EXPERIENTIA 25/4

Speeialia

also i n c u b a t e d for 1:/~ h in t h e s a m e i n c u b a t i o n m e d i u m a n d were e x a m i n e d u n d e r t h e l i g h t microscope. D F P (Dii s o p r o p y l f l u o r o p h o s p h a t e ) a n d 62 C 47 [1,5-bis(4-trim e t h y l a m m o n i u m p h e n y l ) p e n t a n 3-one iodid I a t conc e n t r a t i o n s of 10 S M c o m p l e t e l y a b o l i s h e d t h e r e a c t i o n of ACHE. No i n h i b i t o r s of nonspecific c h o l i n e s t e r a s e were a d d e d to t h e i n c u b a t i o n m e d i u m as t h e l a t t e r e n z y m e is n o t p r e s e n t in t h e p e r i k a r y a of r a t s e n s o r y ganglia 1,~,1~ Light microscope observations showed a network-like p a t t e r n of t h e r e a c t i o n p r o d u c t m a i n l y in t h e s m a l l n e u r o n e s (Figure 1). Similarly, t h e e l e c t r o n microscope r e v e a l e d a p o s i t i v e r e a c t i o n m a i n l y in t h e s m a l l n e u r o n e s . T h e r e a c t i o n p r o d u c t was d e p o s i t e d in t h e c y s t e r n a e , vesicles a n d v a c u o l e s of t h e Golgi a p p a r a t u s , a n d a t t h e p l a s m a m e m b r a n e (Figures 2, 3). T h e e l e c t r o n microg r a p h s i n d i c a t e d t h a t p r e s e r v a t i o n of t h e tissue is adeq u a t e . No r e a c t i o n p r o d u c t was seen in t h e nuclei, m i t o c h o n d r i a or e n d o p l a s m i c r e t i c n l u m . T h e p r e s e n t d a t a are p a r t i a l l y in a g r e e m e n t w i t h o t h e r r e p o r t s on t h e a c t i v i t y of A C h E in t h e c e n t r a l n e r v o u s s y s t e m a n d s y m p a t h e t i c ganglia ~ 7. I n t h e s e n e u r o n e s , localization of t h e r e a c t i o n p r o d u c t was o b s e r v e d in t h e Golgi a p p a r a t u s a n d p l a s m a m e m b r a n e as well as in t h e r o u g h e n d o p l a s m i c r e t i c u l u m . H o w e v e r , in t h e p r e s e n t

389

s t u d i e s n o p o s i t i v e r e a c t i o n was o b s e r v e d in t h e r o u g h e n d o p l a s m i c r e t i c u l u m of t h e n e u r o n e s . I t is n o t possible a t p r e s e n t t o e x p l a i n t h e a b s e n c e of a c t i v i t y of A C h E in t h e e n d o p l a s m i c r e t i c n l u m . T h e p r e s e n t r e s u l t s m a y a d d i n f o r m a t i o n to t h e a s s u m p t i o n t h a t some of t h e n e u r o n e s of t h e s e n s o r y g a n g l i a are cholinergic 3,11,12.

Resumen. U t i l i z a n d o el m i c r o s c o p i o electr6nico se dem o s t r 6 a c t i v i d a d e n z y m A t i c a de la a c e t i l c o l i n e s t e r a s a en el a p a r a t o de Golgi y a n i v e l de la m e m b r a n a p l a s m A t i c a de las n e u r o n a s del ganglio del t r i g 6 m i n o en r a t a s . ~{. KALINA a n d J. J. B u m s

Department o~ Cell Biology and Histology, Tel Aviv University, Medical School, and Department o/Pathology, Government Hospital, Tel-Hashomer (Israel), 26 June 7968.

10 M. KALINA and M. WOLMAN,unpublished results. 11 E. GIACOBINI,Acta physiol, seand., Suppl. 45, 156 (1959a). 12 E. GIACOBINI,Acta physiol, scand. 45, 238 (1959b).

E l e c t r o n M i c r o s c o p i c L o c a l i z a t i o n of A c e t y l c h o l i n e s t e r a s e in S m a l l M u l t i p l e E n d i n g s in the E x t r a o c u l a r M u s c l e s of the Rat T h e r e are 2 d i f f e r e n t t y p e s of m y o n e u r a l j u n c t i o n s in t h e e x t r a o c u l a r muscles of d i f f e r e n t a n i m a l s (for earlier l i t e r a t u r e see e.g. TERAVKINEN1). One of t h e j u n c t i o n s is f o r m e d f r o m a m y e l i n a t e d n e r v e a n d h a s a s t r u c t u r e c o m p a r a b l e w i t h a n o r m a l m o t o r e n d p l a t e of s t r i a t e d s k e l e t a l muscle fibres in b o t h t h e l i g h t microscope a n d t h e e l e c t r o n microscope. T h e o t h e r t y p e of m y o n e u r a l j u n c t i o n differs s t r i k i n g l y f r o m t h e m o t o r e n d plate, since it is d e r i v e d f r o m a n o n - m y e l i n a t e d n e r v e a n d term i n a t e s in m a n y s m a l l endings. Since t h e l i g h t microscopic d i s t r i b u t i o n of c h o l i n e s t e r a s e a c t i v i t y was closely c o r r e l a t e d w i t h t h e e l e c t r o n m i c r o scopic s t r u c t u r e of t h e j u n c t i o n , i t was s u g g e s t e d t h a t t h e s e ' m u l t i p l e e n d i n g s ' are cholinergic e x c i t a t o r y s y n a p s e s t o e x t r a o c u l a r m u s c l e fibres 4. B e c a u s e t h e d i s t r i b u t i o n of c h o l i n e s t e r a s e h a d b e e n s t u d i e d o n l y b y l i g h t microscopy, i t was decided t o e x a m i n e t h e a c e t y l c h o l i n e s t e r a s e (ACHE) a c t i v i t y of t h e s e m u l t i p l e e n d i n g s a t t h e e l e c t r o n microscope level. Methods. A d u l t S p r a g u e - D a w l e y r a t s were killed u n d e r ether anaesthesia by decapitation, and the rectus medialis m u s c l e was i m m e d i a t e l y r e m o v e d a n d fixed for 20-40 m i n a t 4 ~ w i t h 2.5 % s o l u t i o n of g l u t a r a l d e h y d e b u f f e r e d w i t h p h o s p h a t e a t p H 7.2~. T h e muscles were c u t i n t o s m a l l pieces of a b o u t 0.5 m m t h i c k w i t h a r a z o r b l a d e a n d w a s h e d a t 4 ~ w i t h 0 . 2 5 M sucrose for a b o u t 12 h. T h e p r e s e n c e of a c e t y l c h o l i n e s t e r a s e (EC. 3.1.1.7) was d e m o n s t r a t e d u s i n g a c e t y l t h i o c h o l i n e iodide ( F l u k a AG, Buchs) as s u b s t r a t e t o g e t h e r w i t h 10 5 M t e t r a - i s o p r o p y l p y r o p h o s p h o r a m i d e (iso-OMPA; L. L i g h t & Co. Ltd., Colnbrook) in t h e i n c u b a t i o n s o l u t i o n t o exclude a c t i v i t y d u e t o o t h e r c h o l i n e s t e r a s e s (EC. 3.1.1.8), C o n t r o l s t u d i e s were m a d e w i t h b o t h iso-OMPA a n d 1 0 - ~ M 284C51 ( B u r r o u g h s a n d Wellcome, L o n d o n ) i n t h e i n c u b a t i o n solution. T h e f e r r o - f e r r i c y a n i d e m e t h o d a was u s e d a t 4~ a n d p H 6.0, w i t h a n i n c u b a t i o n t i m e of 60-180 rain.

T h e s p e c i m e n s were t h e n buffer, d e h y d r a t e d w i t h a n d e m b e d d e d in E p o n s t a i n e d w i t h lead c i t r a t e

w a s h e d for 30 m i n in p h o s p h a t e g r a d e d series of e t h y l alcohol 8124. T h e sections were a f t e r 5. Results and discussion. A C h E a c t i v i t y was o b s e r v e d in t h e s m a l l m u l t i p l e e n d i n g s in t h e r e c t u s m e d i a l i s muscle. T h e r e a c t i o n was localized b e t w e e n t h e s y n a p t i c m e m b r a n e s . W i t h longer i n c u b a t i o n times, t h e e n d p r o d u c t of t h e r e a c t i o n t o t a l l y filled t h e s y n a p t i c cleft a n d a c t i v i t y was also o b s e r v e d b e t w e e n t h e m e m b r a n e s of t h e a x o n t e r m i n a l a n d t h e teloglial cell (Figure). T h e s y n a p t i c vesicles were n e g a t i v e . S o m e s c a t t e r e d g r a n u l e s w i t h a d i f f e r e n t f o r m f r o m t h e p r e c i p i t a t i o n g r a n u l e of t h e r e a c t i o n p r o d u c t were o b s e r v e d t h r o u g h o u t t h e sections. T h e y were u n r e l a t e d to p a : t i c u l a r s t r u c t u r e s a n d r e g a r d e d as a r t i f a c t s . The ferro-ferricyanide method has previously been used w i t h some success t o localize c h o l i n e s t e r a s e a c t i v i t y in t h e m o t o r e n d plate, w h e r e t h e e n z y m e a c t i v i t y was i n t e n s e a n d t h e i n c u b a t i o n t i m e s h o r t e r 6. I n t h e p r e s e n t work, t h e m e t h o d d i d n o t a p p e a r t o b e v e r y s u i t a b l e for e l e c t r o n microscopic h i s t o c h e m i s t r y if t h e a c t i v i t y to b e d e m o n s t r a t e d was o n l y m o d e r a t e , as was t h e case in t h e s e s m a l l j u n c t i o n s 1. T h e r e f o r e t h e e x a c t l o c a t i o n of A C h E in r e s p e c t of t h e s y n a p t i c m e m b r a n e of t h e s e s m a l l j u n c t i o n s , r e m a i n s t o b e shown. H o w e v e r , t h e present work demonstrates beyond any doubt that these

1 H. TERXV~INEN,Z. Zellforsch. nfikrosk. Anat. 90, 372 (1968). D. D. SABATINI, K. BENSCH and R. J. BA~RNETT, J. Cell Biol. 17, 19 (1963). M. J. KARNOVSKY, J. Cell Biol. 23, 217 (1964). 4 j. H. LVFT, J. biophys, bioehem. Cytol. 9, 409 (1961). 5 E. S. REYNOLDS, J. Cell Biol. 77, 208 (1963). 6 H. TERXVMNEN, Histoehemie 10, 266 (1967).

390

Specialia

15.4. 1969

e n d i n g s e x h i b i t a n a c e t y l c h o l i n e s t e r a s e a c t i v i t y a n d are t h e r e f o r e likely t o b e cholinergic. The c o n t r a c t i o n of t h e e x t r a o c u l a r muscles b y a d r e n e r g i c a g e n t s 7,8 still a w a i t s an explanation.

Zusammen[assung. E l e k t r o n e n m i k r o s k o p i s c h wird gezeigt, d a s s die aus n i c h t m y e l i n h a l t i g e n N e r v e n f a s e r n s t a m m e n d e n kleinen m o t o r i s c h e n E n d p l a t t e n in d e n A u g e n m u s k e l n der R a t t e Azetylcholinesterase-Aktivit~Lt haben. H. TER)~VXINEN

Department o/A natomy, University of Helsinki, Helsinki 17 (Finland), 2 December 1968.

7 I. S. SASCHVI, Invest. Ophthal. 6, 269 (1967). 8 K. E. EAKINS and R. L. KATZ, Invest. Ophthal. 6, 253 (1967).

AChE activity in the small myoneural junction arising from unmyelinated nerves in the extraocular muscles of the rat. The reaction totally fills the irregular synaptie clefts (SC) and activity is also seen between the teloglial cell (TC) and the axon terminal (A). The scattered precipitate (arrow) is regarded as an artifact (see text). • 17,000.

Cervical S y m p a t h e c t o m y and the Origin of Small Nerve Endings in the Extraocular Muscles of the Rat Two s t r u c t u r a l l y d i f f e r e n t t y p e s of m y o n e u r a l j u n c t i o n s are p r e s e n t in t h e e x t r a o c u l a r muscle fibres. One of t h e e n d i n g s c o m e s f r o m m y e l i n a t e d n e r v e s a n d h a s tile struct u r e of a n o r d i n a r y m y o n e u r a l j u n c t i o n of skeletal s t r i a t e d muscle fibres 1,2. T h e s t r u c t u r e of t h e second t y p e of m y o n e u r a l j u n c t i o n is different. I t is v e r y small (even less t h a n 1 ~z), is d e r i v e d f r o m u n m y e l i n a t e d nerves, a n d possesses a fine s t r u c t u r e r e s e m b l i n g t h a t of t h e cholinergic e x c i t a t o r y s y n a p s e 2. The l o c a t i o n of t h e n e u r o n e s supplying the unmyelinated nerves to these 'multiple endings' 2 is u n k n o w n a n d it is n o t e v e n k n o w n b y w h i c h anatomical p a t h w a y their axons reach the extraocular muscles. A lesion of t h e s u p e r i o r cervical ganglion causes p a r t i a l p a r a l y s i s of t h e l e v a t o r p a l p e b r a e superioris muscle (e.g. DAVSONa). S t i m u l a t i o n of t h i s ganglion can s o m e t i m e s also cause c o n t r a c t i o n in t h e e x t r a o c u l a r muscles 4, w h i c h h a v e b e e n r e p o r t e d t o h a v e n e r v e e n d i n g s f r o m unm y e l i n a t e d n e r v e s of t h e s y m p a t h e t i c p e r i v a s c u l a r p l e x u s 5,6. Therefore, we d e c i d e d to s t u d y t h e fine struct u r e of m u l t i p l e e n d i n g s in t h e e x t r a o c u l a r muscle fibres a f t e r e x p e r i m e n t a l r e m o v a l of t h e superior cervical ganglion. Methods. A d u l t S p r a g u e - D a w l e y l a b o r a t o r y r a t s were used in t h e e x p e r i m e n t s . T h e s u p e r i o r cervical ganglion w a s r e m o v e d e i t h e r uni- or b i l a t e r a l l y u n d e r e t h e r a n a e s t h e s i a . T h e r e c t u s superior, lateralis a n d medialis muscles were p r e p a r e d 12 h, 2, 4 a n d 17 d a y s a f t e r t h e o p e r a t i o n a f t e r d e c a p i t a t i o n of t h e a n i m a l s u n d e r e t h e r a n a e s t h e s i a . The muscles were fixed a t 4~ w i t h 2.5% g l u t a r a l d e h y d e in p h o s p h a t e b u f f e r a t p H 7.2 7 a n d p o s t f i x e d w i t h 1% OsO 4 in t h e p h o s p h a t e buffer. A f t e r de-

h y d r a t i o n w i t h g r a d e d series of e t h y l alcohol, E p o n 812 8 was used for e m b e d d i n g . T h e sections were c o u n t e r s t a i n e d w i t h lead c i t r a t e ~

Fig. 1. Myoneural junction derived from unmyelinated nerves (N) in the unoperated rat. 2 axon terminals (TAX) apposed to the muscle plasma membrane and 2 vesiculated axon processes are seen. • 15,000.