Dyskinetic effects of intrastriatally injected GABA-transaminase inhibitors

Pergamon Press

Life Sciences, Vol. 25, pp . 1103-1110 Printed in the U.S .A .

DYSRINETIC EFFECTS OF INTRASTRIATALLY INJECTED GABA-TRANSAMINASE INHIBITORS . M .M . Robin, M.G . Palfreyman and P .J . Schechter Centre de Recherche Merrell International - 16, rue d'Ankara 67084 - Strasbourg Cedex, France, (Received in final form August 14, 1979) Summary Injection of GAZA antagonists into the striatum of rats induces abnormal involuntary movements that are blocked by increasing GABA levels in this area . Attempts to increase GARA by intrastriatal (i .s .) injection of GABA-transaminase (GABA-T) inhibitors surprisingly induced identical dyskinesias . This property was shared by all GABA-T inhibitors tested except ethanolamine-0-sulphate . This dyskinesia is easily blocked by i .s . injection of GARA and muscimol, as well as by intraperitoneal pretreatment with the GABA-T inhibitors themselves . These observations suggest that some GABA-T inhibitors may behave as GABA antagonists when locally applied in the brain at high concentrations . Dyskinetic disorders in man have been attributed to a hyperdopaminergic activity or to cholinergic-dopaminergic imbalances in the striatum (1,2) . In fact, very little is known of the etiology of such syndromes, and attention has recently been drawn to the role of GARA in dyskinesias (6,7) . Postmortem studies in humans clearly indicate that gabaergic and cholinergic functions are significantly reduced in the striatum of patients with Huntington's chorea, whereas dopaminergic activity is generally unchanged . GARA involvement in dyskinesia is further supported by animal studies where intrastriatal (i .s .) injection of GARA antagonists, e .g . picrotoxin, was shown to induce dyskinesia whereas GAZA and GABA agonists blocked this effect (8,9,10,11) . Correlation between dyskinetic disorders in humans and rats is not yet clear ; nevertheless an involvement of GABA in the human dysfunction is probable . To increase gabaergic function is now considered a promising approach to suppressing dyskinesia in man. Two potent enzyme-activated inhibitors of GABA-transaminase (GAZA-T), Y-acetylenic GARA and Y-vinyl GARA, have been synthesized in our center and shown to be active anticonvulsants in several animal models (12,13,14) . We found that intraperitoneal (i .p .) pretreatment with these two compounds was without effect on picrotoxiuinduced dyskinesia . Expecting a greater local GARA increase we injected them directly into the striatum of rats ; these developed abnormal movements identical to those observed after picrotoxin injection . Material and Methods Y-Acetylenic GARA (GAG, RMI 71,645), Y-vinyl GARA (GVG, RMI 71,754), gabaculine HC1, isogabaculine trifluoromethane sulfonate (RMI 71,932) and ethanolamineO-sulphate (EOS) were-synthesized in our center as previously described (15,16, 17,18,19) . Aminooxyacetic acid hemichloride (AOAA) and GARA were purchased from Sigma Chemical Company, St Louis, U.S .A . Picrotoxin was obtained from Aldrich-

0024-3205/79/131103-07502 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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Europe, Beerse, Belgium and .muscimol monohydrate from Research Organics Inc ., Cleveland, USA, All drugs were dissolved either in saline or distilled water and the pH adjusted in the case of aminooxyacetic acid to 7 .5 . Male Sprague-Dawley rats (Charles River, France) weighing 300-350 g were implanted bilaterally with plastic guide-cannulae at stereotaxix coordinates A +7,4, L ±3 .0, V +1 .0 according to Pellegrino and Cushman's stereotaxic atlas (20) . After a recovery period of at least 5 days, freely moving animals received an injection into the striatum using a Hamilton microsyringe connected via a long polyethylene catheter to a needle of 0 .3 mm external diameter . Volumes of 2 or 3 Pl, sometimes 4 pl when drug solubility required it, were injected at a rate of 1 1t1 per 30 sec . The needle was withdrawn 15 sec after completion of injection. Animals were then placed in individual plastic cages for observation . Three different scoring systems were used . They were chosen according to the needs for a qualitative or accurate quantitative observation, and to the short or long-lasting duration of the drug action . Dose-response curves for the dyskinetic effects of GAG and GVG (Fig .1) were obtained with a quantitative scoring : eight different symptoms including facial signs and body signs were estimated from 0 to 3 points over a 6 hour observation divided into 9 periods . The maximal theoretical score was 216 points per animal . When a precise estimation was not required (Fig . 2 and Table 1), dyskinesia was described as "no", "mild" or "high" and antagonism was described as "no", "partial" or "complete" blockade . Antagonism of GAG or GVG-induced dyekinesia (Fig . 3 and Table 2)was studied during a 3 hour period beginning just after the intrastriatal injection and divided into six periods of 30 min . Each animal was scored from 0 to 6 according to the nature, intensity and frequency of the movements . The scores obtained within the first 30 min were computed separately and termed "initial dyekinesia': The scores obtained during the whole observation were termed "global dyskinesia" . Protection was expressed as percent of control scores . All quantitative effects were analysed using the Mann-Whitney U test for nonparametric values . Results Dyskinetic effects of unilateral intrastriatal injection of GAG and GVG : At low doses, both GARA-T inhibitors induce oro-facial dyekinesia consisting of grimacing, chewing, gnawing, cheek tremor and sometimes tongue protrusion . Higher doses affect the whole body and signs are generally localized to the side opposite to the injection . Body dyskinesia includes contralateral front limb tremor which develops into myoclonic twitches, at times extending to the hind limb . Lateral head and/or body jerks appear and may induce falling of the rat onto its injected side and tonic rolling around its longitudinal axis . Salivation is sometimes observed with high doses . Dose-response curves were established for GAG and for GVG (Fig . 1) . GVG appears to be approximately 12 times less potent than GAG in inducing abnormal movements when injected intrastriatally . Even with highly dyskinetic doses, most animals recovered normal behaviour within 4 days and very few deaths were observed .

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70 60 e0 40 a u F

30

Y p

20

G

12 8 4 0

O GVG

2

S

.L. l0

20 DOSE

à 90 100 200 ( ymol x 10"= )

500

1000

FIG . 1 Dose-response of unilateral intrastriatal injection of GAG and GVG in rats (Each point - mean ± S .E .M . of 7 to 10 rats)

WINN aw ~tr

ßew aaylnd +OABA 2ymo1

rw O No bleo(ods

. ß~ß ody~a+~ bbxiein~d oOl~wld

L.~ OW 2.Symd+ OABA 4ymo1

OVO 9yn1d + OAlA 4yng1

® Rrlld eloelads

a Cas0Yls bbdlef

aw cay~a + AAusebed oo0ynd

L OVO end r IAu~oOßynel

FIG . 2 Antagonism of dyskineeia induced by intrastriatal injection of GAG and GVG by subsequent local injection of GABA and muscimol .

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Protection against dyskinesia by intrastriatally injected drugs (Fig .2) Using t e ose-response curves, we chose doses of the two compounds which induced approximately the same intensity of dyskinetic signs . Thus, GAG was injected in 0.5 limol amounts (63 .5 pg/3 pl) and GVG was used in doses 5-18 fold greater i .e . 2.5-9 pmol (322 .5-1161 jig) . Picrotoxin-induced dyskinesia has been reported to be antagonised by concomitant i .s . injection of GABA (10) . We confirmed this observation and found that muscimol, a GARA agonist (21), was also effective in blocking picrotoxin-induced dyskinesias . GABA (2-4 pmol) and muscimol (0 .01-0 .03 pmol) injected i .s . were also effective against both GAG and GVG-induced dyskinesias at a time when these were clearly established (Fig . 2) . Abnormal movements always disappeared within 1 to 5 minutes but sometimes reappeared after 15 min to 2 h .

PROTECTION AGAINST GAG-INDUCED DYSKINESIA BY GAG 100 m0/k0 Lp.

PROTECTION AGAINST GVG INDUCED DYSKNcd1A BY GVG IOOOmg/k014 .

z

0 u

W Kd

TIME AFTER

PRETREATMENT (hr)

FIG . 3 Antagonism of intrastriatal GAG (0 .5 umol) and GVG (6 pmol) induced dyskinesia by pretreatment with intraperitoneal GAG or GVG . (n-7 to 20 rats for each point) . Control values for "initial dyskinesia" (o) are 1 .36 t 0 .24 for GAG (mean ± S .E .M . ; n-36) and 2 .60 ±0 .23 for GVG (n=47) Control values for "global dyakinesia" (e) are 15 .03 ±2 .08 for GAG and 16 .02 ±1 .10 for GVG. * p < 0.05 * * p < 0 .01

Protection against dyskinesia by intraperitoneal pretreatment with GAG and GVG (Fig . 3) : Since local application of GABA protects against dyskinesias, we tried to achieve protection by systemic administration of compounds increasing brain GABA . Intraperitoneal injection of GAG and GVG produces a large and sustained increase in brain GABA (22,23), they were thus tested against their own dyskinetic effects . Rats were injected i .p . with GAG 100 mg/kg or GVG 1000 mg/kg at various times before i .s . injection of 0 .5 pmol GAG or 6 pmol GVG . Dyakinesia was then compared to control rats injected i .p . with saline at the same times . Both GAG and GVG protected against their own dyskinetic effects caused by i .s . injection . A 3 to 6 fold increase in total brain GABA was as effective as 2-4 umol of GABA (206-412 pg) applied intrastriatally .

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Effect of intrastriatal injection of other GABA T inhibitors (Table 1) Gabaculine, isogabaculine and AOAA injected unilaterally into the striatum were found to induce the same dyskinesia .a s GAG and GVG . Ethanolamine-0-sulphate, in doses up to 375 1tg (2 .66 ]fmol), never induced any abnormal movements . Neither AOAA nor EOS caused obvious sedation, but high doses of the former induced convulsions in some animals . TABLE ] Dyskinetic Effect of Intrastriatal Injection of GARA-T Inhibitors . GABA-T Inhibitor

Dose

(hmol)

Gabaculine

1 .14x10 1 .14x10

No . of dyskinetic/ No . of treated rats

3

Intensity

0/3

-2

0/3

1 .14x10 1 1 .4

0/3 4/9

High

Isogabaculine

0.69

5/6

High

Aminooxyacetic acid

0 .23 0 .92 4 .6

0/3 1/3 1/6 t2/6 4/7 2/4 t2/4

Mild Mild High High Mild High

5 .5 6 .9 Ethanolamine-0sulphate

up to 2 .66

0/24

Protection against dyskinesia by intrastriatal injection of GAB&-T inhibi tors (Table 2) : EOS, which does not induce dyakinesia when injected into the striatum, was found to be effective against GVG-induced dyakinesias when applied intrastriatally 24 hours prior to the i .s . injection of 6 }imol GVG . Similarly, a low (0 .5 Pmol) dose of GVG intrastriatally, which induces only a slight and shortlasting dyskinesia, was shown, when injected 28 hours before the next injection, to protect against the dyakinesia induced by a subsequent high dose of GVG. TABLE 2 Protection Against Dyskinesia Induced by GVG (6pmol i .s .) by Prior Intrastriatal Injection of GARA-T Inhibitors .

Pretreatment

N

Saline EOS 2 .66 Umol

5 5

Saline GVG 0 .5 Umol

7 10

N-No . of rats

Initial d a inesia Protection Scores 4 .8t0 .7(SEM) 0 2 .1 0 .1

t 0.8 t 0.1

* * p < 0 .01

Glo al d skin Scores Protection

100 . .2 **

24 .8 t 1 .4 7 .6 t 2 .4

69 Z **

95 %**

16 .6 t 3 .3 5 .3 t 2 .0

68 Z

* p <

0 .05

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Discussion A variety of substances have been reported to induce dyskinetic movements upon intrastriatal injection in rats (3,4,5,8,9,10,11) . Most of these agents are known or suspected to be antagonists of gabaergic mechanisms (9,24,25,26,27, 28,29) . The remainder influence other brain neurotransmitters and in some cases dyskinetic properties may be rationalized on the basis of indirect inhibitory effects on the gabaergic system via known neuronal interactions involving different neurotransmitters . Indeed, the dyskinesia produced by picrotoxin (5,8, 10,11), bicuculline (10), D-tubocurarine (5), 1-(3,4-dihydroxyphenyl) piperazine (9), allylglycine (8,11), and carbachol (5,10) could be reversed by augmenting GARA function e .g . intrastriatal injection of GARA . The appearance of dyskinetic movements after intrastriatal application of potent inhibitors of GAGA-T seems paradoxical, particularly since this could always be reduced or blocked by increasing striatal gabaergic function . This could be achieved by subsequent i .s . injection of GARA and muscimol, by i .s . pretreatment with EOS or with a low dose of GVG, or by i .p . pretreatment with GABA-T inhibitors . One possible explanation for the dyskinetic effects of the GARA-T inhibitors might involve their effects on glutmmic acid decarboxylase (GAD) activity . AOAA and GAG have been shown to inhibit this enzyme in vitro and in vivo (30,31,22) . r On the other hand, GVG, gabaculine and isogabaculí nehave no effects on GAD the activity in vitro (16,32,33) but can decrease activity of this enzyme when large doses are given in vivo (23,34) . Hence, similar to allylglycine and thiosemicarbazide, the GABA-T inhibitors could produce dyskinesia via GAD inhibition . This is unlikely, however, since we found in preliminary experiments, that i .s . injection of EOS produces identical low degrees of GAD inhibition as GVG in areas surrounding the injection site (Robin et al ., unpublished observations) but does not provoke dyskinetic movements . In addition, the GARA-T inhibitors produce these abnormal movements within a few minutes after injection, similar to intrastriatal picrotoxin and bicuculline (5,11), whereas local injection of GAD inhibitors requires approximately one hour before dyskinetic signs appear (5,11) . These results rather suggest that GABA-T inhibitors have two opposite actions on the gabaergic system : they increase brain GABA concentrations by inhibition of its catabolism and block gabaergic function, perhaps by an action on the GARA receptor, this latter effect occurs at higher doses and is reversible . A GARA receptor antagonism is also suggested by the finding that the inhibition of nerve cell firing by GARA could be reversed by iontophoretic application of GAG (35) . The GARA antagonistic properties of the GABA-T inhibitors may be related to their close structural similarity to GARA . However, EOS, whose structure is also similar to that of GABA, is devoid of this antagonistic property . Systemic administration of GAG, GVG, gabaculine, isogabaculine and AOAA produce sedation in laboratory animals (36) . When high doses of GAG, gabaculine, isogabaculine and AOAA are given, this sedation is interspersed with periods of an excitatory syndrome (36), many elements of which resemble the dyskinetic movements described here . GVG rarely produces this excitation and then only after chronic treatment (at least 3 mionths) at very high doses (J .T . YARRINGTON and J.P . GIBSON, unpublished) . These paradoxical behavioral effects of systemically administered GABA-T inhibitors may also be related to their ability to antagonize GARA at its receptor . Acknowledgements We are grateful to Drs J. Koch-Weser, N . Seiler and J .R . Fozard for helpful comments on this article and thank Mrs C. Kratz for typing the manuscript .

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