904
Specialia
tion against the two neuropharmaca but not against the detergent. At the same time, monosialo-ganglioside and monosialo-ganglioside-like fractions from the blastulae were n o t a c t i v e a g a i n s t n e u r o p h a r m a c a , b u t s h o w e d p r o t e c t i v e a c t i o n a g a i n s t t h e d e t e r g e n t OP-10. T h e effective p r o t e c t i v e c o n c e n t r a t i o n s of t h e gangliosides are g i v e n i n t h e Table. T h e s e c o n c e n t r a t i o n s are f r o m 20 t o 1000 t i m e s lower t h a n t h e t o x i c c o n c e n t r a t i o n s of t h e n e u r o p h a r m a c a a n d 10-20 t i m e s lower t h a n t h e c o n c e n t r a t i o n s of t h e d e t e r g e n t . A t t h e s a m e t i m e , t h e di- a n d trisialoganglioside-like f r a c t i o n s i s o l a t e d f r o m sea u r c h i n e m b r y o s were n o t effective e i t h e r a g a i n s t n e u r o p h a r m a c a or a g a i n s t t h e d e t e r g e n t OP-10. T h e f a c t o r d e c r e a s i n g t h e s e n s i t i v i t y of e m b r y o s t o n e u r o p h a r m a c a w a s s h o w n also t o b e p r e s e n t in t h e c h l o r o f o r m - m e t h a n o l e x t r a c t of c o n d i t i o n e d sea w a t e r (CSW), i.e. sea water, i n w h i c h dense s u s p e n s i o n s ( a b o u t 30,000 e m b r y o s / m l ) of S. intermedius e m b r y o s w e r e incubated. T L C of t h e C S W c h l o r o f o r m - m e t h a n o l e x t r a c t r e v e a l e d t h e p r e s e n c e of r e s o r c i n e - p o s i t i v e s u b s t a n c e s coinciding, by their Rf-values, with the hematoside-like fraction of sea u r c h i n e m b r y o s a n d w i t h r a t l i v e r h e m a t o s i d e (Figure). W e f o u n d t h a t t h e sialic acid c o n c e n t r a t i o n in C S W was a b o u t 1 • .5 M . N e i t h e r t h e gangliosides f r o m t h e sea u r c h i n e m b r y o s or f r o m r a t l i v e r n o r t h e c h l o r o f o r m - m e t h a n o l e x t r a c t of t h e C S W or C S W itself a f f e c t e d t h e s e n s i t i v i t y of t h e e m b r y o s t o p u r o m y c i n . I n p r e v i o u s e x p e r i m e n t s ~, i t was s h o w n t h a t t h e s e n s i t i v i t y of t h e e m b r y o s t o t h i s a n t i b i o t i c did n o t d e p e n d o n t h e i r c o n c e n t r a t i o n (in t h e c o n c e n t r a t i o n i n t e r v a l f r o m 10 * t o 104 e m b r y o s / m l ) ; c o n s e q u e n t l y t h e An~- a n d A , - f a c t o r s were n o t e f f e c t i v e in t h i s case. As c a n b e seen f r o m t h e s e results, t h e A n - f a c t o r s m a y c o m p r i s e e i t h e r t h e gangliosides t h e m s e l v e s (i.e. A n 1-
EXPERIENTIA 31/8
h e m a t o s i d e , A n 2 - m o n o s i a l o g a n g l i o s i d e ) or m i x t u r e s cont a i n i n g t h e s e gangliosides. To solve t h i s p r o b l e m it will b e n e c e s s a r y t o e s t i m a t e t h e c o n c e n t r a t i o n of t h e gangliosides in CSW. S u c h e x p e r i m e n t s m a y b e of i n t e r e s t in s t u d i e s of t h e biological role of gangliosides. T h e y also could b e h e l p f u l for u n d e r s t a n d i n g t h e i n t r a c e l l u l a r r e g u l a t o r y f u n c t i o n s of a c e t y l c h o l i n e a n d m o n o a m i n e s ~a. B b l B O j ~ b l . LIyBCTBIITeJIblIOCTb paHH~X aM6p~0HOB MopcK~x e>Kefi K 3M6pHOTOIZCHqec~cHMHefip0~apmalKaeTc~ np~ NOBbImen a ~ K0mleHTpa~HH 3M6pHoHoB. 9TOT 30p~eKT, n0-BH/IH0my, 06ycn0B~eH BS~/IeaeHaem B nHKy6atmoHnym cpe/Iy raHraa03H}IOB H3 3M6pHOHaJIbHBIX KHeTOK. l-[pH HCIIbITaHHH 3an[HTHOF0 j~e~CTBHfl ~paKI~Hfi FaHPJ-IH03H~0B, BbIAeneHHbIX H3
am6pu0HOB M0pCKIIX e>~eti, Han6onee 9qb(lleKTI4BIlbIMnaHTIIJIOTaMH IIp0THB HefipoOpapMaKoaoraqec~nx npenapaToB 6bma reMaTO3g//,ono/lo6Hag ~ppaKt~H~l H IIpOTt4a /leTepreHTOBqbpaKr~n~ MOHOCHanoraHrJ~Hoan~loB/ G. A. BUZNIKOV, N. D. ZVEZDINA, lX]'. V. PROKAZOVA, B. N. MANUKHIN a n d L. D. BERGELSON
Institute o/ Developmental Biology and Shemyakin Institute o/Bioorganic Chemistry USS R Academy o/ Sciences, Vavilov Street 26, Moskwa B-334 ( U S S R 777334), 77 December 1973.
13 The authors are deeply grateful to Drs E. V. DYATLOVITZKAYA, A. M. NOVIKOV (Shemyakin Institute of Bioorganic Chemistry, Moscow), V. l~. VASKOVSKY and V. V. Sov~, (Marine Biology Institute, Vladivostok) as well as to Prof. Dr. RAKId and his collaborators (International Laboratory for Brain Research, Kotor, Yugoslavia) for their assistance.
Intracellular Recording of Secretory Potentials in a 'Mixed' Salivary Gland Secretory potentials evoked by nerve stimulation have b e e n r e c o r d e d in m a m m a l i a n 1 , ~ a n d i n s e c t s s a l i v a r y glands. T h e s e responses, w h i c h c a n also b e elicited b y a p p l i c a t i o n s of n e u r o t r a n s m i t t e r s 4,5, are a p p a r e n t l y h y p e r p o l a r i z a t i o n s of t h e b a s a l cell m e m b r a n e Of a c i n a r cells. R e c e n t l y b i p h a s i e r e s p o n s e s ( d e p o l a r i z a t i o n followed by hyperpolarization) have been noted in cat and rabbit s u b m a x i l l a r y g l a n d s s. I t h a s b e e n a s s u m e d w i t h some j u s t i f i c a t i o n 7 t h a t s u c h s e c r e t o r y p o t e n t i a l s are g e n u i n e m e m b r a n e r e s p o n s e s a n d are r e c o r d e d w i t h t h e m i c r o e l e c t r o d e t i p inside a g l a n d cell. D i r e c t e v i d e n c e is lacking, h o w e v e r , t h a t u n d e r t h e s e c i r c u m s t a n c e s t h e m i c r o e l e c t r o d e is i n t r a c e l l u l a r . Moreover, in m o s t g l a n d s so f a r e x a m i n e d e l e c t r o p h y s i o logically t h e r e are a t l e a s t t w o cell t y p e s in e a c h acinus. F o r e x a m p l e , i n t h e c o c k r o a c h s a l i v a r y gland, w h i c h shows secretory potentials presumably homologous with t h o s e i n m a m m a l s , t h e a c i n u s consists of p e r i p h e r a l cells a n d c e n t r a l ceils. T h e m a i n f e a t u r e s of t h e s e cells h a v e b e e n d e s c r i b e d 8. T h e p e r i p h e r a l cell is n o t a b l e for its large i n t r a c e l l u l a r d u c t u l e ( c o n t i g u o u s w i t h t h e e x c r e t o r y duct) a n d i t s n u m e r o u s m i t o c h o n d r i a w h e r e a s t h e c e n t r a l cell h a s large g r a n u l e s p r o b a b l y c o n t a i n i n g e n z y m e s secreted by this gland. T h e a i m s of t h e p r e s e n t e x p e r i m e n t s were to e s t a b l i s h , first, w h e t h e r t h e t i p of t h e e l e c t r o d e h a d a n i n t r a c e l l u l a r location when a secretory potential was observed and, secondly, w h e t h e r s u c h r e s p o n s e s could b e r e c o r d e d f r o m b o t h p e r i p h e r a l a n d c e n t r a l cells.
Methods. S a l i v a r y g l a n d s were dissected f r o m cockroaches, Nauphoeta cinerea, k e p t u n d e r c o n d i t i o n s described p r e v i o u s l y s. T h e p r e p a r a t i o n w a s m o u n t e d i n a c h a m b e r ~ a n d p e r f u s e d w i t h a s o l u t i o n c o n t a i n i n g 160 m M NaC1, 1 m M KC1, 5 m M CaCI,, 1 m M N a H 2 P O * a n d 1 m M NaHCOa. T h e s a l i v a r y d u c t n e r v e s 10 w e r e d r a w n i n t o a s u c t i o n electrotle a n d s t i m u l a t e d w i t h pulses (0.5 msec, 10-60 V) f r o m a s q u a r e pulse s t i m u l a t o r . M i e r o e l e c t r o d e s were filled w i t h 5 % P r o c i o n Yellow (M-41~) b y 2 m e t h o d s . I n one, t h e electrodes were p u l l e d c o n v e n t i o n a l l y , t h e i r t i p s were b r o k e n b y gentle p r e s s u r e a g a i n s t tissue p a p e r a n d t h e y were t h e n back-filled f r o m a syringe w i t h a T o u h y - B o r s t a d a p t e r (Becton, D i c k i n s o n & Co.). T h e s e e l e c t r o d e s h a d r e s i s t a n c e s i n t h e r a n g e 1 0 30 MYJ. A l t e r n a t i v e l y n o r m a l u n b r o k e n electrodes were filled b y t h e m e t h o d d e s c r i b e d b y THOMASn; s u c h 1 A. LUNDBERG, Acta physiol, scand. 35, 1 (1955). A. LUNDBERG, Acta physiol, scand, d0, 21 (1957). 3 C. R. HOUSE, J. exp. Bid, 58, 29 (1973). 40. H. PETERSEN and J. H. POULSEN, Experientia 24, 919 (1968). C. R. HOUSE, B. g. GINSBORGand E. M. SILINSKY, Nature, New Biol. 245, 63 (1973). A. NISm'ZAMA and M. KACA'ZAMA,Experientia 29, 161 (1973). O. H. PETERSEN, Experientia 30, 130 (1974). s K. P. BLAND and C. R. HOUSE, J. Insect Physiol. 17, 2069 (1971). B. L. GINSBORG, C. R. HOUSE and E. M. SILINSKY, J. Physiol., Lond. 236, 723 (1974). l0 A. T. WHITEHEAD, J. Morph. 135, 483 (1971). 11 R. C. THOMAS, J. Physiol., Lond. 220, 55 (1972).
Peripheral cell
mV
0
b
Central cell
c
b
C
j
o~
50-E
-100 25 sec
Secretory p o t e n t i a l s recorded from peripheral and central cells, a) and d) are respectively fluorescent and l i g h t microscope pictures of identical sections showing t h a t each cell type has been stained w i t h Procion. In d) the borders of the ceils h a v e been a c c e n t u a t e d b y the i n t e r r u p t e d w h i t e lines, b) and c) show the secretory p o t e n t i a l s recorded from these ceils before and 10 rain after dye injection. To elicit the p e r i p h e r a l cell responses the nerve was s t i m u l a t e d w i t h 10 pulses a t 100 Hz; the central cell responses were e v o k e d b y a single stimulus. I t has been d e m o n s t r a t e d 1~ t h a t Procion Yellow ejected from high resistance (30-100 M ~ ) electrodes usually forms a p r e c i p i t a t e at their tips whereas t h a t from low resistance electrodes does not. These features of dye injection are e v i d e n t in a), as the peripheral cell was i m p a l e d w i t h a 20 M,Q electrode and has filled more e v e n l y t h a n the central cell i m p a l e d w i t h a 80 M~'~ electrode. However, the electrical records indicate t h a t smaller r e s t i n g and secretory p o t e n t i a l s were observed w i t h the b r o k e n tip electrodes (peripheral cell; b, c) t h a n those o b t a i n e d w i t h the higher resistance electrode (central cell; b, c). I t is considered t h a t the difference in m e m b r a n e p o t e n t i a l s is due to the different type of electrodes r a t h e r t h a n to some p r o p e r t y of the cell types.
906
Specialia
electrodes h a d r e s i s t a n c e s in t h e r a n g e of 50-100 Mr2 a n d r e c o r d e d larger r e s t i n g a n d s e c r e t o r y p o t e n t i a l s t h a n t h e other. T h e m e t h o d s of r e c o r d i n g m e m b r a n e p o t e n t i a l h a v e b e e n d e s c r i b e d p r e v i o u s l y 3, 9. T h e e x p e r i m e n t s were p e r f o r m e d in t h e following m a n n e r . A n a c i n u s was i m p a l e d w i t h a P r o c i o n electrode and, p r o v i d e d t h e r e s t i n g p o t e n t i a l did n o t decline, t h e s a l i v a r y d u c t n e r v e s were s t i m u l a t e d t o elicit a s e c r e t o r y p o t e n t i a l . P r o c i o n was i n j e c t e d i n t o t h e a c i n u s b y p a s s i n g h y p e r p o l a r i z i n g c u r r e n t pulses (10-20 nA, 100 msec) a t 5 H z t h r o u g h t h e m i c r o e l e c t r o d e . P r o c i o n i n j e c t i o n was c o n t i n u e d for periods of 5 m i n , i n t e r r u p t e d for a b o u t 1 m i n solely t o m o n i t o r r e s t i n g a n d s e c r e t o r y p o t e n t i a l s . T h e m i c r o e l e c t r o d e r e m a i n e d inside t h e cell for u p to 30 m i n as j u d g e d b y t h e s e criteria. T h e s a l i v a r y g l a n d was r e m o v e d f r o m t h e c h a m b e r a b o u t 30 m i n a f t e r t h e e n d of t h e e x p e r i m e n t a n d fixed i n 1 0 % formalin. A f t e r w a x e m b e d d i n g , 10 ~ m s e c t i o n s were p r e p a r e d a n d m o u n t e d in G u r r ' s U v i n e r t m o u n t a n t for e x a m i n a t i o n b y fluorescence m i c r o s c o p y a n d t h e n p h o t o g r a p h e d . T h e c o v e r slips were f l o a t e d off t h e slides b y p r o l o n g e d i m m e r s i o n in x y l e n e a n d t h e tissue sections s t a i n e d w i t h E h r l i c h ' s H a e m a t o x y lin a n d E o s i n a n d f i n a l l y m o u n t e d in G u r r ' s X A M for e x a m i n a t i o n b y l i g h t microscopy. Results and discussion. I n 11 e x p e r i m e n t s i t was d e m o n s t r a t e d t h a t t h e site of m i c r o e l e c t r o d e r e c o r d i n g of s e c r e t o r y p o t e n t i a l s was t h e i n t e r i o r of g l a n d cells. T h e F i g u r e shows r e p r e s e n t a t i v e results f r o m 2 e x p e r i m e n t s w h e r e r e s p o n s e s were o b t a i n e d f r o m e a c h of t h e t w o cell t y p e s p r e s e n t in t h e acinus. E x a m i n a t i o n w i t h l i g h t m i c r o s c o p y (d) e s t a b l i s h e d t h a t t h e cells s t a i n e d w i t h P r o c i o n Under fluorescence m i c r o s c o p y (a) were p e r i p h e r a l a n d c e n t r a l cells. I n sections s t a i n e d w i t h H a e m a t o x y l i n a n d E o s i n t h e p e r i p h e r a l cells a p p e a r pink, are g e n e r a l l y p y r a m i d a l in s h a p e a n d s h o w a p r o m i n e n t b r u s h b o r d e r lining t h e l u m e n of a n i n t r a c e l l u l a r d u c t u l e . I n F i g u r e a) ( p e r i p h e r a l cell) t h e l u m e n of t h i s d u c t u l e c a n be seen to b e d i s t e n d e d a n d free of P r o c i o n Yellow. Some of t h e dye a p p e a r s to h a v e s p r e a d f r o m t h e p e r i p h e r a l cell across its a p i c a l m a r g i n s to n e i g h b o u r i n g c e n t r a l ceils. T h e c e n t r a l cells s t a i n p u r p l e to v a r y i n g degrees, s h o w t h e p r e s e n c e of large g r a n u l e s a n d do n o t c o n t a i n a n i n t r a c e l l u l a r
A Preliminary
Report on the Fine Structure of
EXPERIENTIA 31/8
d u c t u l e since t h e y d i s c h a r g e t h e i r s e c r e t i o n i n t o t h e c e n t r a l l u m e n of t h e a c i n u s s. I t c a n b e seen i n F i g u r e a) ( c e n t r a l cell) t h a t P r o c i o n Yellow h a s b e e n d e p o s i t e d p r e f e r e n t i a l l y in t h e nucleus, a l t h o u g h t h i s m a y be fortuitous. I n 9 o t h e r e x p e r i m e n t s i t was f o u n d t h a t 3 of t h e cells were p e r i p h e r a l a n d 6 were c e n t r a l . T h u s i t seems t h a t r e s p o n s e s c a n b e r e c o r d e d f r o m e i t h e r k i n d of cell a n d t h e r e f o r e , i n t h e l i g h t of t h e p r e v i o u s e v i d e n c e 3, f r o m a n y cell in a n acinus. I t seems u n l i k e l y t h a t e a c h cell h a s its o w n i n n e r v a t i o n i n v i e w of t h e sparse n u m b e r of n e r v e fibres r u n n i n g to acini 10. A l t h o u g h t h i s p o s s i b i l i t y c a n n o t b e e x c l u d e d b y t h e p r e s e n t e x p e r i m e n t s , i t seems m o r e p r o b a b l e t h a t a c i n a r cells, e v e n of d i f f e r e n t kinds, are coupled electrically. I t m i g h t b e t h a t a s e c r e t o r y p o t e n t i a l is e v o k e d in a single cell, or p e r h a p s a few cells, a n d spreads electrotonically. Electrophysiological evidence for electrical c o u p l i n g in t h i s g l a n d h a s a l r e a d y b e e n r e p o r t e d 9 a n d is c o n s i s t e n t w i t h t h e p r e s e n c e of s e p t a t e d e s m o s o m e s s b e t w e e n a c i n a r cells. Also c o n s i s t e n t w i t h t h i s s u g g e s t i o n is t h e o b s e r v a t i o n t h a t in t h e e x p e r i m e n t s d e s c r i b e d a b o v e ( p e r i p h e r a l cell, a), P r o c i o n Yellow a p p e a r s able t o s p r e a d f r o m one cell t o a n o t h e r ; h o w e v e r , f u r t h e r e x p e r i m e n t s are r e q u i r e d t o i n v e s t i g a t e t h i s p o i n t .
Summary. S e c r e t o r y p o t e n t i a l s e v o k e d b y n e r v e s t i m u l a t i o n h a v e b e e n r e c o r d e d f r o m b o t h t y p e s of cell (per i p h e r a l a n d c e n t r a l ) p r e s e n t in t h e acini of c o c k r o a c h s a l i v a r y glands. C. R. HOUSE 13 Department o[ Veterinary Physiology, University o/ Edinburgh, Edinburgh EH9 1QH (Scotland), 28th February 7975.
12 j. N. BARRETT, in Intracellular Staining in Neurobiology (Eds. S. B. I{ATER and C. NICHOLSON; Springer-Verlag, New York 1973), p. 283. 13 Acknowledgments. I am indebted to Mr. S. ROBERTSON for histological work and Dr. B. L. GIIVSBORGfor comments on this paper. I also wish to thank I.C.I. for a gift of Procion Yellow.
Tripneustes esculentus
T h e p a s t d e c a d e h a s g i v e n us m u c h i n f o r m a t i o n a b o u t t h e s t r u c t u r a l o r g a n i z a t i o n of unfertilized, fertilized a n d c e n t r i f u g e d sea u r c h i n eggs 2-4, b u t t h e m e t h o d s of f i x a t i o n e m p l o y e d in t h o s e s t u d i e s r e s u l t e d m o s t l y in g e n e r a l coarse p r e s e r v a t i o n of c y t o p l a s m i c organelles. I t is e s s e n t i a l l y b e c a u s e of t h e i m p o r t a n c e of t h e sea u r c h i n egg in t h e s t u d y of e m b r y o l o g y t h a t w o r k i n v o l v i n g m o d e r n f i x a t i o n m e t h o d s of t h i s m a t e r i a l for e l e c t r o n m i c r o s c o p y o b s e r v a t i o n h a s b e e n in progress in o u r l a b o r a t o r y for some t i m e . O u r a i m was t o a r r i v e a t a s u i t a b l e f i x a t i o n p r o c e d u r e t h a t e n a b l e us to m a k e m o r e m e a n i n g f u l i n t e r p r e t a t i o n s of t h e u l t r a s t r u c t u r e of t h e eggs in n o r m a l or e x p e r i m e n t a l d e v e l o p m e n t . T h e p u r p o s e of t h i s c o m m u n i c a t i o n is t o r e p o r t t h e b e s t p r e s e r v a t i o n for eggs of t h e sea u r c h i n Tripneustes esculentus so far o b t a i n e d t h r o u g h e x t e n s i v e screening a n d m o d i f i c a t i o n of v a r i o u s m e t h o d s . Methodology. Sea u r c h i n s of t h e species Tripneustes esculentus a n d sea w a t e r were o b t a i n e d f r o m t h e C a r r i b e a n R e s e a r c h Centre, M a r t i n i q u e , a n d k e p t a t 22 ~ for a few hours. T h e eggs, o b t a i n e d b y i n j e c t i o n of 0.53 M KC1 in t h e v i c i n i t y of t h e oral region, were g e n t l y filtered t h r o u g h
Eggs 1
c h e e s e - c l o t h a n d r e p e a t e d l y w a s h e d w i t h milliporefiltered sea water. T h e eggs were t h e n fixed in t h e following p H 7.4 m i x t u r e : G l u t a r a l d e h y d e - p a r a f o r m a l d e h y d e (50%), p r e p a r e d in M a r t i n i q u e sea w a t e r . T h e c h e m i c a l s were m i x e d p r e v i o u s to use, a n d t h e w a s h e d cells were s u s p e n d e d in t h i s s o l u t i o n for o n l y 5 m i n a t r o o m t e m p e r a t u r e a p p r o x i m a t e l y (23 ~ Since longer f i x a t i o n periods c a n r e s u l t in l e a c h i n g o u t of t h e p i g m e n t granules, t h e f i x a t i o n t i m e w a s k e p t a t a m i n i m u m , a n d 5 mill was f o u n d to b e m o s t a p p r o p r i a t e . T h i s was f o l l o w e d b y t r e a t m e n t of t h e eggs w i t h a 1 % s o l u t i o n of o s m i u m t e t r o x i d e (OsO4) in sea w a t e r a t 4~ T h e eggs were t h e n d e h y d r a t e d in E p o n u s i n g s t a n d a r d t e c h n i q u e s . To i m p r o v e c o n t r a s t , t h e sections were doublestained with uranyl nitrate and citrate. The preparations were e x a m i n e d w i t h a J E M 7 - A e l e c t r o n microscope. i This work was supported by a grant from the National Research - Council of Canada. 2 F. W. FLITNEY, J. R. microsc. Soc. 85, 353 (1966). a IV[. GOUSKENS, Exp1 Cell Res. 39, 413 (1965). 4 j. j . PASTEELS, P. CASTIAUX and G. VANDERNE~RSSCRE, Arch.
Biol. 69, 627 (1958).
Specialia
tion against the two neuropharmaca but not against the detergent. At the same time, monosialo-ganglioside and monosialo-ganglioside-like fractions from the blastulae were n o t a c t i v e a g a i n s t n e u r o p h a r m a c a , b u t s h o w e d p r o t e c t i v e a c t i o n a g a i n s t t h e d e t e r g e n t OP-10. T h e effective p r o t e c t i v e c o n c e n t r a t i o n s of t h e gangliosides are g i v e n i n t h e Table. T h e s e c o n c e n t r a t i o n s are f r o m 20 t o 1000 t i m e s lower t h a n t h e t o x i c c o n c e n t r a t i o n s of t h e n e u r o p h a r m a c a a n d 10-20 t i m e s lower t h a n t h e c o n c e n t r a t i o n s of t h e d e t e r g e n t . A t t h e s a m e t i m e , t h e di- a n d trisialoganglioside-like f r a c t i o n s i s o l a t e d f r o m sea u r c h i n e m b r y o s were n o t effective e i t h e r a g a i n s t n e u r o p h a r m a c a or a g a i n s t t h e d e t e r g e n t OP-10. T h e f a c t o r d e c r e a s i n g t h e s e n s i t i v i t y of e m b r y o s t o n e u r o p h a r m a c a w a s s h o w n also t o b e p r e s e n t in t h e c h l o r o f o r m - m e t h a n o l e x t r a c t of c o n d i t i o n e d sea w a t e r (CSW), i.e. sea water, i n w h i c h dense s u s p e n s i o n s ( a b o u t 30,000 e m b r y o s / m l ) of S. intermedius e m b r y o s w e r e incubated. T L C of t h e C S W c h l o r o f o r m - m e t h a n o l e x t r a c t r e v e a l e d t h e p r e s e n c e of r e s o r c i n e - p o s i t i v e s u b s t a n c e s coinciding, by their Rf-values, with the hematoside-like fraction of sea u r c h i n e m b r y o s a n d w i t h r a t l i v e r h e m a t o s i d e (Figure). W e f o u n d t h a t t h e sialic acid c o n c e n t r a t i o n in C S W was a b o u t 1 • .5 M . N e i t h e r t h e gangliosides f r o m t h e sea u r c h i n e m b r y o s or f r o m r a t l i v e r n o r t h e c h l o r o f o r m - m e t h a n o l e x t r a c t of t h e C S W or C S W itself a f f e c t e d t h e s e n s i t i v i t y of t h e e m b r y o s t o p u r o m y c i n . I n p r e v i o u s e x p e r i m e n t s ~, i t was s h o w n t h a t t h e s e n s i t i v i t y of t h e e m b r y o s t o t h i s a n t i b i o t i c did n o t d e p e n d o n t h e i r c o n c e n t r a t i o n (in t h e c o n c e n t r a t i o n i n t e r v a l f r o m 10 * t o 104 e m b r y o s / m l ) ; c o n s e q u e n t l y t h e An~- a n d A , - f a c t o r s were n o t e f f e c t i v e in t h i s case. As c a n b e seen f r o m t h e s e results, t h e A n - f a c t o r s m a y c o m p r i s e e i t h e r t h e gangliosides t h e m s e l v e s (i.e. A n 1-
EXPERIENTIA 31/8
h e m a t o s i d e , A n 2 - m o n o s i a l o g a n g l i o s i d e ) or m i x t u r e s cont a i n i n g t h e s e gangliosides. To solve t h i s p r o b l e m it will b e n e c e s s a r y t o e s t i m a t e t h e c o n c e n t r a t i o n of t h e gangliosides in CSW. S u c h e x p e r i m e n t s m a y b e of i n t e r e s t in s t u d i e s of t h e biological role of gangliosides. T h e y also could b e h e l p f u l for u n d e r s t a n d i n g t h e i n t r a c e l l u l a r r e g u l a t o r y f u n c t i o n s of a c e t y l c h o l i n e a n d m o n o a m i n e s ~a. B b l B O j ~ b l . LIyBCTBIITeJIblIOCTb paHH~X aM6p~0HOB MopcK~x e>Kefi K 3M6pHOTOIZCHqec~cHMHefip0~apmalKaeTc~ np~ NOBbImen a ~ K0mleHTpa~HH 3M6pHoHoB. 9TOT 30p~eKT, n0-BH/IH0my, 06ycn0B~eH BS~/IeaeHaem B nHKy6atmoHnym cpe/Iy raHraa03H}IOB H3 3M6pHOHaJIbHBIX KHeTOK. l-[pH HCIIbITaHHH 3an[HTHOF0 j~e~CTBHfl ~paKI~Hfi FaHPJ-IH03H~0B, BbIAeneHHbIX H3
am6pu0HOB M0pCKIIX e>~eti, Han6onee 9qb(lleKTI4BIlbIMnaHTIIJIOTaMH IIp0THB HefipoOpapMaKoaoraqec~nx npenapaToB 6bma reMaTO3g//,ono/lo6Hag ~ppaKt~H~l H IIpOTt4a /leTepreHTOBqbpaKr~n~ MOHOCHanoraHrJ~Hoan~loB/ G. A. BUZNIKOV, N. D. ZVEZDINA, lX]'. V. PROKAZOVA, B. N. MANUKHIN a n d L. D. BERGELSON
Institute o/ Developmental Biology and Shemyakin Institute o/Bioorganic Chemistry USS R Academy o/ Sciences, Vavilov Street 26, Moskwa B-334 ( U S S R 777334), 77 December 1973.
13 The authors are deeply grateful to Drs E. V. DYATLOVITZKAYA, A. M. NOVIKOV (Shemyakin Institute of Bioorganic Chemistry, Moscow), V. l~. VASKOVSKY and V. V. Sov~, (Marine Biology Institute, Vladivostok) as well as to Prof. Dr. RAKId and his collaborators (International Laboratory for Brain Research, Kotor, Yugoslavia) for their assistance.
Intracellular Recording of Secretory Potentials in a 'Mixed' Salivary Gland Secretory potentials evoked by nerve stimulation have b e e n r e c o r d e d in m a m m a l i a n 1 , ~ a n d i n s e c t s s a l i v a r y glands. T h e s e responses, w h i c h c a n also b e elicited b y a p p l i c a t i o n s of n e u r o t r a n s m i t t e r s 4,5, are a p p a r e n t l y h y p e r p o l a r i z a t i o n s of t h e b a s a l cell m e m b r a n e Of a c i n a r cells. R e c e n t l y b i p h a s i e r e s p o n s e s ( d e p o l a r i z a t i o n followed by hyperpolarization) have been noted in cat and rabbit s u b m a x i l l a r y g l a n d s s. I t h a s b e e n a s s u m e d w i t h some j u s t i f i c a t i o n 7 t h a t s u c h s e c r e t o r y p o t e n t i a l s are g e n u i n e m e m b r a n e r e s p o n s e s a n d are r e c o r d e d w i t h t h e m i c r o e l e c t r o d e t i p inside a g l a n d cell. D i r e c t e v i d e n c e is lacking, h o w e v e r , t h a t u n d e r t h e s e c i r c u m s t a n c e s t h e m i c r o e l e c t r o d e is i n t r a c e l l u l a r . Moreover, in m o s t g l a n d s so f a r e x a m i n e d e l e c t r o p h y s i o logically t h e r e are a t l e a s t t w o cell t y p e s in e a c h acinus. F o r e x a m p l e , i n t h e c o c k r o a c h s a l i v a r y gland, w h i c h shows secretory potentials presumably homologous with t h o s e i n m a m m a l s , t h e a c i n u s consists of p e r i p h e r a l cells a n d c e n t r a l ceils. T h e m a i n f e a t u r e s of t h e s e cells h a v e b e e n d e s c r i b e d 8. T h e p e r i p h e r a l cell is n o t a b l e for its large i n t r a c e l l u l a r d u c t u l e ( c o n t i g u o u s w i t h t h e e x c r e t o r y duct) a n d i t s n u m e r o u s m i t o c h o n d r i a w h e r e a s t h e c e n t r a l cell h a s large g r a n u l e s p r o b a b l y c o n t a i n i n g e n z y m e s secreted by this gland. T h e a i m s of t h e p r e s e n t e x p e r i m e n t s were to e s t a b l i s h , first, w h e t h e r t h e t i p of t h e e l e c t r o d e h a d a n i n t r a c e l l u l a r location when a secretory potential was observed and, secondly, w h e t h e r s u c h r e s p o n s e s could b e r e c o r d e d f r o m b o t h p e r i p h e r a l a n d c e n t r a l cells.
Methods. S a l i v a r y g l a n d s were dissected f r o m cockroaches, Nauphoeta cinerea, k e p t u n d e r c o n d i t i o n s described p r e v i o u s l y s. T h e p r e p a r a t i o n w a s m o u n t e d i n a c h a m b e r ~ a n d p e r f u s e d w i t h a s o l u t i o n c o n t a i n i n g 160 m M NaC1, 1 m M KC1, 5 m M CaCI,, 1 m M N a H 2 P O * a n d 1 m M NaHCOa. T h e s a l i v a r y d u c t n e r v e s 10 w e r e d r a w n i n t o a s u c t i o n electrotle a n d s t i m u l a t e d w i t h pulses (0.5 msec, 10-60 V) f r o m a s q u a r e pulse s t i m u l a t o r . M i e r o e l e c t r o d e s were filled w i t h 5 % P r o c i o n Yellow (M-41~) b y 2 m e t h o d s . I n one, t h e electrodes were p u l l e d c o n v e n t i o n a l l y , t h e i r t i p s were b r o k e n b y gentle p r e s s u r e a g a i n s t tissue p a p e r a n d t h e y were t h e n back-filled f r o m a syringe w i t h a T o u h y - B o r s t a d a p t e r (Becton, D i c k i n s o n & Co.). T h e s e e l e c t r o d e s h a d r e s i s t a n c e s i n t h e r a n g e 1 0 30 MYJ. A l t e r n a t i v e l y n o r m a l u n b r o k e n electrodes were filled b y t h e m e t h o d d e s c r i b e d b y THOMASn; s u c h 1 A. LUNDBERG, Acta physiol, scand. 35, 1 (1955). A. LUNDBERG, Acta physiol, scand, d0, 21 (1957). 3 C. R. HOUSE, J. exp. Bid, 58, 29 (1973). 40. H. PETERSEN and J. H. POULSEN, Experientia 24, 919 (1968). C. R. HOUSE, B. g. GINSBORGand E. M. SILINSKY, Nature, New Biol. 245, 63 (1973). A. NISm'ZAMA and M. KACA'ZAMA,Experientia 29, 161 (1973). O. H. PETERSEN, Experientia 30, 130 (1974). s K. P. BLAND and C. R. HOUSE, J. Insect Physiol. 17, 2069 (1971). B. L. GINSBORG, C. R. HOUSE and E. M. SILINSKY, J. Physiol., Lond. 236, 723 (1974). l0 A. T. WHITEHEAD, J. Morph. 135, 483 (1971). 11 R. C. THOMAS, J. Physiol., Lond. 220, 55 (1972).
Peripheral cell
mV
0
b
Central cell
c
b
C
j
o~
50-E
-100 25 sec
Secretory p o t e n t i a l s recorded from peripheral and central cells, a) and d) are respectively fluorescent and l i g h t microscope pictures of identical sections showing t h a t each cell type has been stained w i t h Procion. In d) the borders of the ceils h a v e been a c c e n t u a t e d b y the i n t e r r u p t e d w h i t e lines, b) and c) show the secretory p o t e n t i a l s recorded from these ceils before and 10 rain after dye injection. To elicit the p e r i p h e r a l cell responses the nerve was s t i m u l a t e d w i t h 10 pulses a t 100 Hz; the central cell responses were e v o k e d b y a single stimulus. I t has been d e m o n s t r a t e d 1~ t h a t Procion Yellow ejected from high resistance (30-100 M ~ ) electrodes usually forms a p r e c i p i t a t e at their tips whereas t h a t from low resistance electrodes does not. These features of dye injection are e v i d e n t in a), as the peripheral cell was i m p a l e d w i t h a 20 M,Q electrode and has filled more e v e n l y t h a n the central cell i m p a l e d w i t h a 80 M~'~ electrode. However, the electrical records indicate t h a t smaller r e s t i n g and secretory p o t e n t i a l s were observed w i t h the b r o k e n tip electrodes (peripheral cell; b, c) t h a n those o b t a i n e d w i t h the higher resistance electrode (central cell; b, c). I t is considered t h a t the difference in m e m b r a n e p o t e n t i a l s is due to the different type of electrodes r a t h e r t h a n to some p r o p e r t y of the cell types.
906
Specialia
electrodes h a d r e s i s t a n c e s in t h e r a n g e of 50-100 Mr2 a n d r e c o r d e d larger r e s t i n g a n d s e c r e t o r y p o t e n t i a l s t h a n t h e other. T h e m e t h o d s of r e c o r d i n g m e m b r a n e p o t e n t i a l h a v e b e e n d e s c r i b e d p r e v i o u s l y 3, 9. T h e e x p e r i m e n t s were p e r f o r m e d in t h e following m a n n e r . A n a c i n u s was i m p a l e d w i t h a P r o c i o n electrode and, p r o v i d e d t h e r e s t i n g p o t e n t i a l did n o t decline, t h e s a l i v a r y d u c t n e r v e s were s t i m u l a t e d t o elicit a s e c r e t o r y p o t e n t i a l . P r o c i o n was i n j e c t e d i n t o t h e a c i n u s b y p a s s i n g h y p e r p o l a r i z i n g c u r r e n t pulses (10-20 nA, 100 msec) a t 5 H z t h r o u g h t h e m i c r o e l e c t r o d e . P r o c i o n i n j e c t i o n was c o n t i n u e d for periods of 5 m i n , i n t e r r u p t e d for a b o u t 1 m i n solely t o m o n i t o r r e s t i n g a n d s e c r e t o r y p o t e n t i a l s . T h e m i c r o e l e c t r o d e r e m a i n e d inside t h e cell for u p to 30 m i n as j u d g e d b y t h e s e criteria. T h e s a l i v a r y g l a n d was r e m o v e d f r o m t h e c h a m b e r a b o u t 30 m i n a f t e r t h e e n d of t h e e x p e r i m e n t a n d fixed i n 1 0 % formalin. A f t e r w a x e m b e d d i n g , 10 ~ m s e c t i o n s were p r e p a r e d a n d m o u n t e d in G u r r ' s U v i n e r t m o u n t a n t for e x a m i n a t i o n b y fluorescence m i c r o s c o p y a n d t h e n p h o t o g r a p h e d . T h e c o v e r slips were f l o a t e d off t h e slides b y p r o l o n g e d i m m e r s i o n in x y l e n e a n d t h e tissue sections s t a i n e d w i t h E h r l i c h ' s H a e m a t o x y lin a n d E o s i n a n d f i n a l l y m o u n t e d in G u r r ' s X A M for e x a m i n a t i o n b y l i g h t microscopy. Results and discussion. I n 11 e x p e r i m e n t s i t was d e m o n s t r a t e d t h a t t h e site of m i c r o e l e c t r o d e r e c o r d i n g of s e c r e t o r y p o t e n t i a l s was t h e i n t e r i o r of g l a n d cells. T h e F i g u r e shows r e p r e s e n t a t i v e results f r o m 2 e x p e r i m e n t s w h e r e r e s p o n s e s were o b t a i n e d f r o m e a c h of t h e t w o cell t y p e s p r e s e n t in t h e acinus. E x a m i n a t i o n w i t h l i g h t m i c r o s c o p y (d) e s t a b l i s h e d t h a t t h e cells s t a i n e d w i t h P r o c i o n Under fluorescence m i c r o s c o p y (a) were p e r i p h e r a l a n d c e n t r a l cells. I n sections s t a i n e d w i t h H a e m a t o x y l i n a n d E o s i n t h e p e r i p h e r a l cells a p p e a r pink, are g e n e r a l l y p y r a m i d a l in s h a p e a n d s h o w a p r o m i n e n t b r u s h b o r d e r lining t h e l u m e n of a n i n t r a c e l l u l a r d u c t u l e . I n F i g u r e a) ( p e r i p h e r a l cell) t h e l u m e n of t h i s d u c t u l e c a n be seen to b e d i s t e n d e d a n d free of P r o c i o n Yellow. Some of t h e dye a p p e a r s to h a v e s p r e a d f r o m t h e p e r i p h e r a l cell across its a p i c a l m a r g i n s to n e i g h b o u r i n g c e n t r a l ceils. T h e c e n t r a l cells s t a i n p u r p l e to v a r y i n g degrees, s h o w t h e p r e s e n c e of large g r a n u l e s a n d do n o t c o n t a i n a n i n t r a c e l l u l a r
A Preliminary
Report on the Fine Structure of
EXPERIENTIA 31/8
d u c t u l e since t h e y d i s c h a r g e t h e i r s e c r e t i o n i n t o t h e c e n t r a l l u m e n of t h e a c i n u s s. I t c a n b e seen i n F i g u r e a) ( c e n t r a l cell) t h a t P r o c i o n Yellow h a s b e e n d e p o s i t e d p r e f e r e n t i a l l y in t h e nucleus, a l t h o u g h t h i s m a y be fortuitous. I n 9 o t h e r e x p e r i m e n t s i t was f o u n d t h a t 3 of t h e cells were p e r i p h e r a l a n d 6 were c e n t r a l . T h u s i t seems t h a t r e s p o n s e s c a n b e r e c o r d e d f r o m e i t h e r k i n d of cell a n d t h e r e f o r e , i n t h e l i g h t of t h e p r e v i o u s e v i d e n c e 3, f r o m a n y cell in a n acinus. I t seems u n l i k e l y t h a t e a c h cell h a s its o w n i n n e r v a t i o n i n v i e w of t h e sparse n u m b e r of n e r v e fibres r u n n i n g to acini 10. A l t h o u g h t h i s p o s s i b i l i t y c a n n o t b e e x c l u d e d b y t h e p r e s e n t e x p e r i m e n t s , i t seems m o r e p r o b a b l e t h a t a c i n a r cells, e v e n of d i f f e r e n t kinds, are coupled electrically. I t m i g h t b e t h a t a s e c r e t o r y p o t e n t i a l is e v o k e d in a single cell, or p e r h a p s a few cells, a n d spreads electrotonically. Electrophysiological evidence for electrical c o u p l i n g in t h i s g l a n d h a s a l r e a d y b e e n r e p o r t e d 9 a n d is c o n s i s t e n t w i t h t h e p r e s e n c e of s e p t a t e d e s m o s o m e s s b e t w e e n a c i n a r cells. Also c o n s i s t e n t w i t h t h i s s u g g e s t i o n is t h e o b s e r v a t i o n t h a t in t h e e x p e r i m e n t s d e s c r i b e d a b o v e ( p e r i p h e r a l cell, a), P r o c i o n Yellow a p p e a r s able t o s p r e a d f r o m one cell t o a n o t h e r ; h o w e v e r , f u r t h e r e x p e r i m e n t s are r e q u i r e d t o i n v e s t i g a t e t h i s p o i n t .
Summary. S e c r e t o r y p o t e n t i a l s e v o k e d b y n e r v e s t i m u l a t i o n h a v e b e e n r e c o r d e d f r o m b o t h t y p e s of cell (per i p h e r a l a n d c e n t r a l ) p r e s e n t in t h e acini of c o c k r o a c h s a l i v a r y glands. C. R. HOUSE 13 Department o[ Veterinary Physiology, University o/ Edinburgh, Edinburgh EH9 1QH (Scotland), 28th February 7975.
12 j. N. BARRETT, in Intracellular Staining in Neurobiology (Eds. S. B. I{ATER and C. NICHOLSON; Springer-Verlag, New York 1973), p. 283. 13 Acknowledgments. I am indebted to Mr. S. ROBERTSON for histological work and Dr. B. L. GIIVSBORGfor comments on this paper. I also wish to thank I.C.I. for a gift of Procion Yellow.
Tripneustes esculentus
T h e p a s t d e c a d e h a s g i v e n us m u c h i n f o r m a t i o n a b o u t t h e s t r u c t u r a l o r g a n i z a t i o n of unfertilized, fertilized a n d c e n t r i f u g e d sea u r c h i n eggs 2-4, b u t t h e m e t h o d s of f i x a t i o n e m p l o y e d in t h o s e s t u d i e s r e s u l t e d m o s t l y in g e n e r a l coarse p r e s e r v a t i o n of c y t o p l a s m i c organelles. I t is e s s e n t i a l l y b e c a u s e of t h e i m p o r t a n c e of t h e sea u r c h i n egg in t h e s t u d y of e m b r y o l o g y t h a t w o r k i n v o l v i n g m o d e r n f i x a t i o n m e t h o d s of t h i s m a t e r i a l for e l e c t r o n m i c r o s c o p y o b s e r v a t i o n h a s b e e n in progress in o u r l a b o r a t o r y for some t i m e . O u r a i m was t o a r r i v e a t a s u i t a b l e f i x a t i o n p r o c e d u r e t h a t e n a b l e us to m a k e m o r e m e a n i n g f u l i n t e r p r e t a t i o n s of t h e u l t r a s t r u c t u r e of t h e eggs in n o r m a l or e x p e r i m e n t a l d e v e l o p m e n t . T h e p u r p o s e of t h i s c o m m u n i c a t i o n is t o r e p o r t t h e b e s t p r e s e r v a t i o n for eggs of t h e sea u r c h i n Tripneustes esculentus so far o b t a i n e d t h r o u g h e x t e n s i v e screening a n d m o d i f i c a t i o n of v a r i o u s m e t h o d s . Methodology. Sea u r c h i n s of t h e species Tripneustes esculentus a n d sea w a t e r were o b t a i n e d f r o m t h e C a r r i b e a n R e s e a r c h Centre, M a r t i n i q u e , a n d k e p t a t 22 ~ for a few hours. T h e eggs, o b t a i n e d b y i n j e c t i o n of 0.53 M KC1 in t h e v i c i n i t y of t h e oral region, were g e n t l y filtered t h r o u g h
Eggs 1
c h e e s e - c l o t h a n d r e p e a t e d l y w a s h e d w i t h milliporefiltered sea water. T h e eggs were t h e n fixed in t h e following p H 7.4 m i x t u r e : G l u t a r a l d e h y d e - p a r a f o r m a l d e h y d e (50%), p r e p a r e d in M a r t i n i q u e sea w a t e r . T h e c h e m i c a l s were m i x e d p r e v i o u s to use, a n d t h e w a s h e d cells were s u s p e n d e d in t h i s s o l u t i o n for o n l y 5 m i n a t r o o m t e m p e r a t u r e a p p r o x i m a t e l y (23 ~ Since longer f i x a t i o n periods c a n r e s u l t in l e a c h i n g o u t of t h e p i g m e n t granules, t h e f i x a t i o n t i m e w a s k e p t a t a m i n i m u m , a n d 5 mill was f o u n d to b e m o s t a p p r o p r i a t e . T h i s was f o l l o w e d b y t r e a t m e n t of t h e eggs w i t h a 1 % s o l u t i o n of o s m i u m t e t r o x i d e (OsO4) in sea w a t e r a t 4~ T h e eggs were t h e n d e h y d r a t e d in E p o n u s i n g s t a n d a r d t e c h n i q u e s . To i m p r o v e c o n t r a s t , t h e sections were doublestained with uranyl nitrate and citrate. The preparations were e x a m i n e d w i t h a J E M 7 - A e l e c t r o n microscope. i This work was supported by a grant from the National Research - Council of Canada. 2 F. W. FLITNEY, J. R. microsc. Soc. 85, 353 (1966). a IV[. GOUSKENS, Exp1 Cell Res. 39, 413 (1965). 4 j. j . PASTEELS, P. CASTIAUX and G. VANDERNE~RSSCRE, Arch.
Biol. 69, 627 (1958).
