15. 3. 1975
309
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a c t i v i t y . Cerebellar e x p l a n t s (Figure 1) c h a r a c t e r i s t i c a l l y g a v e r a p i d b u r s t s of spikes o c c u r r i n g e v e r y 100 msec a n d h a v i n g a d u r a t i o n of 50 msec. Some cerebellar c u l t u r e s were o b s e r v e d t o h a v e periodicities of a longer n a t u r e : e v e r y m i n u t e w i t h b u r s t s of 30 sec. T h e l o n g e s t p e r i o d s were o b s e r v e d in e x p l a n t s of t h e m i d b r a i n area (Figure 2) h a v i n g i n t e r v a l s of g e n e r a l l y 4 t o 7 m i n b u t as long as 10 rain y i e l d i n g a c a s c a d i n g firing p a t t e r n of o v e r 1.0 r a i n d u r a t i o n while t h e r e s t of t h e p e r i o d r e m a i n e d e s s e n t i a l l y quiet. T h e s e c a s c a d e s could b e d r i v e n b u t e v i d e n c e d a r e l a t i v e r e f r a c t o r i n e s s t o successive electric shocks. E x p l a n t s t r o m t h e colliculi g a v e p e a k s of a c t i v i t y h a v i n g a s e r r a t e d a p p e a r a n c e t o t h e spike d i s c h a r g e p a t t e r n w i t h periods of a p p r o x i m a t e l y 4 m i n in length. N o t e v e r y e x p l a n t p r o d u c e d a r h y t h m i c p a t t e r n of neuronal activity but when present the type and pattern of spike d i s c h a r g e was s u f f i c i e n t l y c o n s i s t e n t to b e a reliable c h a r a c t e r i s t i c of e x p l a n t s t a k e n f r o m a p a r t i c u l a r a r e a of b r a i n . E x p l a n t s f r o m t h e m i d b r a i n a r e a p r o d u c e d r h y t h m i c a l d i s c h a r g e s 2 0 % of t h e t i m e , c e r e b e l l u m 10%,
D.c.
P
5 min
Fig. 2. Cascades of neuronal activity occurring every 6.5 min in an explant from midbrain 16 days of age. The upper trace indicates the quantity of neuronal spikes counted by the digital frequency meter and the lower trace indicates the de difference between the fluid in the upper and lower chambers. This pattern of activity has been continuous for more than 45 min. It was only altered by shocks at a, b and c where a current density of 9.5 • i 0 -~ Amps per ~xm~ was delivered for 0.05, 0.5 and 5 reset. It can be seen that successive shocks with lengthened duration had less affect indicating refractoriness of the tissue. Hole measured 200 ~xnl in diameter.
a n d colliculus 5 %. E x p l a n t s n o t i n i t i a l l y s h o w i n g r h y t h m i c n e u r o n a l firing w o u l d o c c a s i o n a l l y do so l a t e r e i t h e r s p o n t a n e o u s l y or would b e i n d u c e d t o d o so b y small, brief shocks of t h e o r d e r of 10 -~ Amp/Ezm e g i v e n for a d u r a t i o n of 1.0 msec. T h e r h y t h m i c p a t t e r n once i n i t i a t e d w o u l d c o n t i n u e for o v e r 1 h w i t h l i t t l e v a r i a t i o n . E v e n t u ally t h e spike p r o d u c t i o n failed, i n d i c a t e d b y g r a d u a l d i m i n u t i o n of t h e n u m b e r of spikes g e n e r a t e d d u r i n g each episode u n t i l f i n a l l y n o n e was p r o d u c e d . T h e p a t t e r n could b e r e v i v e d b r i e f l y b y e x c h a n g i n g t h e fluid in t h e r e c o r d i n g c h a m b e r . I n v e s t i g a t i o n s were m a d e a t r o o m t e m p e r a t u r e (27 ~ Of t h e b r a i n areas s t u d i e d c e r e b r a l cortical tissue a l o n e d i d n o t d e m o n s t r a t e a r e g u l a r p a t t e r n of a c t i v i t y ; h o w e v e r , t h e r e was o b s e r v e a i r r e g u l a r slow w a v e a c t i v i t y h a v i n g a d u r a t i o n of 100 msec a n d a n a m p l i t u d e of 500 ~V ( a p p r o x i m a t e l y 5 t i m e s t h e a m p l i t u d e of spikes) w h i c h a p p e a r e d t o i n i t i a t e n e u r o n a l spikes. T h e a c t i v i t y h a s b e e n r e p o r t e d b y CALVET 5 w h o suggests t h a t i t m a y r e s u l t f r o m t h e s i m u l t a n e o u s a c t i v a t i o n of p o s t s y n a p t i c p o t e n t i a l s , l u a n y case, t h e r e m u s t be a genetic c o m p o n e n t to a c c o u n t for t h e u n i f o r m i t y of a c t i v i t y in a g i v e n b r a i n a r e a t h a t is m a i n t a i n e d in t i s s u e culture. T h e r a p i d r h y t h m s r e p o r t e d could b e e x p l a i n e d b y i n t e r n e u r o n a l circuitry. More likely t h e s e r h y t h m s are d r i v e n b y p a c e m a k e r cells n o t u n l i k e t h e p a c e m a k i n g P u r k i n j e cells d e s c r i b e d b y GXHWlL~R'in c e r e b e l l a r e x p l a n t s ! S m a l l a l t e r a t i o n s in m e a s u r e d i m p e d a n c e h a v e o c c a s i o n a l l y been o b s e r v e d r e f l e c t i n g t r a n s i e n t c h a n g e s in e x t r a c e l l u l a r space p r o b a b l y r e s u l t i n g f r o m n e u r o g l i a l i n t e r a c t i o n .
Rdsumd. Les e n r e g i s t r e m e n t s 6lectriques n o n - p e r t u r b a n t s des n e u r o n e s m a i n t e n u s en culture, o a t m o n t r 6 des d6charges s p o n t a n 6 e s p r o t o t y p e s des aires d u s y s t ~ m e n e r v e u x c e n t r a l desquelles les groupes de cellules cultiv6es a v a i e n t 6t6 o b t e n u e s . Les g r o u p e s de n e u r o n e s d o n t les e n r e g i s t r e m e n t s f i g u r e n t ci-dessus, o u t 6t6 e x t r a i t s d u t61enc6phale, m6senc6phale, cervelet, e t des t u b e r c u l e s q u a d r i j u m e a u x de souris n o u v e a u - n 6 e s . Seuls les n e u r o n e s p r o v e n a n t d u t 6 1 e n c @ h a t e n ' o n t p a s p r o d u i t de d 6 c h a r g e 61ectrique r b y t h m i q u e . F. D.
WALKER
Institute o/Psychiatrw Research, Indiana University Medical Center, 7700 West Mwhigan Street, Indianapolis (Indiana 46202, USA), 79 August 797d.
Recording of Electrical Activity from Microscopically Identified Neurons of the M a m m a l i a n Brain I n d i s s e c t e d p r e p a r a t i o n s of t h e p e r i p h e r a l a n d i n v e r t e b r a t e n e r v o u s tissue, a single n e u r o n or a s y n a p t i c site c a n b e v i s u a l i z e d m i c r o s c o p i c a l l y w i t h its f u n c t i o n s i n t a c t . I m p u l s e t r a n s m i s s i o n in t h e s e s y n a p s e s was, therefore, i n t e n s i v e l y s t u d i e d a n d u n e q u i v o c a l r e s u l t s were o b t a i n ed 1-3. I n t h e m a m m a l i a n b r a i n , h o w e v e r , r e c o r d i n g of electrical a c t i v i t y f r o m a m i c r o s c o p i c a l l y i d e n t i f i e d n e u r o n h a s n o t b e e n successful. A l t h o u g h b r a i n sections of 0.3-0.4 m m t h i c k h a v e b e e n f o u n d to e x h i b i t s p o n t a n e o u s a n d e v o k e d p o t e n t i a l a c t i v i t i e s in t h e artificial m e d i a 4-~, t h e y are still too t h i c k for o b s e r v a t i o n of n e u r o n s t h e r e i n . In the present experiments, I have attempted to prepare m u c h t h i n n e r sections of t h e g u i n e a - p i g c e r e b e l l u m a n d r e c o r d a c t i o n p o t e n t i a l s f r o m t h e P u r k i n j e ceils while o b s e r v i n g t h e m w i t h t h e aid of a microscope. Material and methods. P r o c e d u r e s for p r e p a r a t i o n of t h e tissue were i d e n t i c a l w i t h t h o s e d e s c r i b e d p r e v i o u s l y e x c e p t for t h e t h i c k n e s s of s e c t i o n s L G u i n e a - p i g s were
s t u n n e d a n d killed b y blows on t h e b a c k of t h e n e c k a n d t h o r a x . T h e b r a i n was t a k e n o u t of t h e skull a n d t h e v e r m i s of t h e c e r e b e l l u m was isolated o n a piece of filter p a p e r covered w i t h t h e m e d i u m . F r o m t h e n o d u l u s a n d u v u l a , b l o c k s of t h e c o r t e x of a b o u t 1 m m t h i c k were p r e p a r e d a n d k e p t in t h e m e d i u m a t 0 - 4 ~ for 5-20 m i n
1 A. TAKEUCHI and N. TAKEUCHI, J. Physiol., Load. 170, 296 (1964). 2 B. I~ATZand R. MILEDI, Proc. R. Soe. B 161, 496 (1965). M. J. DENNIS, A. J. HARRIS and S. W. KUFFLER, Proc. R. Soc. B 777, 509 (1971). 4 C. YAMAMOTOand FI. MCILWAII%Nature, Lond. 270, 1055 (1966). 5 C. YAMAMOTOand N. KAWAI, Experientia 23, 821 (1967). H. KATO, Z. ITO, S. MATSUOKA and Y. SAKURAI, EEG clin. Neurophysioi. 35, 457 (1973). 7 C. YAMAMOTO, Jap. J. Physiol. 2d, 177 (1974).
310
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EXPERIENTIA 31/3 A) Photomicrograph of a eerebellar section of 80 jim thick. Single and double arrows indicate Purkinje cell bodies and dendrite, respectively. B) Climbing fibre response recorded extraeellularly from cell body. Superimposed records. C) Increase in discharge rate of a Purkinje cell during application of glutamate to cell soma. Solid line, anionic current (5 hA) was passed through a glutamatepipette. Retaining current was + 10 hA.
u n t i l t h e y a d h e r e d one b y one o n t o a s m a l l m e t a l d i s h w i t h A r o n - A l p h a ( s - a c r y l a t e m o n o m e r , T o a Gosei K a g a k u , Tokyo). T h e n t h e tissue blocks were i m m e r s e d in t h e chilled m e d i u m a n d c u t to a t h i c k n e s s of 70-80 [xm w i t h t h e V i b r o t o m e (Oxford L a b o r a t o r i e s , California) a t r i g h t angles t o t h e long axis of t h e Iolium. T h e s e c t i o n i n g was p e r f o r m e d a t a v i b r a t i o n r a t e of 6.5-7 scale u n i t s a n d a feeding speed of 2-3 scale u n i t s w i t h a b l a d e i n c l i n e d a t a b o u t 17 degrees. T h e sections p r e p a r e d in t h i s w a y were i n c u b a t e d a t 37 ~ for m o r e t h e n 45 m i n a n d t h e n t r a n s ferred i n t o t h e o b s e r v a t i o n vessel, w h i c h was s i m i l a r t o t h a t d e s c r i b e d before s, a n d m o u n t e d o n a m i c r o s c o p e stage. Sections were o b s e r v e d f r o m a b o v e w i t h a n o b j e c t i v e lens of long w o r k i n g d i s t a n c e ( • U D 40/0.65 C, Carl Zeiss, W e s t G e r m a n y ) . C o n v e n t i o n a l glass m i c r o e l e e t r o d e s of 3 [~m t i p d i a m e t e r filled w i t h 4 M NaC1 were used for e x t r a c e l l u l a r r e c o r d i n g of single cell discharges a n d for electric s t i m u l a t i o n . F o r a p p l i c a t i o n of g l u t a m a t e , single glass p i p e t t e s were filled w i t h 1 M s o l u t i o n of t h e a m i n o acid (pH 8.0). T h e t i p of t h e p i p e t t e was p o s i t i o n e d in close v i c i n i t y of a n e r v e cell a n d a n i o n i c c u r r e n t was p a s s e d t h r o u g h t h e p i p e t t e . T h e c o m p o s i t i o n of t h e m e d i u m was ( m M ) ; NaC1, 124; KC1, 5; IKH2PO4, 1.24; MgSOa, 1.3; CaC12, 2.4; N a H C O 3, 26 a n d glucose, 10. Results and discussion. "When t h e s e c t i o n s were obs e r v e d microscopically, P u r k i n j e cell bodies, were well discernible, especially w i t h t h e c o n d e n s e r d i a p h r a g m p a r t i a l l y closed (Figure A, arrows). B y a d j u s t i n g t h e focus of t h e m i c r o s c o p e finely, it was possible t o i d e n t i f y t h e o u t l i n e of t h e cell b o d y . I n some cells, c o n t o u r of t h e d e n d r i t e could b e followed in t h e m o l e c u l a r l a y e r (double arrow). As o b s e r v e d before in t h i c k e r slices, m o s t of t h e P u r k i n j e cells g e n e r a t e d s p o n t a n e o u s discharges, w h i c h were 5-80 cps i n f r e q u e n c y a n d e i t h e r a l m o s t r e g u l a r or i n t e r r u p t e d b y t h e s i l e n t p e r i o d of v a r i o u s d u r a t i o n s 7, 9 A l t h o u g h d e n d r i t i c b r a n c h e s were t h o u g h t t o b e p a r t l y d a m a g e d d u r i n g p r e p a r a t i o n of t h e sections, o n l y a few cells in sections f r o m t h e u v u l a a n d n o d u l n s s h o w e d signs of excessive d e p o l a r i z a t i o n , s u c h as t h e b u r s t discharges w i t h a r e v e r s a l of spike polarity~ 7. F o r s y n a p t i c a c t i v a t i o n of t h e P u r k i n j e ceils, t h e t i p of a NaCl-filled glass p i p e t t e w a s p o s i t i o n e d close t o t h e d e n d r i t e of a P u r k i n j e cell, electrical a c t i v i t y
of w h i c h was r e c o r d e d f r o m t h e cell b o d y w i t h a n o t h e r p i p e t t e . A n e g a t i v e pulse of 0.2 msec d u r a t i o n p a s s e d t h r o u g h t h e f o r m e r p i p e t t e i n d u c e d in a n a l l - o r - n o t h i n g m a n n e r a b u r s t of d i s c h a r g e s c o n s i s t i n g of 2 - 4 spikes (Figure t3). T h i s b u r s t d i s c h a r g e seems t o b e t h e c l i m b ing f i b e r (CF) response. T h e n e g a t i v e pulse p r o b a b l y excited a C F w h i c h in t u r n s y n a p t i c a l l y a c t i v a t e d t h e n e a r - b y d e n d r i t e . A l r e a d y , ECCLES et al. 1~ r e p o r t e d t h a t n o t o n l y s t i m u l a t i o n t o t h e inferior olive n u c l e u s a n d e x t r a f a s t i g i a l fibres b u t also d i r e c t s t i m u l a t i o n t o t h e c e r e b e l l a r c o r t e x i n d u c e d t h e C F response. T h e C F r e s p o n s e a n d s p o n t a n e o u s d i s c h a r g e were r e c o r d e d for m o r e t h a n several h o u r s a f t e r p r e p a r a t i o n of t h e tissues. I n a n o t h e r series of e x p e r i m e n t s , t h e t i p of a g l u t a m a t e - f i l l e d electrode was b r o u g h t close t o t h e cell b o d y or a p i c a l d e n d r i t e of a P u r k i n j e cell, t h e s p o n t a n e o u s d i s c h a r g e of w h i c h was p i c k e d u p w i t h a n o t h e r NaCI-filled p i p e t t e . W h e n anionic c u r r e n t was p a s s e d t h r o u g h t h e g l u t a m a t e - f i l l e d electrode, t h e d i s c h a r g e r a t e i n c r e a s e d w i t h a l a t e n c y of 20-200 msec a n d r e t u r n e d t o t h e c o n t r o l levet as t h e c u r r e n t was t u r n e d off (Figure C). FIDONE et al. a l r e a d y succeeded in r e c o r d i n g a c t i o n p o t e n t i a l s f r o m n e u r o n s in slices of t h e frog s y m p a t h e t i c g a n g l i o n w h i c h were m a d e so t h i n t h a t e a c h n e u r o n was m i c r o s c o p i c a l l y o b s e r v e d n . T h e f i n d i n g s of t h e p r e s e n t e x p e r i m e n t s s h o w t h a t e s s e n t i a l l y t h e s a m e t e c h n i q u e is a p p l i c a b l e t o t h e m a m m a l i a n b r a i n . G e n e r a t i o n of t h e CF r e s p o n s e b y electric s t i m u l a t i o n i n d i c a t e s t h a t t h e s y n a p t i c t r a n s m i s s i o n is m a i n t a i n e d in s u c h a t h i n section. Since t h e CFs were m u t i l a t e d in t h e sections, n o C F d i s c h a r g e s o c c u r r e d s p o n t a n e o u s l y . A l t h o u g h t h e m o s s y fibres were also sectioned, P u r k i n j e cells still g e n e r a t e d s p o n t a n e o u s discharges. I t r e m a i n s t o b e clarified w h e t h e r t h e excitat i o n o r i g i n a t e d in t h e P u r k i n j e cells or g r a n u l e cells. G l u t a m a t e ejected w i t h w e a k c u r r e n t s i n d u c e d s t r o n g e x c i t a t i o n w h i c h was q u i c k in o n s e t a n d t e r m i n a t i o n . 8 C. YAMAMOTO,Expl Brain Res. lg, 423 (1972). 9 K. OKAMOTOand J. H. QUAST~L, Proe. R. Soc. B 18g, 83 (1973). lo j . C. ]~CCLES, R. LLINASand K. SASAKI,J. Physiol., Lond. 182, 268 (i966). 11 S. ~'IDONE, T. O'LEARY and C. EYZAGUIRRE, Brain Res. 30, 401 (1971).
15.3. 1975
Specialia
T h i s was in a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n m a d e in v i v o 12. Since r e c o r d i n g a n d s t i m u l a t i n g electrodes c a n be a p p l i e d i n d e p e n d e n t l y t o p o r t i o n s of a n e u r o n i d e n t i f i e d microscopically, t h i n sections as used in t h e p r e s e n t experi m e n t s are e x p e c t e d t o serve as e x c e l l e n t p r e p a r a t i o n s for s t u d i e s o n c h e m o s e n s i t i v i t y of t h e n e u r o n a l m e m b r a n e as well as o n n e u r o n - n e u r o n a n d n e u r o n - g i l a i n t e r a c t i o n in the brain. 12 i-I. KAWAMURAand L. PROVINI,Brain Res. 24, 293 (1970). 18 I thank Profs. H. MA~NE~ and K. SASAKIfor valuable discussion.
311
Zusammen/assung. I n D t i n n s c h n i t t e n v o m K l e i n h i r n des M e e r s c h w e i n c h e n s w u r d e n in k f i n s t l i c h e m M e d i u m Purkinjezellen identifiziert und Spontanentladungen der Z e l l k 6 r p e r registriert. E l e k t r o p h o r e t i s c h e r A n t r a n s p o r t y o n G l u t a m i n s / i u r e zu Zellk6rper oder D e n t r i t e n rief jeweils s t a r k e E r r e g u n g h e r v o r . C. YAMAMOTO 13
Behavior Research Institute, University o/ Gunma Medical School, Showa-machi, Maebashi (Japan), 73 September 7974.
Neurosecretory Cells in the Hypocerebral Ganglion of Gryllus bimaculatus de Geer (Orthoptera: Gryllidae) T h e o c c u r r e n c e of v a r i o u s t y p e s of n e u r o s e c r e t o r y ceils (NSC) in t h e b r a i n a n d t h e v e n t r a l ganglia, w h i c h c o n t r o l all t h e m a j o r p h y s i o l o g i c a l e v e n t s in t h e life cycle of insects, is well k n o w n . T h e r e is n o p r e v i o u s r e p o r t of t h e p r e s e n c e of n e u r o s e c r e t o r y cells in t h e h y p o c e r e b r a l g a n g l i o n b e y o n d t h e r e c o r d i n g of B cells in
Schizodactylus 1. T h e ]3ouin-fixed p a r a f f i n e m b e d d e d m a t e r i a l was serially c u t a t 6 [zm a n d s t a i n e d w i t h C h r o m e H a e m a t o x y l i n - P h l o x i l l e (CHP), P a r a l d e h y d e - F u c h s i n (PF) a n d H e i d e n h a i n ' s A z a n (Azan). T h e h y p o c e r e b r a l g a n g l i o n h a s 2 t y p e s of n e u r o s e c r e t o r y cells w h i c h c a n b e classified as A a n d 13 ceils on t h e basis of t h e i r t i n c t o r i a l affinities w i t h P F , C H P a n d A z a n (Figures 1-3). T h e first t y p e , d e s i g n a t e d as 'A' cells, is c h a r a c t e r i z e d b y c y t o p l a s m i c inclusions s t a i n i n g deep
Fig. 1. Diagrammatic representation of the distribution of neurosecretory cells in the hypocerebral ganglion of Gryllus bimaculatus. A and B, neurosecretory cell types; NCCI, nervus corporis cardiaci; NH, nervus hypocerebrii; NOE, nervus oesophagei; NR, nervus reeurrentus.
p u r p l e w i t h P F , b l u e - b l a c k w i t h C H P a n d red w i t h Azan. T h e fine h o m o g e n o u s inclusions of t h e second t y p e of cells, t h e ']3' cells s t a i n g r e e n i s h w i t h l i g h t g r e e n - o r a n g e G, red w i t h p h l o x i n e of C H P a n d f a i n t blue w i t h t h e a n i l i n e b l u e - o r a n g e G c o u n t e r s t a i n s of Azan. E a c h h a l f of t h e h y p o c e r e b r a l g a n g l i o n h a s 8 to 10 A cells a n d 4 to 6 ]3 cells f o u n d a l o n g w i t h a large n u m b e r of n e r v e cells. T h e a n t e r i o r p a r t of t h e g a n g l i o n h a s 2 cells in t h e m e d i a n a n d m i d v e n t r a l position, 2 ceils in t h e a n t e r i o r r e g i o n a n d 4 t o 6 cells in its p o s t e r o l a t e r a l p a r t (Figure 1). T h e a x o n s a r i z i n g f r o m t h e A cells r u n t o w a r d s t h e l a t e r a l b o r d e r of t h e g a n g l i o n (Figure 3) f r o m w h e r e they enter the corpora cardiaea through the nervi hypoc e r e b r i i (NH, F i g u r e 1). I t is e a s y to t r a c e t h e a x o n a l p a t h w a y s (AX) d u e t o t h e p r e s e n c e of s t a i n e d n e u r o s e c r e t o r y m a t e r i a l in t h e m w h i c h s t a i n s deep p u r p l e w i t h PF, red with Azan and blue-black with CHP. T h e m i d - d o r s a l region of t h e a n t e r i o r h a l f of t h e h y p o c e r e b r a l g a n g l i o n h a s 4 t o 6 t3 ceils w h i c h s t a i n g r e e n i s h w i t h P F , red w i t h C H P a n d f a i n t blue w i t h Azan. T h e a x o n s of t h e s e cells c a n b e t r a c e d o n l y for a s h o r t d i s t a n c e d u e t o lack of s t a i n i n g colloids. A large a m o u n t of n e u r o s e c r e t o r y m a t e r i a l (NSM) h a s b e e n o b s e r v e d l a t e r a l l y in t h e h y p o c e r e b r a l g a n g l i o n of m a l e s as t h e t w o NCC I p a s s t h r o u g h its l a t e r a l m a r g i n s , w h e r e a s t h e a m o u n t of N S M is c o m p a r a t i v e l y less in females as t h e NCC. I are free a n d h a v e n o c o n n e c t i o n w i t h h y p o c e r e b r a l ganglion. T h e s m a l l a m o u n t of NSM, w h i c h is p a r t i c u l a r l y seen before c o p u l a t i o n a n d ovip o s i t i o n in females, is t h e p r o d u c t of t h e n e u r o s e c r e t o r y cells of t h i s ganglion. T h u s t h e g r e a t e r a m o u n t of N S M in m a l e s is t h e p r o d u c t of t h e n e u r o s e c r e t o r y cells of t h e h y p o c e r e b r a l g a n g l i o n as well as t h e p a r s i n t e r c e r e b r a l i s of t h e b r a i n . T h u s t h e p r e s e n t w o r k records for t h e first t i m e in insects t h e p r e s e n c e of b o t h A a n d ]3 cell t y p e s a n d n e u r o s e c r e t o r y p a t h w a y s u p to t h e n e u r o h a e m a l o r g a n s (corpora eardiaca).
1 N. KHATTA~, Bull. Soe. Zool. ft. 93, 225 (1968).
309
Speeialia
a c t i v i t y . Cerebellar e x p l a n t s (Figure 1) c h a r a c t e r i s t i c a l l y g a v e r a p i d b u r s t s of spikes o c c u r r i n g e v e r y 100 msec a n d h a v i n g a d u r a t i o n of 50 msec. Some cerebellar c u l t u r e s were o b s e r v e d t o h a v e periodicities of a longer n a t u r e : e v e r y m i n u t e w i t h b u r s t s of 30 sec. T h e l o n g e s t p e r i o d s were o b s e r v e d in e x p l a n t s of t h e m i d b r a i n area (Figure 2) h a v i n g i n t e r v a l s of g e n e r a l l y 4 t o 7 m i n b u t as long as 10 rain y i e l d i n g a c a s c a d i n g firing p a t t e r n of o v e r 1.0 r a i n d u r a t i o n while t h e r e s t of t h e p e r i o d r e m a i n e d e s s e n t i a l l y quiet. T h e s e c a s c a d e s could b e d r i v e n b u t e v i d e n c e d a r e l a t i v e r e f r a c t o r i n e s s t o successive electric shocks. E x p l a n t s t r o m t h e colliculi g a v e p e a k s of a c t i v i t y h a v i n g a s e r r a t e d a p p e a r a n c e t o t h e spike d i s c h a r g e p a t t e r n w i t h periods of a p p r o x i m a t e l y 4 m i n in length. N o t e v e r y e x p l a n t p r o d u c e d a r h y t h m i c p a t t e r n of neuronal activity but when present the type and pattern of spike d i s c h a r g e was s u f f i c i e n t l y c o n s i s t e n t to b e a reliable c h a r a c t e r i s t i c of e x p l a n t s t a k e n f r o m a p a r t i c u l a r a r e a of b r a i n . E x p l a n t s f r o m t h e m i d b r a i n a r e a p r o d u c e d r h y t h m i c a l d i s c h a r g e s 2 0 % of t h e t i m e , c e r e b e l l u m 10%,
D.c.
P
5 min
Fig. 2. Cascades of neuronal activity occurring every 6.5 min in an explant from midbrain 16 days of age. The upper trace indicates the quantity of neuronal spikes counted by the digital frequency meter and the lower trace indicates the de difference between the fluid in the upper and lower chambers. This pattern of activity has been continuous for more than 45 min. It was only altered by shocks at a, b and c where a current density of 9.5 • i 0 -~ Amps per ~xm~ was delivered for 0.05, 0.5 and 5 reset. It can be seen that successive shocks with lengthened duration had less affect indicating refractoriness of the tissue. Hole measured 200 ~xnl in diameter.
a n d colliculus 5 %. E x p l a n t s n o t i n i t i a l l y s h o w i n g r h y t h m i c n e u r o n a l firing w o u l d o c c a s i o n a l l y do so l a t e r e i t h e r s p o n t a n e o u s l y or would b e i n d u c e d t o d o so b y small, brief shocks of t h e o r d e r of 10 -~ Amp/Ezm e g i v e n for a d u r a t i o n of 1.0 msec. T h e r h y t h m i c p a t t e r n once i n i t i a t e d w o u l d c o n t i n u e for o v e r 1 h w i t h l i t t l e v a r i a t i o n . E v e n t u ally t h e spike p r o d u c t i o n failed, i n d i c a t e d b y g r a d u a l d i m i n u t i o n of t h e n u m b e r of spikes g e n e r a t e d d u r i n g each episode u n t i l f i n a l l y n o n e was p r o d u c e d . T h e p a t t e r n could b e r e v i v e d b r i e f l y b y e x c h a n g i n g t h e fluid in t h e r e c o r d i n g c h a m b e r . I n v e s t i g a t i o n s were m a d e a t r o o m t e m p e r a t u r e (27 ~ Of t h e b r a i n areas s t u d i e d c e r e b r a l cortical tissue a l o n e d i d n o t d e m o n s t r a t e a r e g u l a r p a t t e r n of a c t i v i t y ; h o w e v e r , t h e r e was o b s e r v e a i r r e g u l a r slow w a v e a c t i v i t y h a v i n g a d u r a t i o n of 100 msec a n d a n a m p l i t u d e of 500 ~V ( a p p r o x i m a t e l y 5 t i m e s t h e a m p l i t u d e of spikes) w h i c h a p p e a r e d t o i n i t i a t e n e u r o n a l spikes. T h e a c t i v i t y h a s b e e n r e p o r t e d b y CALVET 5 w h o suggests t h a t i t m a y r e s u l t f r o m t h e s i m u l t a n e o u s a c t i v a t i o n of p o s t s y n a p t i c p o t e n t i a l s , l u a n y case, t h e r e m u s t be a genetic c o m p o n e n t to a c c o u n t for t h e u n i f o r m i t y of a c t i v i t y in a g i v e n b r a i n a r e a t h a t is m a i n t a i n e d in t i s s u e culture. T h e r a p i d r h y t h m s r e p o r t e d could b e e x p l a i n e d b y i n t e r n e u r o n a l circuitry. More likely t h e s e r h y t h m s are d r i v e n b y p a c e m a k e r cells n o t u n l i k e t h e p a c e m a k i n g P u r k i n j e cells d e s c r i b e d b y GXHWlL~R'in c e r e b e l l a r e x p l a n t s ! S m a l l a l t e r a t i o n s in m e a s u r e d i m p e d a n c e h a v e o c c a s i o n a l l y been o b s e r v e d r e f l e c t i n g t r a n s i e n t c h a n g e s in e x t r a c e l l u l a r space p r o b a b l y r e s u l t i n g f r o m n e u r o g l i a l i n t e r a c t i o n .
Rdsumd. Les e n r e g i s t r e m e n t s 6lectriques n o n - p e r t u r b a n t s des n e u r o n e s m a i n t e n u s en culture, o a t m o n t r 6 des d6charges s p o n t a n 6 e s p r o t o t y p e s des aires d u s y s t ~ m e n e r v e u x c e n t r a l desquelles les groupes de cellules cultiv6es a v a i e n t 6t6 o b t e n u e s . Les g r o u p e s de n e u r o n e s d o n t les e n r e g i s t r e m e n t s f i g u r e n t ci-dessus, o u t 6t6 e x t r a i t s d u t61enc6phale, m6senc6phale, cervelet, e t des t u b e r c u l e s q u a d r i j u m e a u x de souris n o u v e a u - n 6 e s . Seuls les n e u r o n e s p r o v e n a n t d u t 6 1 e n c @ h a t e n ' o n t p a s p r o d u i t de d 6 c h a r g e 61ectrique r b y t h m i q u e . F. D.
WALKER
Institute o/Psychiatrw Research, Indiana University Medical Center, 7700 West Mwhigan Street, Indianapolis (Indiana 46202, USA), 79 August 797d.
Recording of Electrical Activity from Microscopically Identified Neurons of the M a m m a l i a n Brain I n d i s s e c t e d p r e p a r a t i o n s of t h e p e r i p h e r a l a n d i n v e r t e b r a t e n e r v o u s tissue, a single n e u r o n or a s y n a p t i c site c a n b e v i s u a l i z e d m i c r o s c o p i c a l l y w i t h its f u n c t i o n s i n t a c t . I m p u l s e t r a n s m i s s i o n in t h e s e s y n a p s e s was, therefore, i n t e n s i v e l y s t u d i e d a n d u n e q u i v o c a l r e s u l t s were o b t a i n ed 1-3. I n t h e m a m m a l i a n b r a i n , h o w e v e r , r e c o r d i n g of electrical a c t i v i t y f r o m a m i c r o s c o p i c a l l y i d e n t i f i e d n e u r o n h a s n o t b e e n successful. A l t h o u g h b r a i n sections of 0.3-0.4 m m t h i c k h a v e b e e n f o u n d to e x h i b i t s p o n t a n e o u s a n d e v o k e d p o t e n t i a l a c t i v i t i e s in t h e artificial m e d i a 4-~, t h e y are still too t h i c k for o b s e r v a t i o n of n e u r o n s t h e r e i n . In the present experiments, I have attempted to prepare m u c h t h i n n e r sections of t h e g u i n e a - p i g c e r e b e l l u m a n d r e c o r d a c t i o n p o t e n t i a l s f r o m t h e P u r k i n j e ceils while o b s e r v i n g t h e m w i t h t h e aid of a microscope. Material and methods. P r o c e d u r e s for p r e p a r a t i o n of t h e tissue were i d e n t i c a l w i t h t h o s e d e s c r i b e d p r e v i o u s l y e x c e p t for t h e t h i c k n e s s of s e c t i o n s L G u i n e a - p i g s were
s t u n n e d a n d killed b y blows on t h e b a c k of t h e n e c k a n d t h o r a x . T h e b r a i n was t a k e n o u t of t h e skull a n d t h e v e r m i s of t h e c e r e b e l l u m was isolated o n a piece of filter p a p e r covered w i t h t h e m e d i u m . F r o m t h e n o d u l u s a n d u v u l a , b l o c k s of t h e c o r t e x of a b o u t 1 m m t h i c k were p r e p a r e d a n d k e p t in t h e m e d i u m a t 0 - 4 ~ for 5-20 m i n
1 A. TAKEUCHI and N. TAKEUCHI, J. Physiol., Load. 170, 296 (1964). 2 B. I~ATZand R. MILEDI, Proc. R. Soe. B 161, 496 (1965). M. J. DENNIS, A. J. HARRIS and S. W. KUFFLER, Proc. R. Soc. B 777, 509 (1971). 4 C. YAMAMOTOand FI. MCILWAII%Nature, Lond. 270, 1055 (1966). 5 C. YAMAMOTOand N. KAWAI, Experientia 23, 821 (1967). H. KATO, Z. ITO, S. MATSUOKA and Y. SAKURAI, EEG clin. Neurophysioi. 35, 457 (1973). 7 C. YAMAMOTO, Jap. J. Physiol. 2d, 177 (1974).
310
Specialia
EXPERIENTIA 31/3 A) Photomicrograph of a eerebellar section of 80 jim thick. Single and double arrows indicate Purkinje cell bodies and dendrite, respectively. B) Climbing fibre response recorded extraeellularly from cell body. Superimposed records. C) Increase in discharge rate of a Purkinje cell during application of glutamate to cell soma. Solid line, anionic current (5 hA) was passed through a glutamatepipette. Retaining current was + 10 hA.
u n t i l t h e y a d h e r e d one b y one o n t o a s m a l l m e t a l d i s h w i t h A r o n - A l p h a ( s - a c r y l a t e m o n o m e r , T o a Gosei K a g a k u , Tokyo). T h e n t h e tissue blocks were i m m e r s e d in t h e chilled m e d i u m a n d c u t to a t h i c k n e s s of 70-80 [xm w i t h t h e V i b r o t o m e (Oxford L a b o r a t o r i e s , California) a t r i g h t angles t o t h e long axis of t h e Iolium. T h e s e c t i o n i n g was p e r f o r m e d a t a v i b r a t i o n r a t e of 6.5-7 scale u n i t s a n d a feeding speed of 2-3 scale u n i t s w i t h a b l a d e i n c l i n e d a t a b o u t 17 degrees. T h e sections p r e p a r e d in t h i s w a y were i n c u b a t e d a t 37 ~ for m o r e t h e n 45 m i n a n d t h e n t r a n s ferred i n t o t h e o b s e r v a t i o n vessel, w h i c h was s i m i l a r t o t h a t d e s c r i b e d before s, a n d m o u n t e d o n a m i c r o s c o p e stage. Sections were o b s e r v e d f r o m a b o v e w i t h a n o b j e c t i v e lens of long w o r k i n g d i s t a n c e ( • U D 40/0.65 C, Carl Zeiss, W e s t G e r m a n y ) . C o n v e n t i o n a l glass m i c r o e l e e t r o d e s of 3 [~m t i p d i a m e t e r filled w i t h 4 M NaC1 were used for e x t r a c e l l u l a r r e c o r d i n g of single cell discharges a n d for electric s t i m u l a t i o n . F o r a p p l i c a t i o n of g l u t a m a t e , single glass p i p e t t e s were filled w i t h 1 M s o l u t i o n of t h e a m i n o acid (pH 8.0). T h e t i p of t h e p i p e t t e was p o s i t i o n e d in close v i c i n i t y of a n e r v e cell a n d a n i o n i c c u r r e n t was p a s s e d t h r o u g h t h e p i p e t t e . T h e c o m p o s i t i o n of t h e m e d i u m was ( m M ) ; NaC1, 124; KC1, 5; IKH2PO4, 1.24; MgSOa, 1.3; CaC12, 2.4; N a H C O 3, 26 a n d glucose, 10. Results and discussion. "When t h e s e c t i o n s were obs e r v e d microscopically, P u r k i n j e cell bodies, were well discernible, especially w i t h t h e c o n d e n s e r d i a p h r a g m p a r t i a l l y closed (Figure A, arrows). B y a d j u s t i n g t h e focus of t h e m i c r o s c o p e finely, it was possible t o i d e n t i f y t h e o u t l i n e of t h e cell b o d y . I n some cells, c o n t o u r of t h e d e n d r i t e could b e followed in t h e m o l e c u l a r l a y e r (double arrow). As o b s e r v e d before in t h i c k e r slices, m o s t of t h e P u r k i n j e cells g e n e r a t e d s p o n t a n e o u s discharges, w h i c h were 5-80 cps i n f r e q u e n c y a n d e i t h e r a l m o s t r e g u l a r or i n t e r r u p t e d b y t h e s i l e n t p e r i o d of v a r i o u s d u r a t i o n s 7, 9 A l t h o u g h d e n d r i t i c b r a n c h e s were t h o u g h t t o b e p a r t l y d a m a g e d d u r i n g p r e p a r a t i o n of t h e sections, o n l y a few cells in sections f r o m t h e u v u l a a n d n o d u l n s s h o w e d signs of excessive d e p o l a r i z a t i o n , s u c h as t h e b u r s t discharges w i t h a r e v e r s a l of spike polarity~ 7. F o r s y n a p t i c a c t i v a t i o n of t h e P u r k i n j e ceils, t h e t i p of a NaCl-filled glass p i p e t t e w a s p o s i t i o n e d close t o t h e d e n d r i t e of a P u r k i n j e cell, electrical a c t i v i t y
of w h i c h was r e c o r d e d f r o m t h e cell b o d y w i t h a n o t h e r p i p e t t e . A n e g a t i v e pulse of 0.2 msec d u r a t i o n p a s s e d t h r o u g h t h e f o r m e r p i p e t t e i n d u c e d in a n a l l - o r - n o t h i n g m a n n e r a b u r s t of d i s c h a r g e s c o n s i s t i n g of 2 - 4 spikes (Figure t3). T h i s b u r s t d i s c h a r g e seems t o b e t h e c l i m b ing f i b e r (CF) response. T h e n e g a t i v e pulse p r o b a b l y excited a C F w h i c h in t u r n s y n a p t i c a l l y a c t i v a t e d t h e n e a r - b y d e n d r i t e . A l r e a d y , ECCLES et al. 1~ r e p o r t e d t h a t n o t o n l y s t i m u l a t i o n t o t h e inferior olive n u c l e u s a n d e x t r a f a s t i g i a l fibres b u t also d i r e c t s t i m u l a t i o n t o t h e c e r e b e l l a r c o r t e x i n d u c e d t h e C F response. T h e C F r e s p o n s e a n d s p o n t a n e o u s d i s c h a r g e were r e c o r d e d for m o r e t h a n several h o u r s a f t e r p r e p a r a t i o n of t h e tissues. I n a n o t h e r series of e x p e r i m e n t s , t h e t i p of a g l u t a m a t e - f i l l e d electrode was b r o u g h t close t o t h e cell b o d y or a p i c a l d e n d r i t e of a P u r k i n j e cell, t h e s p o n t a n e o u s d i s c h a r g e of w h i c h was p i c k e d u p w i t h a n o t h e r NaCI-filled p i p e t t e . W h e n anionic c u r r e n t was p a s s e d t h r o u g h t h e g l u t a m a t e - f i l l e d electrode, t h e d i s c h a r g e r a t e i n c r e a s e d w i t h a l a t e n c y of 20-200 msec a n d r e t u r n e d t o t h e c o n t r o l levet as t h e c u r r e n t was t u r n e d off (Figure C). FIDONE et al. a l r e a d y succeeded in r e c o r d i n g a c t i o n p o t e n t i a l s f r o m n e u r o n s in slices of t h e frog s y m p a t h e t i c g a n g l i o n w h i c h were m a d e so t h i n t h a t e a c h n e u r o n was m i c r o s c o p i c a l l y o b s e r v e d n . T h e f i n d i n g s of t h e p r e s e n t e x p e r i m e n t s s h o w t h a t e s s e n t i a l l y t h e s a m e t e c h n i q u e is a p p l i c a b l e t o t h e m a m m a l i a n b r a i n . G e n e r a t i o n of t h e CF r e s p o n s e b y electric s t i m u l a t i o n i n d i c a t e s t h a t t h e s y n a p t i c t r a n s m i s s i o n is m a i n t a i n e d in s u c h a t h i n section. Since t h e CFs were m u t i l a t e d in t h e sections, n o C F d i s c h a r g e s o c c u r r e d s p o n t a n e o u s l y . A l t h o u g h t h e m o s s y fibres were also sectioned, P u r k i n j e cells still g e n e r a t e d s p o n t a n e o u s discharges. I t r e m a i n s t o b e clarified w h e t h e r t h e excitat i o n o r i g i n a t e d in t h e P u r k i n j e cells or g r a n u l e cells. G l u t a m a t e ejected w i t h w e a k c u r r e n t s i n d u c e d s t r o n g e x c i t a t i o n w h i c h was q u i c k in o n s e t a n d t e r m i n a t i o n . 8 C. YAMAMOTO,Expl Brain Res. lg, 423 (1972). 9 K. OKAMOTOand J. H. QUAST~L, Proe. R. Soc. B 18g, 83 (1973). lo j . C. ]~CCLES, R. LLINASand K. SASAKI,J. Physiol., Lond. 182, 268 (i966). 11 S. ~'IDONE, T. O'LEARY and C. EYZAGUIRRE, Brain Res. 30, 401 (1971).
15.3. 1975
Specialia
T h i s was in a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n m a d e in v i v o 12. Since r e c o r d i n g a n d s t i m u l a t i n g electrodes c a n be a p p l i e d i n d e p e n d e n t l y t o p o r t i o n s of a n e u r o n i d e n t i f i e d microscopically, t h i n sections as used in t h e p r e s e n t experi m e n t s are e x p e c t e d t o serve as e x c e l l e n t p r e p a r a t i o n s for s t u d i e s o n c h e m o s e n s i t i v i t y of t h e n e u r o n a l m e m b r a n e as well as o n n e u r o n - n e u r o n a n d n e u r o n - g i l a i n t e r a c t i o n in the brain. 12 i-I. KAWAMURAand L. PROVINI,Brain Res. 24, 293 (1970). 18 I thank Profs. H. MA~NE~ and K. SASAKIfor valuable discussion.
311
Zusammen/assung. I n D t i n n s c h n i t t e n v o m K l e i n h i r n des M e e r s c h w e i n c h e n s w u r d e n in k f i n s t l i c h e m M e d i u m Purkinjezellen identifiziert und Spontanentladungen der Z e l l k 6 r p e r registriert. E l e k t r o p h o r e t i s c h e r A n t r a n s p o r t y o n G l u t a m i n s / i u r e zu Zellk6rper oder D e n t r i t e n rief jeweils s t a r k e E r r e g u n g h e r v o r . C. YAMAMOTO 13
Behavior Research Institute, University o/ Gunma Medical School, Showa-machi, Maebashi (Japan), 73 September 7974.
Neurosecretory Cells in the Hypocerebral Ganglion of Gryllus bimaculatus de Geer (Orthoptera: Gryllidae) T h e o c c u r r e n c e of v a r i o u s t y p e s of n e u r o s e c r e t o r y ceils (NSC) in t h e b r a i n a n d t h e v e n t r a l ganglia, w h i c h c o n t r o l all t h e m a j o r p h y s i o l o g i c a l e v e n t s in t h e life cycle of insects, is well k n o w n . T h e r e is n o p r e v i o u s r e p o r t of t h e p r e s e n c e of n e u r o s e c r e t o r y cells in t h e h y p o c e r e b r a l g a n g l i o n b e y o n d t h e r e c o r d i n g of B cells in
Schizodactylus 1. T h e ]3ouin-fixed p a r a f f i n e m b e d d e d m a t e r i a l was serially c u t a t 6 [zm a n d s t a i n e d w i t h C h r o m e H a e m a t o x y l i n - P h l o x i l l e (CHP), P a r a l d e h y d e - F u c h s i n (PF) a n d H e i d e n h a i n ' s A z a n (Azan). T h e h y p o c e r e b r a l g a n g l i o n h a s 2 t y p e s of n e u r o s e c r e t o r y cells w h i c h c a n b e classified as A a n d 13 ceils on t h e basis of t h e i r t i n c t o r i a l affinities w i t h P F , C H P a n d A z a n (Figures 1-3). T h e first t y p e , d e s i g n a t e d as 'A' cells, is c h a r a c t e r i z e d b y c y t o p l a s m i c inclusions s t a i n i n g deep
Fig. 1. Diagrammatic representation of the distribution of neurosecretory cells in the hypocerebral ganglion of Gryllus bimaculatus. A and B, neurosecretory cell types; NCCI, nervus corporis cardiaci; NH, nervus hypocerebrii; NOE, nervus oesophagei; NR, nervus reeurrentus.
p u r p l e w i t h P F , b l u e - b l a c k w i t h C H P a n d red w i t h Azan. T h e fine h o m o g e n o u s inclusions of t h e second t y p e of cells, t h e ']3' cells s t a i n g r e e n i s h w i t h l i g h t g r e e n - o r a n g e G, red w i t h p h l o x i n e of C H P a n d f a i n t blue w i t h t h e a n i l i n e b l u e - o r a n g e G c o u n t e r s t a i n s of Azan. E a c h h a l f of t h e h y p o c e r e b r a l g a n g l i o n h a s 8 to 10 A cells a n d 4 to 6 ]3 cells f o u n d a l o n g w i t h a large n u m b e r of n e r v e cells. T h e a n t e r i o r p a r t of t h e g a n g l i o n h a s 2 cells in t h e m e d i a n a n d m i d v e n t r a l position, 2 ceils in t h e a n t e r i o r r e g i o n a n d 4 t o 6 cells in its p o s t e r o l a t e r a l p a r t (Figure 1). T h e a x o n s a r i z i n g f r o m t h e A cells r u n t o w a r d s t h e l a t e r a l b o r d e r of t h e g a n g l i o n (Figure 3) f r o m w h e r e they enter the corpora cardiaea through the nervi hypoc e r e b r i i (NH, F i g u r e 1). I t is e a s y to t r a c e t h e a x o n a l p a t h w a y s (AX) d u e t o t h e p r e s e n c e of s t a i n e d n e u r o s e c r e t o r y m a t e r i a l in t h e m w h i c h s t a i n s deep p u r p l e w i t h PF, red with Azan and blue-black with CHP. T h e m i d - d o r s a l region of t h e a n t e r i o r h a l f of t h e h y p o c e r e b r a l g a n g l i o n h a s 4 t o 6 t3 ceils w h i c h s t a i n g r e e n i s h w i t h P F , red w i t h C H P a n d f a i n t blue w i t h Azan. T h e a x o n s of t h e s e cells c a n b e t r a c e d o n l y for a s h o r t d i s t a n c e d u e t o lack of s t a i n i n g colloids. A large a m o u n t of n e u r o s e c r e t o r y m a t e r i a l (NSM) h a s b e e n o b s e r v e d l a t e r a l l y in t h e h y p o c e r e b r a l g a n g l i o n of m a l e s as t h e t w o NCC I p a s s t h r o u g h its l a t e r a l m a r g i n s , w h e r e a s t h e a m o u n t of N S M is c o m p a r a t i v e l y less in females as t h e NCC. I are free a n d h a v e n o c o n n e c t i o n w i t h h y p o c e r e b r a l ganglion. T h e s m a l l a m o u n t of NSM, w h i c h is p a r t i c u l a r l y seen before c o p u l a t i o n a n d ovip o s i t i o n in females, is t h e p r o d u c t of t h e n e u r o s e c r e t o r y cells of t h i s ganglion. T h u s t h e g r e a t e r a m o u n t of N S M in m a l e s is t h e p r o d u c t of t h e n e u r o s e c r e t o r y cells of t h e h y p o c e r e b r a l g a n g l i o n as well as t h e p a r s i n t e r c e r e b r a l i s of t h e b r a i n . T h u s t h e p r e s e n t w o r k records for t h e first t i m e in insects t h e p r e s e n c e of b o t h A a n d ]3 cell t y p e s a n d n e u r o s e c r e t o r y p a t h w a y s u p to t h e n e u r o h a e m a l o r g a n s (corpora eardiaca).
1 N. KHATTA~, Bull. Soe. Zool. ft. 93, 225 (1968).
