopamine deficiency is involved in the pathophysiology of the dystonic syndrome in these mutant hamsters. The results of several clinical studies suggest that a state of excessive dopamine activity and additional cholinergic overactivity might bc involved in some forms of dystonia (Lang, 1988); however, direct neurochemical evidence for this suggestion is sparse. The commonest lesions disclosed by brain scan with computed tomography or magnetic resonance imaging or by pathological examination in patients with symptomatic dystonia lie in the basal ganglia, particularly in the putamen (Marsdcn and Quinn, 1990). PET scanning of a patient with hemidystonia with a well-defined left basal ganglia lesion showed enlarged radioactive uptake of [11C] spiperone in that area, suggesting an increased density of dopamine receptors (Quinn ct al., 1985). Postmortem brain studies showed elevations of dopamine (and noradrenaline) levels in the red nucleus (Jankovic and Svendsen, 1987), a brain region which may also be of importance in dystonia (McGeer and McGeer, 1988). The prodystonic effect of the cholinomimetic pilocarpine in mutant hamsters is consistent with the hypothesis that dystonia, at least in part, might be due to an excess of cholinergic transmission (McGeer and McGeer, 1988). It was hypothesized that an anomaly in the degradation of acetylcholine, i.e. in the activity of acetylcholinesterase, may underlie dystonic symptoms. However, no difference in the activity of this enzyme could be detected in CSF from dystonic patients (Ruberg et al., 1988). Furthermore, in postmortem brain studies, biochemical markers for cholinergic neuroncs were within the range of control values (Hornykiewicz et al., 1988). Nevertheless, there is increasing evidence that high doses of anticholinergic drugs, such as trihexyphenidyl, should be considered as the treatment of choice for patients with dystonia (Fahn and Marsden, 1987; Marsden and Quinn, 1990). The efficacy of these compounds can be explained by an imbalance in basal ganglia dopamine/acetylcholine systems (McGeer and McGeer, 1988). However, anticholinergic drugs are not effective in all cases, but only in about 40-50% of

patients with dystonia (McGeer and McGeer, 1988). In the mutant hamsters, trihexyphenidyl and biperiden were not capable of attenuating the dystonic attack, which was in contrast to the potent antidystonic effect of haloperidol, suggesting that excess of dopaminergic rather than cholinergic transmission might be involved in dystonia in this model. In this respect, it should be noted that dystonia is transient in dt sz mutant hamsters, i.e. spontaneous remission and even total disappearance of the disorder occurs after the age of 40-60 days in animals of both sexes (I_/Sscher et al., 1989; Fredow and l_/ischer, 1991). Therefore, as recently suggested for transient paroxysmal dystonia in infancy (Angelini et al., 1988), it seems reasonable that the overall pattern of dystonia in hamsters could be correlated with an imbalance of cortical and basal ganglia synaptogenesis involving the respective neurotransmitters. For instance, experimental data, obtained by means of immunocytochemical procedures, demonstrate in the striatum a continuous modification of the ratio between cholinergic and dopaminergic systems during prenatal and postnatal ontogeny (Graybiel and Newman-Gagc, 1987). A temporary impairment of morphofunctional organization could thus be a reason for transient paroxysmal dystonia as observed in mutant hamstcrs. In conclusion, dystonia in mutant dt sz hamsters is pharmacologically characterized by a high susceptibility to drugs which manipulate dopaminergic and, as previously shown (L6scher et al., 1989; Fredow and L6scher, 1991) GABAergic neurones. Furthermore, we recently reported that antagonists of excitatory amino acidergic neurotransmission exert antidystonic effects in this model (Richter et al., 1991). Neurochemical studies are under way to examine the state of the dopaminergic, GABAergic and glutamatergic systems in the brain of dystonic hamsters in order to identify the sites of neurochemical dysfunction that generate or mediate the condition in these animals. Although the validity of the hamster as a model for human dystonia has to be proven further, the present data indicate that dt sz mutant hamsters can be used to obtain valuable information on the effects of pharmacological agents on dystonic postures and movements.

Ackowledgement The authors are grateful to Mrs. (3. Walkling for technical assistance during the studies.

References Angelini, L., V. Rumi, E. Lamperti and N. Nardocci, 1988, Transient paroxysmal dystonia in infancy, Neuropediatrics 19, 171.

39 Bressman, S.B., S. Fahn and R.E. Burke, 1988, Paroxysmal nonkinesigenic dystonia, in: Dystonia 2, Advances in Neurology, Vol. 50, eds. S. Fahn, C.D. Marsden and D.B. Calne (Raven Press, New York) p. 403. Carvey, R.M., L.C. Kao, C.M. Tanner, C.G. Goetz and H.L. Klawans, 1986, The effect of antimuscarinic agents on haloperidol induced behavioural hypersensitivity, Eur. J. Pharmacol. 120, 193. De Yebenes, J.G., A. Vazquez, A. Martinez, M.A. Mena, R.M. Del Rio, C. De Felipe and J. Del Rio, 1988, Biochemical findings in symptomatic dystonias, in: Dystonia 2, Advances in Neurology, Vol. 50, eds. S. Fahn. C.D. Marsden and D.B. Calne (Raven Press, New York) p. 167. Fahn. S. and C.D. Marsden, 1987, The treatment of dystonia, in: Movement Disorders 2, eds. C.D. Marsden and S. Fahn (Butterworths, London) p. 359. Fahn. S., C.D. Marsden and D.B. Calne, 1987, Classification and investigation of dystonia, in: Movement Disorders 2, eds. C.D. Marsden and S. Fahn (Butterwortths, London) p. 332. Fredow, G. and W. L/Sscher, 1991, Effects of pharmacological manipulation of GABAergic neurotransmission in a new mutant hamster model of paroxysmal dystonia, Eur. J. Pharmacol. 192, 207. Graybiel, A.M. and H. Newman-Gage, 1987, Ontogeny of dopaminergic and cholinergic systems in the basal ganglia, in: Extrapyramidal Disorders in Childhood, eds. L. Angelini, G. Lanzi, U. Balottin and N. Nardocci (Exerpta Medica, Amsterdam) p. I. Hornykiewicz, O., S.J. Kish, L.E. Becker, I. Farley and K. Shannak, Biochemical evidence for brain neurotransmitter changes in idiopathic torsion dystonia (dystonia musculorum deformans), in: Dystonia 2, Advances in Neurology, Vol. 50, eds. S. Fahn, C.D. Marsden and D.B. Calne (Raven Press, New York) p. 157. Jancovic, J. and C.N. Svendsen, 1987, Brain neurotransmitters in dystonia, N. Engl. J. Med. 316, 278. Lang, A.E., 1988, Dopamine agonists and antagonists in the treatment of idiopathic dystonia, in: Dystonia 2, Advances in Neurology, Vol. 50, eds. S. Fahn, C.D. Marsden and D.B. Calne (Raven Press, New York) p. 561. Lorden, J.F., G.A. Oltmans, S. Stratton and L.E. Mays. 1988, Neuropharmacological correlates of the motor syndrome of the genetically dystonic (dt) rat, in: Dystonia 2, Advances in Neurology, Vol 50, eds. S. Fahn, C.D. Marsden and D.B. Calne (Raven Press, New York) p. 277. L/~scher, W., 1985, Influence of pharmacological manipulation of inhibitory, and excitatory neurotransmitter systems on seizure

behavior in the Mongolian gerbil. J. Pharmacol. Exp. Ther. 233, 204. L6scher, W., J.E. Fisher, Jr., D. Schmidt, G. Fredow, D. H6nack and W.B. lturrian, 1989, The sz mutant hamster: a genetic model of epilepsy or of paroxysmal dystonia?, Movement Dis. 4, 219. Marsden, C.D. and N.P. Quinn, 1990, The dystonias, Br. Med. J. 300, 139. McGeer, E.G. and P.L. McGeer, 1988, The dystonias. Can. J. Neurol. Sci. 15, 447. Mitchell, l.J., R. Luquin, S. Boyce. C.E. Clarke, R.G. Robertson, M.A. Sambrook and A.R. Crossman, 1990, Neural mechanisms of dystonia: Evidence from a 2-deoxyglucose study in a primate model of dopamine agonist-induced dystonia, Movement Dis. 5, 49. Quinn, N., G. Bydder, N. Leenders and C.D. Marsden, 19885. Magnetic resonance imaging to detect deep basal ganglia lesions in hemidystonia that arc missed by computerized tomography, Lancet ii, I(X)7. Richter, A., G. Fredow and W. L6scher, 1991, Antidystonic effects of the NMDA receptor antagonists memantine, MK-801 and CGP 37849 in a mutant hamster model of paroxysmal dystonia, Neurosci. Lett. 133, 57. Rubcrg, M., A. Villageois, A.-M. Bonnet, F. Rieger, Y. Agid and S. Fahn, 1988, Acetylcholinesterase and butyrylcholinesterase in cerebrospinal fluid from patients with dystonia, in: Dystonia 2, Advances in Neurology, Vol. 5(I, eds. S. Fahn, C.D. Marsden and D.B. Calne (Raven Press, New York) p. 211. Sanberg, P.R., 1980, Haloperidol-induced catalepsy is mediated by postsynaptic dopamine receptors, Nature 284, 472. Sawlc. G.V., K.L. Leenders, D.J. Brooks, G. Harwood, A.J. Lees, R.S.J. Frackowiak and C.D. Marsden, 1991, Dopa-responsive dystonia - [F-18]Dopa positron emission tomography, Ann. Neurol. 30, 24. Skirboll, L.R., A.A. Grace and B.S. Bunney, 1979, Dopamine autoand post-synaptic receptors: electrophysiological evidence for differential sensitivity to dopamine agonists, Science 206, 80. Turski, W.A., S.J. Czuczwar, Z. Kleinrok and L. Turski, 1983, Cholinomimetics produce seizures and brain damage in rats, Experentia 39, 1408. Wahnschaffe, U., G. Fredow, P. lleintz and W. L6scher, 1990, Neuropathological studies in a mutant hamster model of paroxysmal dystcmia. Movement Dis. 5, 286.