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d-Tubocurarine does not antagonize the inhibitory effects of GABA at the crustacean neuromuscular junction
-156
B~am Re~ea/'(t~..4') ~1973~ -~',: ~5~ , Elsevier Sclentlhc+ Ptibh¢>hlng (,~lnpany
Ambterdam
Printed H, The N~'~hcta.~,,l.
d-Tubocurarine does not amagonize the inhibitory effects of GABA at the crustacean neuromuscular junction
J A N E T F A R L AND W. A L A R G E
Department oJ Pharmacoh)gy, The Sdtool ol Pharmacy, London WCIN 1A,k (Great Btttam) (Accepted October 19th. 1972)
It has been known for many years that D-tubocurarme (DTC) shows convutsant properties when applied topically to the cerebral cortexL Recently, Hill et al 4, using microiontophoreUc techmques in cat cerebral cortex, have suggested that thts acuon of DTC is due, at least m part, to its ability to antagomze the inhtbltory effects of
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2,.v[ Fig I. The effects of D T C and p~crotoxm on neurally-e~oked mh~biUon. Each trace Js a 500 m,c~ tram of EJPs; the horizontal bar m B - F ,ndicates simultaneous stimulat~on (100 msec) of the inhibitory axon A, control excitation alone; B, control excitation plus inhibition. C, 30 min alter addition of 10 ,~ M DTC: D, 5 m m after washing out D T C , E, 4 min after 5 10 ~ M picrotoxm, F, 30 m m after wash Vertical bar: 2 m V cahbratxon.
SHORT COMMUNICATIONS
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Fig. 2 The effects of DTC and pmrotoxm on GABA-mduced depress/on of EJPs Each trace is a 600 msec tram of EJPs. A, control, B, 1 mm after addlnon of 5 > 10 ~ M GABA; C, 5 mm after wash; D, 1 mm after mixture of 2 × 10-4 M DTC + 5 × 10 ~ M GABA, DTC having already been m the bath for 5 mm; E, 5 m l n a f t e r w a s h m g ; F , l mlnafter5 "< 10 ~Mplcrotoxln + 5 \ l0 .5 M GABA, plcrotoxln having already been m the bath for 5 mm Vertical b3r' 2 mV calibration
G A B A . T h e r e is m u c h evidence t h a t G A B A is the i n h i b i t o r y t r a n s m i t t e r at m a n y c r u s t a c e a n p e r i p h e r a l synapses and these p r e p a r a t i o n s have been used as m o d e l s for G A B A - m e d i a t e d inhibition in the m a m m a l i a n central n e r v o u s system. It was t h e r e f o r e o f interest to c o m p a r e , at a n e u r o m u s c u l a r i n h i b i t o r y synapse, the effects o f D T C with those o f picrotoxin, k n o w n to block G A B A in c r u s t a c e a 3,6,7 E x c i t a t o r y j u n c t i o n potentials (EJPs) were r e c o r d e d intracellularly from the a b d u c t o r muscle o f large claws o f h e r m i t crabs (Eupagurus hernhardus). Stimulation ( 1/30 sec) o f the e x c i t a t o r y axon with a 500-700 msec train o f repetitive (20 Hz) pulses e v o k e d EJPs o f a m p l i t u d e 1.5-13 mV. I n h i b i t i o n was elicited by s i m u l t a n e o u s stimulation (90-220 Hz) of the i n h i b i t o r y axon for 100-150 msec d u r i n g the excitatory train a n d was m a n i f e s t e d as a r e d u c t i o n in E J P a m p l i t u d e . In o t h e r e x p e r i m e n t s EJPs were r a p i d l y depressed by a d d i t i o n o f G A B A to the bath. Drugs were i n t r o d u c e d by replacem e n t o f the b a t h solution wath R i n g e r c o n t a i n i n g the c o m p o u n d . T h e Ringer c o m p o sition was NaC1, 445 m M ; KCI, 12.2 r a M ; CaCI2, 29 6 r a M : MgCI2, 5.75 r a M ; N a H C O 3 1.79 m M . In 6 experiments, D T C ( 2 " / 1 0 L I > ' 1 0 -3 M ) had no effect on the neurally e v o k e d i n h i b i t i o n o f the EJPs d u r i n g 15-30 min exposure. On two occasions,
458
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with c o n t i n u e d recording from the same fibre, subsequent a p p h c a t m n of plcrotoxm (5 - 10-5M) rapidly blocked the m h l b m o n . Fig. I shows the results of an e,~pel tin,tit m which 10 3 M D T C was used In the experiments m which EJPs were a t t e n u a t e d by the a d d i u o n of G A B A to the bath, DTC, m an c o n c e n t r a t i o n range of 2 , 10-5--5 ~, 10-4M (7 experiments), proved ineffecUve m blocking G A B A a n d restoring the EJP a m p h t u d e As wHh neurally-evoked mh~bmon, s u b s e q u e n t addition of picrotoxin blocked the actio~ ~f G A B A (F~g. 2). The results show that D T C is meffecttve in blocking the inhibitory effects of G A B A at the n e u r o m u s c u l a r j u n c t m n of the hermit crab. Tbe highest c o n c e n t r a u o n of D T C used was 20 times greater than the c o n c e n t r a t i o n of picrotoxm which tolatl3 blocked these G A B A effects. In a previous report ~t, we d e m o n s t r a t e d that bicuculline, a G A B A antagomst ~n the m a m m a h a n central nervous system'-', was ineffective against G A B A m our preparation. Takeuchi and O n o d e r a '5. studying the crayfish n e u r o m u s c u l a r j u n c u o n , also f o u n d blcuculline to be considerably less potent t h a n plcrotoxm, it would therefore a p p e a r that there is a difference m susceptibility to G A B A - b l o c k i n g drugs between cells at the crustacean n e u r o m u s c u l a r j u n c t i o n and in the m a m m a h a n central ner~ ou~ system
1 CHANG,
H., Similarity in action between curare and strychnine on cortical ncurones, J Ncu, t~-
phy~tol, 16 (1953) 221-233
2 CuRTis, D. R . D U ( , ( , A N . A W . FELIX.. D, JOHNSTON,G A R , AND MCLENXlAN.H . Antagomsm between blcuculhne and GABA m the cat brain, Brain Research, 33 (1971) 57 73 3 EARL, J . ~XND LARGE. W. A . The effects of bicuculhne, picrotoxin and strychnine on neuromu~cula r Inhibition in hermit crabs (Eupagurus bernhardus), J PhyswL fLond.), 224 (1972) 45-46P. 4 HILl., R G , SIMMONDS, M. A., ~,ND STRAUGHAN, D W , The convulsive properties of D-tt~bocu,arine and cortical inhibition, m press. 5 TAKFUCHLA , ANO ONOD~RA, K , Effect of Ncuculhne on the GABA receptor of the crayhsh neuromuscular Junction, Nature New Biol., 236 (1972) 55-56 6 T A K E U C H I , A , AND TAKEUCHI, N , A study, of the action of p~crotoxm on the inhibitory n e l J l O muscular junction of the crayfish, J Phy.~wt (Lond.), 205 (1969) 377-391 7 VaN DER KLOOT, W G , ROBBINS,J , AND COOKe, 1., Blocking by picrotoxtn of peripheral inhibition m ~rayfish, Science, 127 (1958) 521 522.
B~am Re~ea/'(t~..4') ~1973~ -~',: ~5~ , Elsevier Sclentlhc+ Ptibh¢>hlng (,~lnpany
Ambterdam
Printed H, The N~'~hcta.~,,l.
d-Tubocurarine does not amagonize the inhibitory effects of GABA at the crustacean neuromuscular junction
J A N E T F A R L AND W. A L A R G E
Department oJ Pharmacoh)gy, The Sdtool ol Pharmacy, London WCIN 1A,k (Great Btttam) (Accepted October 19th. 1972)
It has been known for many years that D-tubocurarme (DTC) shows convutsant properties when applied topically to the cerebral cortexL Recently, Hill et al 4, using microiontophoreUc techmques in cat cerebral cortex, have suggested that thts acuon of DTC is due, at least m part, to its ability to antagomze the inhtbltory effects of
-A
C
B . i t:
fl
i
{:: i
i--4L ; t ~
:
2
I.]>'k,_ L_,
D i: -~ ~ h ~-:~ iT ~ "t
I
,~l.d
1
E
F
'-i-' -~---~i ~ b ~ .
2,.v[ Fig I. The effects of D T C and p~crotoxm on neurally-e~oked mh~biUon. Each trace Js a 500 m,c~ tram of EJPs; the horizontal bar m B - F ,ndicates simultaneous stimulat~on (100 msec) of the inhibitory axon A, control excitation alone; B, control excitation plus inhibition. C, 30 min alter addition of 10 ,~ M DTC: D, 5 m m after washing out D T C , E, 4 min after 5 10 ~ M picrotoxm, F, 30 m m after wash Vertical bar: 2 m V cahbratxon.
SHORT COMMUNICATIONS
457
A
,
C
f I
........ .l.i~,+],l,~:~]~ }
2mY
.
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;'"
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Fig. 2 The effects of DTC and pmrotoxm on GABA-mduced depress/on of EJPs Each trace is a 600 msec tram of EJPs. A, control, B, 1 mm after addlnon of 5 > 10 ~ M GABA; C, 5 mm after wash; D, 1 mm after mixture of 2 × 10-4 M DTC + 5 × 10 ~ M GABA, DTC having already been m the bath for 5 mm; E, 5 m l n a f t e r w a s h m g ; F , l mlnafter5 "< 10 ~Mplcrotoxln + 5 \ l0 .5 M GABA, plcrotoxln having already been m the bath for 5 mm Vertical b3r' 2 mV calibration
G A B A . T h e r e is m u c h evidence t h a t G A B A is the i n h i b i t o r y t r a n s m i t t e r at m a n y c r u s t a c e a n p e r i p h e r a l synapses and these p r e p a r a t i o n s have been used as m o d e l s for G A B A - m e d i a t e d inhibition in the m a m m a l i a n central n e r v o u s system. It was t h e r e f o r e o f interest to c o m p a r e , at a n e u r o m u s c u l a r i n h i b i t o r y synapse, the effects o f D T C with those o f picrotoxin, k n o w n to block G A B A in c r u s t a c e a 3,6,7 E x c i t a t o r y j u n c t i o n potentials (EJPs) were r e c o r d e d intracellularly from the a b d u c t o r muscle o f large claws o f h e r m i t crabs (Eupagurus hernhardus). Stimulation ( 1/30 sec) o f the e x c i t a t o r y axon with a 500-700 msec train o f repetitive (20 Hz) pulses e v o k e d EJPs o f a m p l i t u d e 1.5-13 mV. I n h i b i t i o n was elicited by s i m u l t a n e o u s stimulation (90-220 Hz) of the i n h i b i t o r y axon for 100-150 msec d u r i n g the excitatory train a n d was m a n i f e s t e d as a r e d u c t i o n in E J P a m p l i t u d e . In o t h e r e x p e r i m e n t s EJPs were r a p i d l y depressed by a d d i t i o n o f G A B A to the bath. Drugs were i n t r o d u c e d by replacem e n t o f the b a t h solution wath R i n g e r c o n t a i n i n g the c o m p o u n d . T h e Ringer c o m p o sition was NaC1, 445 m M ; KCI, 12.2 r a M ; CaCI2, 29 6 r a M : MgCI2, 5.75 r a M ; N a H C O 3 1.79 m M . In 6 experiments, D T C ( 2 " / 1 0 L I > ' 1 0 -3 M ) had no effect on the neurally e v o k e d i n h i b i t i o n o f the EJPs d u r i n g 15-30 min exposure. On two occasions,
458
SHORI
~ ~)MMI,NI(,\It~'-
with c o n t i n u e d recording from the same fibre, subsequent a p p h c a t m n of plcrotoxm (5 - 10-5M) rapidly blocked the m h l b m o n . Fig. I shows the results of an e,~pel tin,tit m which 10 3 M D T C was used In the experiments m which EJPs were a t t e n u a t e d by the a d d i u o n of G A B A to the bath, DTC, m an c o n c e n t r a t i o n range of 2 , 10-5--5 ~, 10-4M (7 experiments), proved ineffecUve m blocking G A B A a n d restoring the EJP a m p h t u d e As wHh neurally-evoked mh~bmon, s u b s e q u e n t addition of picrotoxin blocked the actio~ ~f G A B A (F~g. 2). The results show that D T C is meffecttve in blocking the inhibitory effects of G A B A at the n e u r o m u s c u l a r j u n c t m n of the hermit crab. Tbe highest c o n c e n t r a u o n of D T C used was 20 times greater than the c o n c e n t r a t i o n of picrotoxm which tolatl3 blocked these G A B A effects. In a previous report ~t, we d e m o n s t r a t e d that bicuculline, a G A B A antagomst ~n the m a m m a h a n central nervous system'-', was ineffective against G A B A m our preparation. Takeuchi and O n o d e r a '5. studying the crayfish n e u r o m u s c u l a r j u n c u o n , also f o u n d blcuculline to be considerably less potent t h a n plcrotoxm, it would therefore a p p e a r that there is a difference m susceptibility to G A B A - b l o c k i n g drugs between cells at the crustacean n e u r o m u s c u l a r j u n c t i o n and in the m a m m a h a n central ner~ ou~ system
1 CHANG,
H., Similarity in action between curare and strychnine on cortical ncurones, J Ncu, t~-
phy~tol, 16 (1953) 221-233
2 CuRTis, D. R . D U ( , ( , A N . A W . FELIX.. D, JOHNSTON,G A R , AND MCLENXlAN.H . Antagomsm between blcuculhne and GABA m the cat brain, Brain Research, 33 (1971) 57 73 3 EARL, J . ~XND LARGE. W. A . The effects of bicuculhne, picrotoxin and strychnine on neuromu~cula r Inhibition in hermit crabs (Eupagurus bernhardus), J PhyswL fLond.), 224 (1972) 45-46P. 4 HILl., R G , SIMMONDS, M. A., ~,ND STRAUGHAN, D W , The convulsive properties of D-tt~bocu,arine and cortical inhibition, m press. 5 TAKFUCHLA , ANO ONOD~RA, K , Effect of Ncuculhne on the GABA receptor of the crayhsh neuromuscular Junction, Nature New Biol., 236 (1972) 55-56 6 T A K E U C H I , A , AND TAKEUCHI, N , A study, of the action of p~crotoxm on the inhibitory n e l J l O muscular junction of the crayfish, J Phy.~wt (Lond.), 205 (1969) 377-391 7 VaN DER KLOOT, W G , ROBBINS,J , AND COOKe, 1., Blocking by picrotoxtn of peripheral inhibition m ~rayfish, Science, 127 (1958) 521 522.