1170
Specialia
p e a k value of v e n o u s blood o x y g e n s a t u r a t i o n d u r i n g reaction was n o t m o r e t h a n 68% HbO~, v e n o u s o u t f l o w c o n s t a n t l y increased. W h e n t h e p e a k value r e a c h e d 70 to 85% H b O 2, t h e v e n o u s outflow e i t h e r d e c r e a s e d or in11] 3) P
0.5
~.-.o
\
0
1 N2 AN P1 P2 aP
- 0.5
[
I -38.7%
"N
1I -2Z2 % III-15.5 %
-1s 1.0 b)
0.5 n
PI P2 /~P
C - 0,-~
I - 42.6 % I I - 25.9 %
m- 14.8 %
-1.0
Fig. 3. Results of tbe factor analysis of vasomotor reactions and dynanfies of venous blood oxygen saturation changes in the spleen (a) and intestine (b) under electrical stimulation of sympathetic fibres. Abszissa, analyzed priznacs; ordinate, means of coefficient of correlation. Line, I factor; interrupt line, II factor; point, III factor. Weights of factors: a) I, 38.7% ; II, 27.2% ; III, 15,5%. b) I, 42.6% ; II, 23.9%; III, 14.8%. V, venous outflow (ml); N~, initial level of the venous blood oxygen saturation (%Hb 02) ; N2, peak means of the venous blood oxygen saturation (%Hb 02) ; N, venous blood oxygen saturation changes; Pt, initial level of the perfusion pressure; P2, peak means of the perfusion pressure; P, the perfussion pressure changes.
EXPERIENTIA 32/9
creased. W h e n v e n o u s blood o x y g e n s a t u r a t i o n r e a c h e d , u n d e r s t i m u l a t i o n of s y m p a t h e t i c n e r v e s , t h e v a l u e of 85% H b O 2 or more, t h e v e n o u s o u t f l o w decreased. W e r e v e a l e d as well r e v e r s e d line c o r r e l a t i o n b e t w e e n initial level of v e n o u s b l o o d o x y g e n s a t u r a t i o n a n d t h e c h a r a c t e r of v e n o u s o u t f l o w f r o m an o r g a n u n d e r electrical s t i m u l a t i o n of s y m p a t h e t i c fibres (Figure 2b). I t t u r n e d o u t t h a t if t h e initial level v e n o u s b l o o d o x y g e n s a t u r a t i o n was small, i.e. t h e o x y g e n c o n s u m p t i o n b y t h e tissue of a n o r g a n was high, v e n o u s o u t f l o w as a rule was increased. On t h e c o n t r a r y , t h e m o r e t h e v e n o u s blood o x y g e n satu r a t i o n before s t i m u l a t i o n , t h e m o r e e v i d e n t was t h e t e n d e n c y to t h e decrease of t h e v e n o u s o u t f l o w u n d e r electrical s t i m u l a t i o n of s y m p a t h e t i c fibres. To elucidate t h e r e l a t i o n b e t w e e n b o t h t h e v a l u e a n d c h a r a c t e r of c a p a c i t a n c e vessel r e s p o n s e s of spleen a n d int e s t i n e a n d all t h e p a r a m e t e r s recorded, we used one of t h e m e t h o d s of t h e f a c t o r analysis - m e t h o d of chief c o m p o n e n t s . I t was s h o w n (Figure 3) t h a t t h e m o s t essential f a c t o r was t h e close n e g a t i v e c o r r e l a t i o n b e t w e e n m a g n i t u d e a n d c h a r a c t e r of c a p a c i t a n c e vessel r e s p o n s e s f r o m one side a n d d y n a m i c s of v e n o u s blood o x y g e n saturation from another. T h u s t h e results of t h e analysis p e r m i t us to s u g g e s t t h a t t h e m o r e i n t e n s i v e t h e o x y g e n e x c h a n g e in spleen a n d i n t e s t i n e before a n d d u r i n g t h e s y m p a t h e t i c s t i m u lation, i,e. t h e m o r e is v e n o u s b l o o d d e s a t u r a t e d w i t h o x y g e n , t h e m o r e f r e q u e n t t h e c o n s t r i c t o r y r e s p o n s e of c a p a c i t a n c e vessels. On t h e c o n t r a r y , t h e lower t h e o x y g e n e x c h a n g e in spleen a n d small i n t e s t i n e , i.e. t h e m o r e t h e venous blood saturated with oxygen, the more frequent is d i l a t o r y r e s p o n s e of c a p a c i t a n c e vessels. I t is n e c e s s a r y to e m p h a s i z e , h o w e v e r , t h a t t h i s corr e l a t i o n was o b t a i n e d in e x p e r i m e n t s w i t h s y m p a t h e t i c fibres cut, i.e. w h e n t h e organs i n v e s t i g a t e d were p r a c t i cally d e p r i v e d of n e u r o g e n i c control. W h e t h e r t h i s corr e l a t i o n exists in t h e n o r m a l l y f u n c t i o n i n g spleen a n d small i n t e s t i n e will be s h o w n in f u r t h e r i n v e s t i g a t i o n s .
C o o r d i n a t e d A c t i v a t i o n of M u s c l e F i b r e s b y D i f f e r e n t C o n d u c t i o n V e l o c i t i e s i n B r a n c h e s of a C r u s t a cean Motor Axon C. I~. G O V I N D 1 a n d
F. LANG 2
Scarborough College, University o/ Toronto, West Hill, Ontario, (Canada M1C 1A4); and Boston University Marine Program, Marine Biological Laboratory, Woods Hole (Massachusetts, USA), 23 February 1976. Summary. H i g h e r c o n d u c t i o n velocities in b r a n c h e s of t h e f a s t e x c i t o r a x o n to distal muscle fibres ensure t h a t ttlese fibres are a c t i v a t e d a l m o s t s i m u l t a n e o u s l y with p r o x i m a l fibres in t h e claw closer muscle of lobsters, p r o d u c i n g a c o n t r a c t i o n of m a x i m a l force. I n muscle c o n t r a c t i o n , m a x i m a l force is e x e r t e d w h e n all fibres w i t h i n a muscle (or m o t o r unit) are e x c i t e d s i m u l t a n e o u s l y . The r e s u l t i n g c o o r d i n a t i o n p r o b l e m is a c c e n t u a t e d in c r u s t a c e a n s , in w h i c h entire muscles are i n n e r v a t e d b y few (1-4) e x c i t a t o r y m o t o r axons, so each n e u r o n serves a large n u m b e r of muscle fibres. I n addition, t h e muscle fibres are a r r a n g e d in p i n n a t e fashion resulting in a wide s e p a r a t i o n b e t w e e n p r o x i m a l a n d distal fibres (Figure A). W h a t is t h e m e c h a n i s m for e x c i t i n g t h e fibres w i t h i n s u c h muscles so t h a t t h e y c o n t r a c t a l m o s t s i m u l t a n e o u s l y ? I n t h e l o b s t e r claw closer muscle, diff e r e n t c o n d u c t i o n velocities in t h e b r a n c h e s of a single e x c i t a t o r y a x o n allow w i d e l y s e p a r a t e d muscle fibres to be a c t i v a t e d w i t h i n a few milliseconds of one a n o t h e r .
The claw closer muscle in l o b s t e r s is t h e largest l i m b muscle a n d is i n n e r v a t e d b y 2 e x c i t a t o r y (fast a n d slow) a x o n s a n d an i n h i b i t o r y a x o n 3. The f a s t a x o n is t h e m a j o r m o t o r fibre to t h e dorsal surface os t h e muscle, a l t h o u g h all regions of t h e muscle are i n n e r v a t e d b y b o t h a x o n s t o some e x t e n t ~. F u r t h e r m o r e , it is possible to selectively s t i m u l a t e t h e f a s t axon, since it i n v a r i a b l y has t h e lowest t h r e s h o l d of all 3 a x o n s ~. W e t h e r e f o r e e x a m i n e d t h e t e m p o r a l differences in e x c i t a t i o n of w i d e l y s e p a r a t e d dorsal fibres w h e n t h e f a s t e x c i t o r was s t i m u l a t ed. I n a t y p i c a l e x p e r i m e n t , t h e dorsal surface of t h e closer muscle was e x p o s e d b y r e m o v i n g t h e o p e n e r muscle. A p r o x i m a l a n d a distal fibre were p e n e t r a t e d s i m u l t a n e -
15.9. 1976
Specialia
o u s l y w i t h c o n v e n t i o n a l 3 M KC1 filled glass microelect r o d e s (Figure A). T h e closer n e r v e , w h i c h w a s i s o l a t e d f r o m t h e m a i n leg n e r v e , w a s s t i m u l a t e d w i t h brief ( < 0.1 msec) s q u a r e p u l s e s v i a p l a t i n u m wire electrodes. T h e r e s u l t i n g s p i k e s were m o n i t o r e d d i s t a l to t h e s t i m u l a t i n g e l e c t r o d e s b y a s u c t i o n e l e c t r o d e r e c o r d i n g 'enp a s s a n t ' (Figure A). T h e d i s t a n c e s b e t w e e n e l e c t r o d e s were m e a s u r e d u s i n g a n o c u l a r m i c r o m e t e r . T h i s exp e r i m e n t a l d e s i g n p r o v i d e d a r e c o r d of t h e e x t r a c e l l u l a r s p i k e s in t h e f a s t a x o n a n d t h e r e s u l t a n t e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s ( E P S P s ) in t h e m u s c l e fibres (Figure B). T h e t i m e t a k e n f r o m t h e a x o n spike to t h e b e g i n n i n g of e a c h E P S P , t o g e t h e r w i t h t h e d i s t a n c e s e p a r a t i n g t h e e l e c t r o d e s r e c o r d i n g t h e s e e v e n t s , g a v e a n a v e r a g e cond u c t i o n v e l o c i t y for t h o s e b r a n c h e s of t h e f a s t e x c i t o r a x o n w h i c h s u p p l i e d t h e m u s c l e fibres. T h e s e c o n d u c t i o n velocities e n a b l e d u s to c o m p a r e t h e l a t e n c i e s for a c t i v a t i o n in w i d e l y s e p a r a t e d m u s c l e fibres.
A I If-
DACTYL
\ I-----~
PROPODITE
I
_
CARPOPODITE
B E ....................... RE .....................
i
DF ..................... A) Dorsal surface of the closer muscle in a crusher claw showing location of stimulating electrode (S), extracellular suction electrode (E) on closer nerve and intracellular microelectrodes in proximal (PF) and distal (DF) muscle fibres. Note branching pattern of nerve. Scale mark 1 cm. B) A representative record of the electrical activity monitored by the recording electrodes shown in Figure A. Calibration : horizontal 10 msec; vertical, 1st record 0.4 mV; 2nd, 3rd records, 4 mV.
Comparison of conduction velocities (CV) in the fast exeitor axon to proximal (PF) and distal (DF) fibres in the lobster claw closer muscle CV (m/sec)
Differences in CV between PF and DF
PF
DF-PF DF-PF - -PF- • 100
DF
(%)
1.4 1.1 0.6 0.7 2.0 1.3 0.7 2.0
2.1 1.9 1.2 2.0 2.5 2.4 2.0 3.0
0.7 0.8 0.6 1.3 0.5 1.1 1.3 1.0
50 73 50 186 25 85 186 50
Differences in time of EPSP initiation between PF and DF (msec)
Distance between PI e and DF (nlm)
6.5 4 5.6 5 2.2 2.5 2.5 4
20 16.6 12.5 24.1 10.1 15 15 28
1171
T h e r e s u l t s f r o m 8 s e p a r a t e closer m u s c l e s (5 c r u s h e r a n d 3 c u t t e r claws) are s u m m a r i z e d in t h e T a b l e . I m p u l s e p r o p a g a t i o n in a x o n b r a n c h e s to t h e d i s t a l fibres w a s c o n s i s t e n t l y f a s t e r (25% t o 186%) t h a n in b r a n c h e s t o p r o x i m a l fibres. E v e n t h o u g h t h e fibres were w i d e l y sep a r a t e d (10.1 to 28 m m ) , t h e E P S P to t h e d i s t a l fibre w a s o n l y a few m s e c (2.2 t o 6.5) b e h i n d t h a t in t h e prox i m a l fibre. I n t h e e x a m p l e g i v e n in F i g u r e B, t h o u g h t h e 2 m u s c l e fibres are s e p a r a t e d b y 28 r a m , t h e r e is o n l y a 4 m s e c lag in t h e i n i t i a t i o n of a n E P S P in t h e d i s t a l fibre. W h e n t h e d i s t a n c e b e t w e e n fibres w a s less t h a n 5 m m , t h e c o n d u c t i o n v e l o c i t y in a x o n a l b r a n c h e s t o t h e 2 fibres did n o t differ s i g n i f i c a n t l y . Clearly, f a s t e r i m p u l s e p r o p a g a t i o n t o t h e d i s t a l fibres e n s u r e s t h a t t h e y are a c t i v a t ed a l m o s t s i m u l t a n e o u s l y w i t h t h e p r o x i m a l fibres. T h i s c o o r d i n a t i o n is p a r t i c u l a r l y i m p o r t a n t for t h e f a s t a x o n of t h e c u t t e r claw, w h i c h c a n c a u s e c l a w closure in a p p r o x i m a t e l y 20 m s e c ~. I t s h o u l d be b o r n e in m i n d t h a t w h e r e a s c o n t r a c t i o n , of a v e r t e b r a t e t w i t c h t y p e m u s c l e fibre d e p e n d s o n a n all-or-nothing action potential generated by the EPSP, c o n t r a c t i o n of a c r u s t a c e a n s t r i a t e d m u s c l e fibre is g r a d e d a c c o r d i n g t o t h e r a t e a n d degree of d e p o l a r i z a t i o n of t h e fibre r e s u l t i n g f r o m t h e E P S P . T h e size of E P S P v a r i e s c o n s i d e r a b l y a m o n g s t fibres of t h e d i m o r p h i c claw closer muscle with the fast axon generating relatively small E P S P s in t h e c r u s h e r c l a w (1 3 m V as in F i g u r e B), a n d l a r g e r E P S P s in t h e c u t t e r claw (4-15 mV) ~. F a c i l i t a t i o n a n d s u m m a t i o n of E P S P s leads t o l a r g e r d e p o l a r i z a t i o n of t h e fibre a n d m a y l e a d to a n a l l - o r - n o t h i n g a c t i o n p o t e n t i a l . T h e l a t t e r is c e r t a i n l y t h e case in s o m e fibres of t h e c u t t e i c l a w w h e r e s t i m u l a t i o n of t h e f a s t a x o n w i t h p a i r e d p u l s e s r e s u l t s in a n o v e r s h o o t i n g a c t i o n p o t e n t i a l w h i c h p r o d u c e s a t w i t c h c o n t r a c t i o n ~. I n t h i s case coo r d i n a t e d a c t i v a t i o n of w i d e l y s e p a r a t e d m u s c l e fibres would ensure the maximal twitch contraction. W h a t is t h e b a s i s for t h e d i f f e r e n t r a t e s of i m p u l s e p r o p a g a t i o n in b r a n c h e s of a single m o t o r a x o n ? Conduction velocity varies directly with axon diameter, h e n c e d i f f e r e n c e s in d i a m e t e r m a y e x p l a i n d i f f e r e n c e s in c o n d u c t i o n v e l o c i t y . I t is t h e r e f o r e i n s t r u c t i v e to c o n s i d e r t h e b r a n c h i n g p a t t e r n of t h e closer n e r v e in t h e l o b s t e r c l a w ( F i g u r e A). T h e closer n e r v e d i v i d e s i n t o 2 p r i m a r y b r a n c h e s , w h i c h i n n e r v a t e fibres on e i t h e r side of t h e t e n d o n a l o n g t h e l e n g t h of t h e m u s c l e . P r o x i m a l t o t h i s m a j o r b i f u r c a t i o n is a b r a n c h w h i c h is m u c h t h i n n e r t h a n the primary branches and which supplies the proximal m u s c l e fibres. P r e s u m a b l y i m p u l s e p r o p a g a t i o n a l o n g t h i s fine b r a n c h w o u l d be slower t h a n a l o n g t h e large p r i m a r y b r a n c h e s p r o d u c i n g c o o r d i n a t e d a c t i v a t i o n of p r o x i m a l a n d d i s t a l m u s c l e fibres. Alternatively the excitatory axon may take a more c i r c u i t o u s r o u t e to t h e p r o x i m a l m u s c l e fibres, so t h a t t h e p a t h w a y to t h e s e fibres is s t r i c t l y v i a fine a x o n b r a n c h e s . I n c o n t r a s t t h e d i s t a l fibres m a y be i n n e r v a t e d by small axon branches that travel only short distances f r o m t h e large p r i m a r y b r a n c h .
1 Supported by Muscular Dystrophy Association and National Research Council of Canada. 2 Supported by National Institute of Health and Muscular Dystrophy Association of America. a C. A. G. WIERSMA,Arch. Neerl. Zool. 11, 1 (1955). C. K. GOVlND and F. LANG, J. exp. Zool. 100, 281 (1974).
Specialia
p e a k value of v e n o u s blood o x y g e n s a t u r a t i o n d u r i n g reaction was n o t m o r e t h a n 68% HbO~, v e n o u s o u t f l o w c o n s t a n t l y increased. W h e n t h e p e a k value r e a c h e d 70 to 85% H b O 2, t h e v e n o u s outflow e i t h e r d e c r e a s e d or in11] 3) P
0.5
~.-.o
\
0
1 N2 AN P1 P2 aP
- 0.5
[
I -38.7%
"N
1I -2Z2 % III-15.5 %
-1s 1.0 b)
0.5 n
PI P2 /~P
C - 0,-~
I - 42.6 % I I - 25.9 %
m- 14.8 %
-1.0
Fig. 3. Results of tbe factor analysis of vasomotor reactions and dynanfies of venous blood oxygen saturation changes in the spleen (a) and intestine (b) under electrical stimulation of sympathetic fibres. Abszissa, analyzed priznacs; ordinate, means of coefficient of correlation. Line, I factor; interrupt line, II factor; point, III factor. Weights of factors: a) I, 38.7% ; II, 27.2% ; III, 15,5%. b) I, 42.6% ; II, 23.9%; III, 14.8%. V, venous outflow (ml); N~, initial level of the venous blood oxygen saturation (%Hb 02) ; N2, peak means of the venous blood oxygen saturation (%Hb 02) ; N, venous blood oxygen saturation changes; Pt, initial level of the perfusion pressure; P2, peak means of the perfusion pressure; P, the perfussion pressure changes.
EXPERIENTIA 32/9
creased. W h e n v e n o u s blood o x y g e n s a t u r a t i o n r e a c h e d , u n d e r s t i m u l a t i o n of s y m p a t h e t i c n e r v e s , t h e v a l u e of 85% H b O 2 or more, t h e v e n o u s o u t f l o w decreased. W e r e v e a l e d as well r e v e r s e d line c o r r e l a t i o n b e t w e e n initial level of v e n o u s b l o o d o x y g e n s a t u r a t i o n a n d t h e c h a r a c t e r of v e n o u s o u t f l o w f r o m an o r g a n u n d e r electrical s t i m u l a t i o n of s y m p a t h e t i c fibres (Figure 2b). I t t u r n e d o u t t h a t if t h e initial level v e n o u s b l o o d o x y g e n s a t u r a t i o n was small, i.e. t h e o x y g e n c o n s u m p t i o n b y t h e tissue of a n o r g a n was high, v e n o u s o u t f l o w as a rule was increased. On t h e c o n t r a r y , t h e m o r e t h e v e n o u s blood o x y g e n satu r a t i o n before s t i m u l a t i o n , t h e m o r e e v i d e n t was t h e t e n d e n c y to t h e decrease of t h e v e n o u s o u t f l o w u n d e r electrical s t i m u l a t i o n of s y m p a t h e t i c fibres. To elucidate t h e r e l a t i o n b e t w e e n b o t h t h e v a l u e a n d c h a r a c t e r of c a p a c i t a n c e vessel r e s p o n s e s of spleen a n d int e s t i n e a n d all t h e p a r a m e t e r s recorded, we used one of t h e m e t h o d s of t h e f a c t o r analysis - m e t h o d of chief c o m p o n e n t s . I t was s h o w n (Figure 3) t h a t t h e m o s t essential f a c t o r was t h e close n e g a t i v e c o r r e l a t i o n b e t w e e n m a g n i t u d e a n d c h a r a c t e r of c a p a c i t a n c e vessel r e s p o n s e s f r o m one side a n d d y n a m i c s of v e n o u s blood o x y g e n saturation from another. T h u s t h e results of t h e analysis p e r m i t us to s u g g e s t t h a t t h e m o r e i n t e n s i v e t h e o x y g e n e x c h a n g e in spleen a n d i n t e s t i n e before a n d d u r i n g t h e s y m p a t h e t i c s t i m u lation, i,e. t h e m o r e is v e n o u s b l o o d d e s a t u r a t e d w i t h o x y g e n , t h e m o r e f r e q u e n t t h e c o n s t r i c t o r y r e s p o n s e of c a p a c i t a n c e vessels. On t h e c o n t r a r y , t h e lower t h e o x y g e n e x c h a n g e in spleen a n d small i n t e s t i n e , i.e. t h e m o r e t h e venous blood saturated with oxygen, the more frequent is d i l a t o r y r e s p o n s e of c a p a c i t a n c e vessels. I t is n e c e s s a r y to e m p h a s i z e , h o w e v e r , t h a t t h i s corr e l a t i o n was o b t a i n e d in e x p e r i m e n t s w i t h s y m p a t h e t i c fibres cut, i.e. w h e n t h e organs i n v e s t i g a t e d were p r a c t i cally d e p r i v e d of n e u r o g e n i c control. W h e t h e r t h i s corr e l a t i o n exists in t h e n o r m a l l y f u n c t i o n i n g spleen a n d small i n t e s t i n e will be s h o w n in f u r t h e r i n v e s t i g a t i o n s .
C o o r d i n a t e d A c t i v a t i o n of M u s c l e F i b r e s b y D i f f e r e n t C o n d u c t i o n V e l o c i t i e s i n B r a n c h e s of a C r u s t a cean Motor Axon C. I~. G O V I N D 1 a n d
F. LANG 2
Scarborough College, University o/ Toronto, West Hill, Ontario, (Canada M1C 1A4); and Boston University Marine Program, Marine Biological Laboratory, Woods Hole (Massachusetts, USA), 23 February 1976. Summary. H i g h e r c o n d u c t i o n velocities in b r a n c h e s of t h e f a s t e x c i t o r a x o n to distal muscle fibres ensure t h a t ttlese fibres are a c t i v a t e d a l m o s t s i m u l t a n e o u s l y with p r o x i m a l fibres in t h e claw closer muscle of lobsters, p r o d u c i n g a c o n t r a c t i o n of m a x i m a l force. I n muscle c o n t r a c t i o n , m a x i m a l force is e x e r t e d w h e n all fibres w i t h i n a muscle (or m o t o r unit) are e x c i t e d s i m u l t a n e o u s l y . The r e s u l t i n g c o o r d i n a t i o n p r o b l e m is a c c e n t u a t e d in c r u s t a c e a n s , in w h i c h entire muscles are i n n e r v a t e d b y few (1-4) e x c i t a t o r y m o t o r axons, so each n e u r o n serves a large n u m b e r of muscle fibres. I n addition, t h e muscle fibres are a r r a n g e d in p i n n a t e fashion resulting in a wide s e p a r a t i o n b e t w e e n p r o x i m a l a n d distal fibres (Figure A). W h a t is t h e m e c h a n i s m for e x c i t i n g t h e fibres w i t h i n s u c h muscles so t h a t t h e y c o n t r a c t a l m o s t s i m u l t a n e o u s l y ? I n t h e l o b s t e r claw closer muscle, diff e r e n t c o n d u c t i o n velocities in t h e b r a n c h e s of a single e x c i t a t o r y a x o n allow w i d e l y s e p a r a t e d muscle fibres to be a c t i v a t e d w i t h i n a few milliseconds of one a n o t h e r .
The claw closer muscle in l o b s t e r s is t h e largest l i m b muscle a n d is i n n e r v a t e d b y 2 e x c i t a t o r y (fast a n d slow) a x o n s a n d an i n h i b i t o r y a x o n 3. The f a s t a x o n is t h e m a j o r m o t o r fibre to t h e dorsal surface os t h e muscle, a l t h o u g h all regions of t h e muscle are i n n e r v a t e d b y b o t h a x o n s t o some e x t e n t ~. F u r t h e r m o r e , it is possible to selectively s t i m u l a t e t h e f a s t axon, since it i n v a r i a b l y has t h e lowest t h r e s h o l d of all 3 a x o n s ~. W e t h e r e f o r e e x a m i n e d t h e t e m p o r a l differences in e x c i t a t i o n of w i d e l y s e p a r a t e d dorsal fibres w h e n t h e f a s t e x c i t o r was s t i m u l a t ed. I n a t y p i c a l e x p e r i m e n t , t h e dorsal surface of t h e closer muscle was e x p o s e d b y r e m o v i n g t h e o p e n e r muscle. A p r o x i m a l a n d a distal fibre were p e n e t r a t e d s i m u l t a n e -
15.9. 1976
Specialia
o u s l y w i t h c o n v e n t i o n a l 3 M KC1 filled glass microelect r o d e s (Figure A). T h e closer n e r v e , w h i c h w a s i s o l a t e d f r o m t h e m a i n leg n e r v e , w a s s t i m u l a t e d w i t h brief ( < 0.1 msec) s q u a r e p u l s e s v i a p l a t i n u m wire electrodes. T h e r e s u l t i n g s p i k e s were m o n i t o r e d d i s t a l to t h e s t i m u l a t i n g e l e c t r o d e s b y a s u c t i o n e l e c t r o d e r e c o r d i n g 'enp a s s a n t ' (Figure A). T h e d i s t a n c e s b e t w e e n e l e c t r o d e s were m e a s u r e d u s i n g a n o c u l a r m i c r o m e t e r . T h i s exp e r i m e n t a l d e s i g n p r o v i d e d a r e c o r d of t h e e x t r a c e l l u l a r s p i k e s in t h e f a s t a x o n a n d t h e r e s u l t a n t e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s ( E P S P s ) in t h e m u s c l e fibres (Figure B). T h e t i m e t a k e n f r o m t h e a x o n spike to t h e b e g i n n i n g of e a c h E P S P , t o g e t h e r w i t h t h e d i s t a n c e s e p a r a t i n g t h e e l e c t r o d e s r e c o r d i n g t h e s e e v e n t s , g a v e a n a v e r a g e cond u c t i o n v e l o c i t y for t h o s e b r a n c h e s of t h e f a s t e x c i t o r a x o n w h i c h s u p p l i e d t h e m u s c l e fibres. T h e s e c o n d u c t i o n velocities e n a b l e d u s to c o m p a r e t h e l a t e n c i e s for a c t i v a t i o n in w i d e l y s e p a r a t e d m u s c l e fibres.
A I If-
DACTYL
\ I-----~
PROPODITE
I
_
CARPOPODITE
B E ....................... RE .....................
i
DF ..................... A) Dorsal surface of the closer muscle in a crusher claw showing location of stimulating electrode (S), extracellular suction electrode (E) on closer nerve and intracellular microelectrodes in proximal (PF) and distal (DF) muscle fibres. Note branching pattern of nerve. Scale mark 1 cm. B) A representative record of the electrical activity monitored by the recording electrodes shown in Figure A. Calibration : horizontal 10 msec; vertical, 1st record 0.4 mV; 2nd, 3rd records, 4 mV.
Comparison of conduction velocities (CV) in the fast exeitor axon to proximal (PF) and distal (DF) fibres in the lobster claw closer muscle CV (m/sec)
Differences in CV between PF and DF
PF
DF-PF DF-PF - -PF- • 100
DF
(%)
1.4 1.1 0.6 0.7 2.0 1.3 0.7 2.0
2.1 1.9 1.2 2.0 2.5 2.4 2.0 3.0
0.7 0.8 0.6 1.3 0.5 1.1 1.3 1.0
50 73 50 186 25 85 186 50
Differences in time of EPSP initiation between PF and DF (msec)
Distance between PI e and DF (nlm)
6.5 4 5.6 5 2.2 2.5 2.5 4
20 16.6 12.5 24.1 10.1 15 15 28
1171
T h e r e s u l t s f r o m 8 s e p a r a t e closer m u s c l e s (5 c r u s h e r a n d 3 c u t t e r claws) are s u m m a r i z e d in t h e T a b l e . I m p u l s e p r o p a g a t i o n in a x o n b r a n c h e s to t h e d i s t a l fibres w a s c o n s i s t e n t l y f a s t e r (25% t o 186%) t h a n in b r a n c h e s t o p r o x i m a l fibres. E v e n t h o u g h t h e fibres were w i d e l y sep a r a t e d (10.1 to 28 m m ) , t h e E P S P to t h e d i s t a l fibre w a s o n l y a few m s e c (2.2 t o 6.5) b e h i n d t h a t in t h e prox i m a l fibre. I n t h e e x a m p l e g i v e n in F i g u r e B, t h o u g h t h e 2 m u s c l e fibres are s e p a r a t e d b y 28 r a m , t h e r e is o n l y a 4 m s e c lag in t h e i n i t i a t i o n of a n E P S P in t h e d i s t a l fibre. W h e n t h e d i s t a n c e b e t w e e n fibres w a s less t h a n 5 m m , t h e c o n d u c t i o n v e l o c i t y in a x o n a l b r a n c h e s t o t h e 2 fibres did n o t differ s i g n i f i c a n t l y . Clearly, f a s t e r i m p u l s e p r o p a g a t i o n t o t h e d i s t a l fibres e n s u r e s t h a t t h e y are a c t i v a t ed a l m o s t s i m u l t a n e o u s l y w i t h t h e p r o x i m a l fibres. T h i s c o o r d i n a t i o n is p a r t i c u l a r l y i m p o r t a n t for t h e f a s t a x o n of t h e c u t t e r claw, w h i c h c a n c a u s e c l a w closure in a p p r o x i m a t e l y 20 m s e c ~. I t s h o u l d be b o r n e in m i n d t h a t w h e r e a s c o n t r a c t i o n , of a v e r t e b r a t e t w i t c h t y p e m u s c l e fibre d e p e n d s o n a n all-or-nothing action potential generated by the EPSP, c o n t r a c t i o n of a c r u s t a c e a n s t r i a t e d m u s c l e fibre is g r a d e d a c c o r d i n g t o t h e r a t e a n d degree of d e p o l a r i z a t i o n of t h e fibre r e s u l t i n g f r o m t h e E P S P . T h e size of E P S P v a r i e s c o n s i d e r a b l y a m o n g s t fibres of t h e d i m o r p h i c claw closer muscle with the fast axon generating relatively small E P S P s in t h e c r u s h e r c l a w (1 3 m V as in F i g u r e B), a n d l a r g e r E P S P s in t h e c u t t e r claw (4-15 mV) ~. F a c i l i t a t i o n a n d s u m m a t i o n of E P S P s leads t o l a r g e r d e p o l a r i z a t i o n of t h e fibre a n d m a y l e a d to a n a l l - o r - n o t h i n g a c t i o n p o t e n t i a l . T h e l a t t e r is c e r t a i n l y t h e case in s o m e fibres of t h e c u t t e i c l a w w h e r e s t i m u l a t i o n of t h e f a s t a x o n w i t h p a i r e d p u l s e s r e s u l t s in a n o v e r s h o o t i n g a c t i o n p o t e n t i a l w h i c h p r o d u c e s a t w i t c h c o n t r a c t i o n ~. I n t h i s case coo r d i n a t e d a c t i v a t i o n of w i d e l y s e p a r a t e d m u s c l e fibres would ensure the maximal twitch contraction. W h a t is t h e b a s i s for t h e d i f f e r e n t r a t e s of i m p u l s e p r o p a g a t i o n in b r a n c h e s of a single m o t o r a x o n ? Conduction velocity varies directly with axon diameter, h e n c e d i f f e r e n c e s in d i a m e t e r m a y e x p l a i n d i f f e r e n c e s in c o n d u c t i o n v e l o c i t y . I t is t h e r e f o r e i n s t r u c t i v e to c o n s i d e r t h e b r a n c h i n g p a t t e r n of t h e closer n e r v e in t h e l o b s t e r c l a w ( F i g u r e A). T h e closer n e r v e d i v i d e s i n t o 2 p r i m a r y b r a n c h e s , w h i c h i n n e r v a t e fibres on e i t h e r side of t h e t e n d o n a l o n g t h e l e n g t h of t h e m u s c l e . P r o x i m a l t o t h i s m a j o r b i f u r c a t i o n is a b r a n c h w h i c h is m u c h t h i n n e r t h a n the primary branches and which supplies the proximal m u s c l e fibres. P r e s u m a b l y i m p u l s e p r o p a g a t i o n a l o n g t h i s fine b r a n c h w o u l d be slower t h a n a l o n g t h e large p r i m a r y b r a n c h e s p r o d u c i n g c o o r d i n a t e d a c t i v a t i o n of p r o x i m a l a n d d i s t a l m u s c l e fibres. Alternatively the excitatory axon may take a more c i r c u i t o u s r o u t e to t h e p r o x i m a l m u s c l e fibres, so t h a t t h e p a t h w a y to t h e s e fibres is s t r i c t l y v i a fine a x o n b r a n c h e s . I n c o n t r a s t t h e d i s t a l fibres m a y be i n n e r v a t e d by small axon branches that travel only short distances f r o m t h e large p r i m a r y b r a n c h .
1 Supported by Muscular Dystrophy Association and National Research Council of Canada. 2 Supported by National Institute of Health and Muscular Dystrophy Association of America. a C. A. G. WIERSMA,Arch. Neerl. Zool. 11, 1 (1955). C. K. GOVlND and F. LANG, J. exp. Zool. 100, 281 (1974).
