15. 1. 1978
67
Specialia
Modulation of t r a n s m i s s i o n in different electronic junctions by aminopyridine A. I. S h a p o v a l o v a n d ]3. I. S h i r i a e v l
Laboratory o/Physiology o/the Nerve Cell, Sechenov Institute o[ Evolutionary Physiology and Biochemistry, Academy of Sciences o/ USSR, Thorez Pr. zig, Leningrad 194 223 (USSR), 22 June 7977 Summary. 4 d i s t i n c t e l e c t r o n i c i n p u t s c a n be i d e n t i f i e d i n a m p h i b i a n m o t o n e u r o n s p e r f u s e d w i t h c a l c i u m - f r e e s o l u t i o n c o n t a i n i n g m a n g a n e s e . T h e d i f f e r e n t i a l effect of 4 - a m i n o p y r i d i n e o n d i f f e r e n t e l e c t r o t o n i c j u n c t i o n s m a y reflect t h e p e c u l i a r i t i e s of m o l e c u l a r a r c h i t e c t u r e of p o t a s s i u m c h a n n e l s in d i f f e r e n t e l e c t r i c a l l y e x c i t a b l e p r e s y n a p t i c m e m b r a n e s . I n c o n t r a s t t o s y n a p s e s w i t h c h e m i c a l m o d e of t r a n s m i s s i o n in e l e c t r o t o n i c j u n c t i o n s , t h e g e n e r a t o r of t h e p o s t s y n a p t i c c u r r e n t is a) l o c a t e d w i t h i n t h e p r e s y n a p t i c m e m b r a n e a n d b) p r o d u c e d b y t h e o p e n i n g of t h e v o l t a g e s e n s i t i v e m e m b r a n e c h a n n e l s . To f i n d o u t m o r e a b o u t t h e r e l a t i o n b e t w e e n t h e electrical e v e n t s in t h e p r e s y n a p t i c m e m b r a n e a n d e l e c t r o t o n i c e x c i t a t o r y p o s t s y n a p t i c pot e n t i a l ( E P S P ) , i t is d e s i r a b l e to i n t e r f e r e w i t h t h e c h a n g e s of ionic c o n d u c t a n c e p r o d u c e d b y t h e p r e s y n a p t i c i m pulse. In the present investigation, an a t t e m p t was made to affect the t r a n s m i s s i o n process across the different e l e c t r o t o n i c j u n c t i o n s of a m p h i b i a n m o t o n e u r o n e s b y 4 - a m i n o p y r i d i n e (4-AP) w h i c h b l o c k s v o l t a g e s e n s i t i v e K + c u r r e n t s in m a n y n e u r a l t i s s u e s 2 5, i n c l u d i n g frog p e r i p h e r a l n e r v e 8. T h e effect of 4 - A P o n t h e i n t r a c e l l u l a r l y r e c o r d e d E P S P s in s o l u t i o n s c o n t a i n i n g no Ca 2+ b u t 2 m M M n 2+ ( s t a n d a r d saline), or 1.5 m M Ca 2+ a n d 2 m M M n 2+, w a s t e s t e d on t h e i s o l a t e d p e r f u s e d frog s p i n a l cord (Rana ridibunda). P r e t r e a t m e n t w i t h M n ~+ q u i c k l y a n d r e v e r s i b l y a b o l i s h e d chemically mediated synaptic transmission, whereas e l e c t r o t o n i c E P S P s elicited b y s t i m u l a t i o n of d o r s a l a n d v e n t r a l r o o t s ( D R a n d V R ) or m e d u l l a r y r e t i c u l a r form a t i o n (RF) were n o t a l t e r e d a f t e r 1 0 - 1 4 h of p e r f u s i o n . 4 - A P ( 4 - a m i n o p y r i d i n e p u r u m , F l u k a AG, 3 9 10 5_ t o 5 - 10 -4 M) q u i c k l y i n c r e a s e d t h e d u r a t i o n , p e a k a m p l i t u d e a n d t i m e to p e a k of D R E P S P (figure 1). T h i s effect w a s d o s e - d e p e n d e n t a n d in 0.5 m M 4 - A P t h e t o t a l d u r a t i o n of D R E P S P c o u l d r e a c h 3 0 0 - 3 5 0 m s e c (as c o m p a r e d w i t h 30-40 m s e c before t h e t r e a t m e n t ) a n d its a m p l i t u d e -
S
1 2 3 4 5 6
We thank Prof. D. R. Curtis, of National University Canberra, for the gift of 4-aminopyridine. M. Pelnate and Y. Piehon, J. Physiol., Lond. 249, 90P (1974). C.Z. Schlauf, C. A. Colton, J. S. Colton et F. A. Davis, J. Pharm. exp. Ther. 197, 414 (1976). J.Z. Yeh, G. S. Oxford, C. t-I. Wu and T. Narahashi, J. gen. Physiol. 68, 519 (1976). R. Llinas, K. Walton and V. Rohr, Biophys. J. 16, 83 (1976). W. Ulbricht and H. H. Wagner, Pfliigers Arch. 367, 77 (1976).
L
_
.
_ _
-/F
Contro, 54min 310 - 5 M 4 - A P
t
Recovery
A)
'
I 1mY 10 msec
!
Control
/"
24min510.4
M 4-AP
!
1mY
/'~;
DR --!V
4 - 5 m V . S i m u l t a n e o u s l y w i t h tile p r o l o n g a t i o n of t h e E P S P , n u m e r o u s h u m p s a p p e a r e d (figure 1), s u g g e s t i n g t h e r h y t h m i c firing in t h e p r e s y n a p t i c t e r m i n a l s . T h e a n tidromic spike was only m o d e r a t e l y prolonged. Neither the resting membrane potential nor the postsynaptic m e m b r a n e t i m e c o n s t a n t w a s m o d i f i e d . T h e r e w a s no c h a n g e in t h e a v e r a g e s h a p e of t h e s p o n t a n e o u s m i n i a t u r e
Coot,ol ~ 2 L -
4m~.w:
5msec
i~,
Control
!~t~~
13'10-5M 4-AP
]0 msec
Control
;
77min 5.10 - 4 M
4-AP
Recovery
lmV 5msec Fig. 1. Effect of 4-AP on electrotonic DR and RF EPSPs recorded from 3 different motoneurones. The top and the bottom traces are averaged records, obtained in Ringer containing 1.5 inM Ca2+, 2 mM Mn2+. The middle traces were recorded in standard saline (no Ca'2+, 2 mM Mn~+). Temperature during recording of these and all other traces was 16 18~
5mser
50 mV
5msec
Fig. 2. A Records of early VR EPSP ill standard saline (top), after addition of 4-AP (middle) and after washing for 70 min in standard saline (bottom trace). B Effect of 4 AP on late VR EPSP (left) and antidromic action potentials (right) recorded from the same motoneurone. Top are responses recorded after 135 min perfusion by standard saline, middle are responses obtained 20 rain after addition of 4-AP, bottom are responses 54 rain after washing in standard saline.
68
Specialia
E P S P s . T h e s e r e s u l t s rule o u t t h e r a t h e r r e m o t e possib i l i t y t h a t t h e a l t e r a t i o n s of D R E P S P s b y 4 - A P are d u e t o its a c t i o n o n t h e p o s t s y n a p t i c m e m b r a n e . T h e effects of s m a l l c o n c e n t r a t i o n s were reversible, while a f t e r long e x p o s u r e t o large c o n c e n t r a t i o n s of 4 - A P we could n o t a c h i e v e t h e c o m p l e t e r e s t o r a t i o n . T h e r a t e of r e c o v e r y of a n t i d r o m i c spike was clearly m u c h faster. A s i m i l a r d r a m a t i c increase of t h e R F E P S P b y 4 - A P was f o u n d (figure 1), a l t h o u g h t h e l a t t e r were m u c h s m a l l e r in a m p l i t u d e a n d could b e d e t e c t e d o n l y in few m o t o n e u r o n e s i n c o n t r a s t to e l e c t r o t o n i c D R E P S P s w h i c h were p r e s e n t p r a c t i c a l l y in all i m p a l e d ceils. The electrotonic VR EPSPs can be subdivided into 2 s e p a r a t e g r o u p s : e a r l y a n d late. T h e f o r m e r are c h a r a c terized b y v e r y s h o r t l a t e n c y (0.5-2.0 msec) a n d r e l a t i v e l y long d u r a t i o n . T h e s u b t r a c t i o n of t h e e x t r a c e l l u l a r response f r o m t h e i n t r a c e l l u l a r one d e m o n s t r a t e s t h a t t h e e a r l y V R E P S P s a l w a y s b e g i n before t h e p e a k of t h e a n t i d r o m i c spike. A f t e r 4 - A P t r e a t m e n t , t h e e a r l y V R E P S P s were e n h a n c e d a n d p r o l o n g e d in a s i m i l a r w a y as D R a n d R F E P S P s (figure 2, A). T h i s fact a n d t h e s h o r t l a t e n c y of e a r l y E P S P s suggest t h a t t h e y are p r o d u c e d b y m o n o s y n a p t i c c o n n e c t i o n s b e t w e e n r e c u r r e n t collaterals of m o t o r a x o n s a n d m o t o n e u r o n e s . In s t a n d a r d saline, t h e d e l a y e d V R E P S P s were p r e s e n t o n l y in a few cells. T h e y h a d a v e r y f a s t t i m e course, b u t
EXPERIENTIA 34/1
a l w a y s a p p e a r e d a f t e r t h e p e a k of t h e a n t i d r o m i c spike ( l a t e n c y 2.5-4.8 msec). W h e n 4 - A P was applied, t h e del a y e d V R E P S P s a p p e a r e d in m o s t cells a n d t h e i r h e i g h t could r e a c h u p to 8-12 inV. H o w e v e r , t h e d u r a t i o n was o n l y s l i g h t l y a l t e r e d a n d t h e y p r e s e r v e d t h e r a p i d spikelike s h a p e (figure 2, B). T h e long l a t e n c y o f d e l a y e d V R EPSPs and their sudden appearance after 4-AP treatm e n t are c o n s i s t e n t w i t h t h e p r o l o n g a t i o n of t h e s o m a d e n d r i t i c spike a n d suggest t h a t t h i s t y p e of a c t i v i t y is t r a n s f e r r e d f r o m m o t o n e u r o n e to m o t o n e u r o n e v i a elect r i c a l l y o p e r a t i n g d e n d r o - d e n d r i t i c synapses. O u r r e s u l t s show t h a t 4 - A P is a n efficient p o t e n t i a t o r of e x c i t a t o r y t r a n s m i s s i o n in d i f f e r e n t e l e c t r o t o n i c j u n c t i o n s a n d t h a t t h i s effect is p r o d u c e d a t t h e p r e s y n a p t i c level. T h e o b s e r v e d e n h a n c e m e n t of e l e c t r o t o n i c E P S P s b y t h e substance blocking K + conductance further indicates that t h e y are n o t c a u s e d b y K + a c c u m u l a t i o n in p e r i n e u r o n a l space. T h e d i f f e r e n t i a l effect of 4 - A P on d i f f e r e n t electrot o n i c j u n c t i o n s m a y reflect t h e d i s t i n c t s t r u c t u r a l org a n i z a t i o n of r e l e v a n t s y n a p s e s ( a x o - d e n d r i t i c a n d d e n d r o d e n d r i t i c ) , b u t m a y d e p e n d also o n t h e p e c u l i a r i t i e s of m o l e c u l a r a r c h i t e c t u r e of K+ c h a n n e l s in d i f f e r e n t elect r i c a l l y e x c i t a b l e p r e s y n a p t i c m e m b r a n e s . Therefore, t h i s s u b s t a n c e m a y b e used as a p r o b e for a n a l y s i s of pres y n a p t i c e v e n t s which, as a rule, are n o t s u b j e c t to d i r e c t electrophysiological investigation.
P r o j e c t i o n s f r o m n u c l e u s a c c u m b e n s to g l o b u s pallidus and s u b s t a n t i a n i g r a in the rat 1 A. D r a y a n d N. R. O a k l e y
Department o/ Pharmacology, The School o/ Pharmacy, 29/39 Brunswick Square, London W C 1 N I A X (England), 31 M a y 1977 Summary. S t i m u l a t i o n of n u c l e u s a c c u m b e n s p r o d u c e s i n h i b i t i o n a n d f a c i l i t a t i o n of n e u r o n a l a c t i v i t y in t h e i p s i l a t e r a l globus p a l l i d u s a n d s u b s t a n t i a nigra. S m a l l lesions in t h e a c c u m b e n s r e d u c e p a l l i d a l b u t n o t n i g r a l G A B A c o n t e n t . R e c e n t s t u d i e s u s i n g t h e o r t h o g r a d e t r a n s p o r t of labelled a m i n o acids or t h e r e t r o g r a d e t r a n s p o r t of h o r s e r a d i s h p e r o x i d a s e h a v e s h o w n t h a t n e u r o n e s of t h e n u c l e u s a c c u m b e n s send e f f e r e n t p r o j e c t i o n s to t h e g l o b u s p a l l i d u s a n d s u b s t a n t i a n i g r a 2-4. T h i s r e p o r t p r o v i d e s electrop h y s i o l o g i c a l c o n f i r m a t i o n for t h e s e p r o j e c t i o n s a n d discusses t h e possible n e u r o t r a n s m i t t e r s released. Materials and methods. E x p e r i m e n t s were p e r f o r m e d in 10 m a l e a l b i n o r a t s l i g h t l y a n a e s t h e t i z e d w i t h u r e t h a n e (1.2 g/kg, i.p.). T h e effect of n u c l e u s a c c u m b e n s s t i m u l a t i o n was e x a m i n e d o n s p o n t a n e o u s n e u r o n a l a c t i v i t y in t h e i p s i l a t e r a l g l o b u s p a l l i d u s a n d s u b s t a n t i a nigra. T h e c e n t r e b a r r e l of a m u l t i b a r r e l l e d m i c r o p i p e t t e was used to r e c o r d e x t r a c e l l u l a r a c t i o n p o t e n t i a l s . O t h e r b a r r e l s c o n t a i n e d a q u e o u s s o l u t i o n s of s u b s t a n c e s for electrop h o r e s i s , p o n t a m i n e blue, G A B A , d o p a m i n e , 1 M NaC1 ( c u r r e n t control). M e t h o d s for r e c o r d i n g a n d a n a l y s i s h a v e b e e n d e s c r i b e d 5. I n o t h e r a n i m a l s (11) u n i l a t e r a l e l e c t r o l y t i c lesions (300-400 FA for 20 sec) of t h e n u c l e u s a c c u m b e n s were m a d e . T h e c o n c e n t r a t i o n of G A B A ~ was e s t i m a t e d in globus p a l l i d u s a n d s u b s t a n t i a n i g r a 14 d a y s a f t e r w a r d s . Results. T h e effects of n u c l e u s a c c u m b e n s s t i m u l a t i o n are s u m m a r i z e d in t h e table. I n t h e g l o b u s p a l l i d u s 39 of 62 cells t e s t e d were a c t i v a t e d . T h e p r e d o m i n a n t response w a s i n h i b i t i o n of firing (33 cells) (figure 1). T h i s occurred alone with relatively short and constant latency (3.3 msec), b u t in o t h e r cells t h e l a t e n c y was longer (13.0 msec) m o r e v a r i a b l e a n d i n h i b i t i o n w a s of s h o r t e r
d u r a t i o n (table). I n 2 cells s h o r t l a t e n c y i n h i b i t i o n was followed b y a single p h a s e of r e b o u n d e x c i t a t i o n . I n 3 cells f a c i l i t a t i o n p r e c e d e d a p e r i o d of i n h i b i t i o n (figure 1) a n d 6 cells r e s p o n d e d b y f a c i l i t a t i o n alone. F a c i l i t a t i o n was c h a r a c t e r i z e d b y a b u r s t of a c t i o n p o t e n t i a l s (figure 1) a n d n o e v i d e n c e was o b t a i n e d for a n t i d r o m i c a c t i v a t i o n . T h e l a t e n c y of t h e s e e x c i t a t i o n s was similar (2.4 msec) b u t t h e d u r a t i o n of e x c i t a t i o n o c c u r r i n g a l o n e was l o n g e r (45.0 msec) t h a n t h a t w h i c h p r e c e d e d a p e r i o d of inh i b i t i o n (15.7 msec). Histological e x a m i n a t i o n r e v e a l e d t h a t m o s t cells a f f e c t e d (22 i n h i b i t e d , 5 excited) were localized r o s t r o m e d i a l l y in t h e v e n t r a l t w o - t h i r d s of t h e pallidus. B o t h G A B A a n d D A (5-75 nA) d e p r e s s e d all cells t e s t e d in t h e p a l l i d u s (26 cells). I n t h e s u b s t a n t i a n i g r a 13 of 30 cells t e s t e d r e s p o n d e d . O n l y simple i n h i b i t i o n (7 cells, figure 2) or simple facilitat i o n (6 cells) was o b s e r v e d . T h e l a t e n c y of i n h i b i t i o n (6.7 msec) or e x c i t a t i o n (5.2 msec) was similar as were t h e r e s p e c t i v e d u r a t i o n s of t h e e v o k e d effect (table). Of t h e r e s p o n s i v e n e u r o n e s , m o s t (5 i n h i b i t e d , 5 excited) 1 2 3 4 5
Acknowledgment. This work was supported by an MRC grant to Prof. D. W. Straughan. B.S. Bunney and G. K. Aghajanian, Brain Res. 117, 423 (1976). L.C.A. Conrad and D. W. Pfaff, Brain Res. 113, 589 (1976). L.W. Swanson and W. M. Cowan, Brain Res. 92, 324 (1975). A. Dray, T. J. Gonye and N. R. Oakley, J. Physiol. 259, 825
6
(1976). I.P. Lowe, E. Robins and G. S. Eyerman, J. Neuroehem. 3, 8
(1958).
67
Specialia
Modulation of t r a n s m i s s i o n in different electronic junctions by aminopyridine A. I. S h a p o v a l o v a n d ]3. I. S h i r i a e v l
Laboratory o/Physiology o/the Nerve Cell, Sechenov Institute o[ Evolutionary Physiology and Biochemistry, Academy of Sciences o/ USSR, Thorez Pr. zig, Leningrad 194 223 (USSR), 22 June 7977 Summary. 4 d i s t i n c t e l e c t r o n i c i n p u t s c a n be i d e n t i f i e d i n a m p h i b i a n m o t o n e u r o n s p e r f u s e d w i t h c a l c i u m - f r e e s o l u t i o n c o n t a i n i n g m a n g a n e s e . T h e d i f f e r e n t i a l effect of 4 - a m i n o p y r i d i n e o n d i f f e r e n t e l e c t r o t o n i c j u n c t i o n s m a y reflect t h e p e c u l i a r i t i e s of m o l e c u l a r a r c h i t e c t u r e of p o t a s s i u m c h a n n e l s in d i f f e r e n t e l e c t r i c a l l y e x c i t a b l e p r e s y n a p t i c m e m b r a n e s . I n c o n t r a s t t o s y n a p s e s w i t h c h e m i c a l m o d e of t r a n s m i s s i o n in e l e c t r o t o n i c j u n c t i o n s , t h e g e n e r a t o r of t h e p o s t s y n a p t i c c u r r e n t is a) l o c a t e d w i t h i n t h e p r e s y n a p t i c m e m b r a n e a n d b) p r o d u c e d b y t h e o p e n i n g of t h e v o l t a g e s e n s i t i v e m e m b r a n e c h a n n e l s . To f i n d o u t m o r e a b o u t t h e r e l a t i o n b e t w e e n t h e electrical e v e n t s in t h e p r e s y n a p t i c m e m b r a n e a n d e l e c t r o t o n i c e x c i t a t o r y p o s t s y n a p t i c pot e n t i a l ( E P S P ) , i t is d e s i r a b l e to i n t e r f e r e w i t h t h e c h a n g e s of ionic c o n d u c t a n c e p r o d u c e d b y t h e p r e s y n a p t i c i m pulse. In the present investigation, an a t t e m p t was made to affect the t r a n s m i s s i o n process across the different e l e c t r o t o n i c j u n c t i o n s of a m p h i b i a n m o t o n e u r o n e s b y 4 - a m i n o p y r i d i n e (4-AP) w h i c h b l o c k s v o l t a g e s e n s i t i v e K + c u r r e n t s in m a n y n e u r a l t i s s u e s 2 5, i n c l u d i n g frog p e r i p h e r a l n e r v e 8. T h e effect of 4 - A P o n t h e i n t r a c e l l u l a r l y r e c o r d e d E P S P s in s o l u t i o n s c o n t a i n i n g no Ca 2+ b u t 2 m M M n 2+ ( s t a n d a r d saline), or 1.5 m M Ca 2+ a n d 2 m M M n 2+, w a s t e s t e d on t h e i s o l a t e d p e r f u s e d frog s p i n a l cord (Rana ridibunda). P r e t r e a t m e n t w i t h M n ~+ q u i c k l y a n d r e v e r s i b l y a b o l i s h e d chemically mediated synaptic transmission, whereas e l e c t r o t o n i c E P S P s elicited b y s t i m u l a t i o n of d o r s a l a n d v e n t r a l r o o t s ( D R a n d V R ) or m e d u l l a r y r e t i c u l a r form a t i o n (RF) were n o t a l t e r e d a f t e r 1 0 - 1 4 h of p e r f u s i o n . 4 - A P ( 4 - a m i n o p y r i d i n e p u r u m , F l u k a AG, 3 9 10 5_ t o 5 - 10 -4 M) q u i c k l y i n c r e a s e d t h e d u r a t i o n , p e a k a m p l i t u d e a n d t i m e to p e a k of D R E P S P (figure 1). T h i s effect w a s d o s e - d e p e n d e n t a n d in 0.5 m M 4 - A P t h e t o t a l d u r a t i o n of D R E P S P c o u l d r e a c h 3 0 0 - 3 5 0 m s e c (as c o m p a r e d w i t h 30-40 m s e c before t h e t r e a t m e n t ) a n d its a m p l i t u d e -
S
1 2 3 4 5 6
We thank Prof. D. R. Curtis, of National University Canberra, for the gift of 4-aminopyridine. M. Pelnate and Y. Piehon, J. Physiol., Lond. 249, 90P (1974). C.Z. Schlauf, C. A. Colton, J. S. Colton et F. A. Davis, J. Pharm. exp. Ther. 197, 414 (1976). J.Z. Yeh, G. S. Oxford, C. t-I. Wu and T. Narahashi, J. gen. Physiol. 68, 519 (1976). R. Llinas, K. Walton and V. Rohr, Biophys. J. 16, 83 (1976). W. Ulbricht and H. H. Wagner, Pfliigers Arch. 367, 77 (1976).
L
_
.
_ _
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Contro, 54min 310 - 5 M 4 - A P
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I 1mY 10 msec
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24min510.4
M 4-AP
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/'~;
DR --!V
4 - 5 m V . S i m u l t a n e o u s l y w i t h tile p r o l o n g a t i o n of t h e E P S P , n u m e r o u s h u m p s a p p e a r e d (figure 1), s u g g e s t i n g t h e r h y t h m i c firing in t h e p r e s y n a p t i c t e r m i n a l s . T h e a n tidromic spike was only m o d e r a t e l y prolonged. Neither the resting membrane potential nor the postsynaptic m e m b r a n e t i m e c o n s t a n t w a s m o d i f i e d . T h e r e w a s no c h a n g e in t h e a v e r a g e s h a p e of t h e s p o n t a n e o u s m i n i a t u r e
Coot,ol ~ 2 L -
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;
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lmV 5msec Fig. 1. Effect of 4-AP on electrotonic DR and RF EPSPs recorded from 3 different motoneurones. The top and the bottom traces are averaged records, obtained in Ringer containing 1.5 inM Ca2+, 2 mM Mn2+. The middle traces were recorded in standard saline (no Ca'2+, 2 mM Mn~+). Temperature during recording of these and all other traces was 16 18~
5mser
50 mV
5msec
Fig. 2. A Records of early VR EPSP ill standard saline (top), after addition of 4-AP (middle) and after washing for 70 min in standard saline (bottom trace). B Effect of 4 AP on late VR EPSP (left) and antidromic action potentials (right) recorded from the same motoneurone. Top are responses recorded after 135 min perfusion by standard saline, middle are responses obtained 20 rain after addition of 4-AP, bottom are responses 54 rain after washing in standard saline.
68
Specialia
E P S P s . T h e s e r e s u l t s rule o u t t h e r a t h e r r e m o t e possib i l i t y t h a t t h e a l t e r a t i o n s of D R E P S P s b y 4 - A P are d u e t o its a c t i o n o n t h e p o s t s y n a p t i c m e m b r a n e . T h e effects of s m a l l c o n c e n t r a t i o n s were reversible, while a f t e r long e x p o s u r e t o large c o n c e n t r a t i o n s of 4 - A P we could n o t a c h i e v e t h e c o m p l e t e r e s t o r a t i o n . T h e r a t e of r e c o v e r y of a n t i d r o m i c spike was clearly m u c h faster. A s i m i l a r d r a m a t i c increase of t h e R F E P S P b y 4 - A P was f o u n d (figure 1), a l t h o u g h t h e l a t t e r were m u c h s m a l l e r in a m p l i t u d e a n d could b e d e t e c t e d o n l y in few m o t o n e u r o n e s i n c o n t r a s t to e l e c t r o t o n i c D R E P S P s w h i c h were p r e s e n t p r a c t i c a l l y in all i m p a l e d ceils. The electrotonic VR EPSPs can be subdivided into 2 s e p a r a t e g r o u p s : e a r l y a n d late. T h e f o r m e r are c h a r a c terized b y v e r y s h o r t l a t e n c y (0.5-2.0 msec) a n d r e l a t i v e l y long d u r a t i o n . T h e s u b t r a c t i o n of t h e e x t r a c e l l u l a r response f r o m t h e i n t r a c e l l u l a r one d e m o n s t r a t e s t h a t t h e e a r l y V R E P S P s a l w a y s b e g i n before t h e p e a k of t h e a n t i d r o m i c spike. A f t e r 4 - A P t r e a t m e n t , t h e e a r l y V R E P S P s were e n h a n c e d a n d p r o l o n g e d in a s i m i l a r w a y as D R a n d R F E P S P s (figure 2, A). T h i s fact a n d t h e s h o r t l a t e n c y of e a r l y E P S P s suggest t h a t t h e y are p r o d u c e d b y m o n o s y n a p t i c c o n n e c t i o n s b e t w e e n r e c u r r e n t collaterals of m o t o r a x o n s a n d m o t o n e u r o n e s . In s t a n d a r d saline, t h e d e l a y e d V R E P S P s were p r e s e n t o n l y in a few cells. T h e y h a d a v e r y f a s t t i m e course, b u t
EXPERIENTIA 34/1
a l w a y s a p p e a r e d a f t e r t h e p e a k of t h e a n t i d r o m i c spike ( l a t e n c y 2.5-4.8 msec). W h e n 4 - A P was applied, t h e del a y e d V R E P S P s a p p e a r e d in m o s t cells a n d t h e i r h e i g h t could r e a c h u p to 8-12 inV. H o w e v e r , t h e d u r a t i o n was o n l y s l i g h t l y a l t e r e d a n d t h e y p r e s e r v e d t h e r a p i d spikelike s h a p e (figure 2, B). T h e long l a t e n c y o f d e l a y e d V R EPSPs and their sudden appearance after 4-AP treatm e n t are c o n s i s t e n t w i t h t h e p r o l o n g a t i o n of t h e s o m a d e n d r i t i c spike a n d suggest t h a t t h i s t y p e of a c t i v i t y is t r a n s f e r r e d f r o m m o t o n e u r o n e to m o t o n e u r o n e v i a elect r i c a l l y o p e r a t i n g d e n d r o - d e n d r i t i c synapses. O u r r e s u l t s show t h a t 4 - A P is a n efficient p o t e n t i a t o r of e x c i t a t o r y t r a n s m i s s i o n in d i f f e r e n t e l e c t r o t o n i c j u n c t i o n s a n d t h a t t h i s effect is p r o d u c e d a t t h e p r e s y n a p t i c level. T h e o b s e r v e d e n h a n c e m e n t of e l e c t r o t o n i c E P S P s b y t h e substance blocking K + conductance further indicates that t h e y are n o t c a u s e d b y K + a c c u m u l a t i o n in p e r i n e u r o n a l space. T h e d i f f e r e n t i a l effect of 4 - A P on d i f f e r e n t electrot o n i c j u n c t i o n s m a y reflect t h e d i s t i n c t s t r u c t u r a l org a n i z a t i o n of r e l e v a n t s y n a p s e s ( a x o - d e n d r i t i c a n d d e n d r o d e n d r i t i c ) , b u t m a y d e p e n d also o n t h e p e c u l i a r i t i e s of m o l e c u l a r a r c h i t e c t u r e of K+ c h a n n e l s in d i f f e r e n t elect r i c a l l y e x c i t a b l e p r e s y n a p t i c m e m b r a n e s . Therefore, t h i s s u b s t a n c e m a y b e used as a p r o b e for a n a l y s i s of pres y n a p t i c e v e n t s which, as a rule, are n o t s u b j e c t to d i r e c t electrophysiological investigation.
P r o j e c t i o n s f r o m n u c l e u s a c c u m b e n s to g l o b u s pallidus and s u b s t a n t i a n i g r a in the rat 1 A. D r a y a n d N. R. O a k l e y
Department o/ Pharmacology, The School o/ Pharmacy, 29/39 Brunswick Square, London W C 1 N I A X (England), 31 M a y 1977 Summary. S t i m u l a t i o n of n u c l e u s a c c u m b e n s p r o d u c e s i n h i b i t i o n a n d f a c i l i t a t i o n of n e u r o n a l a c t i v i t y in t h e i p s i l a t e r a l globus p a l l i d u s a n d s u b s t a n t i a nigra. S m a l l lesions in t h e a c c u m b e n s r e d u c e p a l l i d a l b u t n o t n i g r a l G A B A c o n t e n t . R e c e n t s t u d i e s u s i n g t h e o r t h o g r a d e t r a n s p o r t of labelled a m i n o acids or t h e r e t r o g r a d e t r a n s p o r t of h o r s e r a d i s h p e r o x i d a s e h a v e s h o w n t h a t n e u r o n e s of t h e n u c l e u s a c c u m b e n s send e f f e r e n t p r o j e c t i o n s to t h e g l o b u s p a l l i d u s a n d s u b s t a n t i a n i g r a 2-4. T h i s r e p o r t p r o v i d e s electrop h y s i o l o g i c a l c o n f i r m a t i o n for t h e s e p r o j e c t i o n s a n d discusses t h e possible n e u r o t r a n s m i t t e r s released. Materials and methods. E x p e r i m e n t s were p e r f o r m e d in 10 m a l e a l b i n o r a t s l i g h t l y a n a e s t h e t i z e d w i t h u r e t h a n e (1.2 g/kg, i.p.). T h e effect of n u c l e u s a c c u m b e n s s t i m u l a t i o n was e x a m i n e d o n s p o n t a n e o u s n e u r o n a l a c t i v i t y in t h e i p s i l a t e r a l g l o b u s p a l l i d u s a n d s u b s t a n t i a nigra. T h e c e n t r e b a r r e l of a m u l t i b a r r e l l e d m i c r o p i p e t t e was used to r e c o r d e x t r a c e l l u l a r a c t i o n p o t e n t i a l s . O t h e r b a r r e l s c o n t a i n e d a q u e o u s s o l u t i o n s of s u b s t a n c e s for electrop h o r e s i s , p o n t a m i n e blue, G A B A , d o p a m i n e , 1 M NaC1 ( c u r r e n t control). M e t h o d s for r e c o r d i n g a n d a n a l y s i s h a v e b e e n d e s c r i b e d 5. I n o t h e r a n i m a l s (11) u n i l a t e r a l e l e c t r o l y t i c lesions (300-400 FA for 20 sec) of t h e n u c l e u s a c c u m b e n s were m a d e . T h e c o n c e n t r a t i o n of G A B A ~ was e s t i m a t e d in globus p a l l i d u s a n d s u b s t a n t i a n i g r a 14 d a y s a f t e r w a r d s . Results. T h e effects of n u c l e u s a c c u m b e n s s t i m u l a t i o n are s u m m a r i z e d in t h e table. I n t h e g l o b u s p a l l i d u s 39 of 62 cells t e s t e d were a c t i v a t e d . T h e p r e d o m i n a n t response w a s i n h i b i t i o n of firing (33 cells) (figure 1). T h i s occurred alone with relatively short and constant latency (3.3 msec), b u t in o t h e r cells t h e l a t e n c y was longer (13.0 msec) m o r e v a r i a b l e a n d i n h i b i t i o n w a s of s h o r t e r
d u r a t i o n (table). I n 2 cells s h o r t l a t e n c y i n h i b i t i o n was followed b y a single p h a s e of r e b o u n d e x c i t a t i o n . I n 3 cells f a c i l i t a t i o n p r e c e d e d a p e r i o d of i n h i b i t i o n (figure 1) a n d 6 cells r e s p o n d e d b y f a c i l i t a t i o n alone. F a c i l i t a t i o n was c h a r a c t e r i z e d b y a b u r s t of a c t i o n p o t e n t i a l s (figure 1) a n d n o e v i d e n c e was o b t a i n e d for a n t i d r o m i c a c t i v a t i o n . T h e l a t e n c y of t h e s e e x c i t a t i o n s was similar (2.4 msec) b u t t h e d u r a t i o n of e x c i t a t i o n o c c u r r i n g a l o n e was l o n g e r (45.0 msec) t h a n t h a t w h i c h p r e c e d e d a p e r i o d of inh i b i t i o n (15.7 msec). Histological e x a m i n a t i o n r e v e a l e d t h a t m o s t cells a f f e c t e d (22 i n h i b i t e d , 5 excited) were localized r o s t r o m e d i a l l y in t h e v e n t r a l t w o - t h i r d s of t h e pallidus. B o t h G A B A a n d D A (5-75 nA) d e p r e s s e d all cells t e s t e d in t h e p a l l i d u s (26 cells). I n t h e s u b s t a n t i a n i g r a 13 of 30 cells t e s t e d r e s p o n d e d . O n l y simple i n h i b i t i o n (7 cells, figure 2) or simple facilitat i o n (6 cells) was o b s e r v e d . T h e l a t e n c y of i n h i b i t i o n (6.7 msec) or e x c i t a t i o n (5.2 msec) was similar as were t h e r e s p e c t i v e d u r a t i o n s of t h e e v o k e d effect (table). Of t h e r e s p o n s i v e n e u r o n e s , m o s t (5 i n h i b i t e d , 5 excited) 1 2 3 4 5
Acknowledgment. This work was supported by an MRC grant to Prof. D. W. Straughan. B.S. Bunney and G. K. Aghajanian, Brain Res. 117, 423 (1976). L.C.A. Conrad and D. W. Pfaff, Brain Res. 113, 589 (1976). L.W. Swanson and W. M. Cowan, Brain Res. 92, 324 (1975). A. Dray, T. J. Gonye and N. R. Oakley, J. Physiol. 259, 825
6
(1976). I.P. Lowe, E. Robins and G. S. Eyerman, J. Neuroehem. 3, 8
(1958).
