Zeitschrift fiir ZeUforschung 54, 613--630 (1961)

From the Zoological Institute of the University of Kyoto (Japan) (Director: Prof. Dr. M. ICn]KAWA) ELECTRON MICROSCOPIC STUDIES ON T H E N E U R O S E C R E T O R Y S Y S T E M I N L E P I D O P T E R A By J. NISHIITSUTSUJI-Uwo * With 12 Figures in the Text

(Received December28, 1960) Introduction Since W~YER (1935) first o b s e r v e d nerve cells h a v i n g a s e c r e t o r y a c t i v i t y in the b r a i n of t h e honey-bee, Apis melli]ica, n e u r o s e c r e t o r y cells h a v e been discovered in t h e n e r v o u s system, especially in t h e p a r s i n t e r c e r e b r a h s of t h e b r a i n of insects belonging to different orders. B. a n d E. SCHARRER (1944) h a v e advoc a t e d from t h e i r c o m p r e h e n s i v e studies t h a t t h e h y p o t h a l a m o - h y p o p h y s e a l s y s t e m in v e r t e b r a t e s is c o m p a r a b l e in m o r p h o l o g y to t h e i n t e r c e r e b r a h s - c a r d i a c u m - a l l a t u m s y s t e m in insects. I n Leucophaea, t h e s e c r e t o r y m a t e r i a l can be t r a c e d from the p e r i k a r y o n v i a a x o n to t h e corpus c a r d i a c u m . The l a t t e r serves as a storage center of the n e u r o s e c r e t o r y m a t e r i a l (B. SCHARR~R 1952). This finding has been c o r r o b o r a t e d s u b s e q u e n t l y in different species of insects. Our e x p e r i m e n t a l results in L e p i d o p t e r a n s f a v o r t h e view of t r a n s p o r t a t i o n of t h e s e c r e t o r y m a t e r i a l . H o w e v e r , the m a j o r storage center for n e u r o s e c r e t o r y m a t e r i a l is t h e corpus a l l a t u m ; t h e corpus c a r d i a c u m plays, if any, a m i n o r role in this r e s p e c t (ICHIKAWA a n d NISHIITSUTSUJI-Uwo 1959, 1960; ICHIKAWA a n d TAKAHASHI 1959). I n our studies t h e n e u r o s e c r e t o r y m a t e r i a l p r o v e d less s t a i n a b l e t h a n in r e p r e s e n t a t i v e s of o t h e r orders ( u n p u b l i s h e d d a t a ) . Therefore, t h e electron microscopic a p p r o a c h was chosen to e l u c i d a t e t h e i n t e r c e r e b r a l i s - c a r d i a c a - a l l a t a system. The p r e s e n t p a p e r records o b s e r v a t i o n s on t h e s e c r e t o r y a c t i v i t y in t h e p e r i k a r y o n s of n e u r o s e c r e t o r y cells a n d on t h e t r a n s p o r t a n d release of t h e secretory product. Materials and Methods The tissues used in the present work are the brain-cardiaca-allata complexes of both the domestic silkworm, Bombyx mori and the Eri-silkworm, Philosamia cynthia ricini. In the mature stage of the last instar of these caterpillars the heads were cut off and the braincardiaca-allata complexes were immediately dissected out and fixed. The fixation was carried 9 The author wishes to express her cordial thank to Prof. Dr. M. Ic~xxwA, under whose supervision the present investigation was performed. She is also indebted to Prof. Dr. Y. SANO of the Anatomical Institute, Kyoto Prefectural Medical College, for his valuable criticism of the electron micrographs. Thanks are also due to Prof. Dr. G. YASUZUMrand other members of the Electron Microscope Research Laboratory, Nara Medical College, as well as to the members of the Electron Microscope Laboratory of Keio-University, for their technical assistance in taking the micrographs. And last but not least, she wishes to acknowledge her indebtedness to Prof. Dr. M. S~IOENAOA of the Nara Women's University and to Prof. Dr. N. SnINKE of the University of Kyoto for their kind aid in the preparation of the electron microscopic sections. Z. Zellforsch., Bd. 54 41

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out in the case of Bombyx at 10~ C for 30 minutes in 1 per cent osmium tetroxide solution adjusted to pH 7.0 with veronal-acetate buffer. Thereafter, without washing in distilled water, the specimens were rapidly dehydrated in a graded series of ethanol (YAsuzuMI and ISHIDA 1957), impregnated with a mixture of 20 per cent methyl and 80 per cent n-butyl methacrylate, and finally embedded in the same resin mixture using 1 per cent 2,6-dichlorobenzoyl peroxide as catalyst for polymerization at 400 C. In the case of Philosamia, the materials were fixed at 50 C for 90 minutes in a mixture composed of 1 per cent osmium tetroxide, 0.22 M. sucrose and 1 per cent dextran, buffered with phosphate solution at pH 7.0 (BA~R, Br.OOM and FRIBERG 1957). After dehydration in a graded series of butanol, the specimens were embedded in a mixture of 75 per cent n-butyl and 25 per cent methyl methacrylate, using 0.2 per cent benzoyl peroxide as catalyst for polymerization at 65 o C. Sections were mounted on grids coated with formvar and examined, without removing the resin, with the AKASm electron microscope, model TRS-50, or with the HITACHIelectron microscope, model HS-5. Electron micrographs were taken at different magnifications ranging from 2200 to 12000, and enlarged photographically as desired. For the light microscopic examination, the materials mentioned above were fixed in BouIN's solution for 2 4 ~ 8 hours. Serial sections were cut at 5/~ and stained with GOMORI'S chrome-alum-hematoxylin-phloxin.

Observations

I. Neurosecretory cell I n t h e m a t u r e larva, t h e n e u r o s e c r e t o r y cells are clearly f o u n d in t h e different p a r t s of t h e brain. The m e d i a l groups of t h e cells are s i t u a t e d in t h e p a r s intercerebralis of t h e p r o t o c e r e b r u m , each g r o u p consisting of two t y p e s of cells. One t y p e consists of large ellipsoid or p o l y g o n a l cells, m e a s u r i n g 3 0 4 0 # in m a j o r d i a m e t e r ; t h e i r nucleus is round, a b o u t 1 0 - - 1 5 # in d i a m e t e r . C y t o p l a s m i c inclusions of these cells are generally s t a i n e d light blue in GOMORI'S chromea l u m - h e m a t o x y l i n - p h l o x i n . Sometimes, a few granules with a t i n t of deep blue (about 1 # in d i a m e t e r ) are f o u n d in t h e c y t o p l a s m . Besides, a n u m b e r of small vacuoles are p r e s e n t in t h e p e r i p h e r a l p a r t of t h e c y t o p l a s m in Philosamia. I n Bombyx a large vacuole is seen a t t h e opposite pole of t h e a x o n hillock. The o t h e r t y p e are small r o u n d cells m e a s u r i n g 1 8 - - 2 5 # in d i a m e t e r ; c y t o p l a s m i c inclusions of these cells are sometimes s t a i n e d deep red a n d sometimes deep blue in GOMORI'S staining. T h e 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 small cells has g e n e r a l l y a fine g r a n u l a r a p p e a r a n c e . U n f o r t u n a t e l y , it was often impossible t o distinguish the large cell from t h e small cell t h r o u g h t h e electron microscopic observations. T h e l a t e r a l groups, consisting of a few n e u r o s e c r e t o r y cells each, are f o u n d in t h e l a t e r a l p a r t s of t h e p r o t o c e r e b r u m . I n the p r e s e n t p a p e r the description will be l i m i t e d to the fine s t r u c t u r e s of t h e cells of t h e m e d i a l groups. 1. Nucleus. The nucleus is s i t u a t e d a t t h e center of t h e cell a n d is, in general, r o u n d or oval, w i t h irregular outline. As seen in Fig. 1, electron m i c r o g r a p h s r e v e a l t h a t t h e nuclear m e m b r a n e (NM) consists of two t h i n layers, t h e i n n e r being t h i c k e r (about 100/~) t h a n t h e o u t e r (about 70 A). The o u t e r l a y e r carries fine granules, p r o b a b l y P a l a d e granules. Sometimes, t h e o u t e r l a y e r is associated with m e m b r a n e s of t h e endoplasmic reticulum. B e t w e e n t h e two layers a space is present (about 180 A). Thus, the nuclear m e m b r a n e as a whole is a b o u t 350 in thickness. The nucleus contains m a n y dense granules (100--250 A), e v e n l y d i s t r i b u t e d or a g g r e g a t e d into small clouds in a r a t h e r homogeneous m a t r i x .

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The nucleolus appears as a dark area, which is irregular in outline and comprises a few masses of fine dense granules connected with each other by filaments. Perhaps these granules and filamentous structures are equivalent to the

F i g . 1. N e u r o s e c r e t o r y cell in the p a r s i n t e r c e r e b r a l i s of P h i l o s a m i a . This cell c o n t a i n s no neuros e c r e t o r y g r a n u l e s , h o w e v e r , it is i d e n t i f i e d as t h e n e u r o s c c r e t o r y cell f r o m its f o r m , d i m e n s i o n , etc. Such n e u r o s e c r e t o r y cells are f r e q u e n t l y e n c o u n t e r e d , especially in Philosam~a. N N u c l e u s ; N 3 I N u c l e a r m e m b r a n e ; M M i t o c h o n d r i a ; L L i p i d e b o d y ; P,~I P l a s m a m e m b r a n e ; G P N e u r o g l i a l cell processes; E R o u g h s u r f a c e d c n d o p l a s m i c r e t i e u l u m . M a g n i f i c a t i o n : 14000 x

"pars amorpha" and nucleolonema respectively found in varying cells of different animals as reported by ESTABL~ and SOT~LO (1955) and HOI~STMANNand K~OOl~ (1957). 2. Perikaryon. The perikaryon of the neurosecretory ceils in the pars intercerebralis harbors mitochondria, endoplasmic reticulum, the ribosomes, and the Golgi apparatus as seen in neurones in general. Besides, as a special feature of these cells, electron-dense granules are often seen. They are referred to as neurosecretory granules, details of which will be stated later. 41"

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a) Mitochondria. In the cytoplasm, there occur numerous filamentous, rodlike or sometimes bead-like mitochondria. They show a slightly greater density than the cytoplasmic matrix. The mitochondria distribute at random or sometimes gather together in crowds (M in Fig. 1). The mitochondria consist of double membranes and cristae. There seems no relationship between the mitochondria and the physiological activity of the cell.

Fig. 2. N e u r o s e c r e t o r y cell of B o m b y x filled w i t h n e u r o s e c r e t o r y e l e m e n t a r y g r a n u l e s . N N N u c l e u s of t h e n e u r o s e c r e t o r y cell; E E n d o p l a s m i c r e t i c u l u m ; E G E l e m e n t a r y g r a n u l e ; G N N u c l e u s of t h e n c u r o g l i a l cell; G P N c u r o g l i a l cell processes; I I n v a g i n a t i o n of n e u r o g l i a l cell p r o c e s s ; P B P i g m e n t b o d y . M a g u i f i c a t i o n : 9000 •

b) Endoplasmic reticulum and ribosomes. The cytoplasm of the neurosecretory cell is filled with endoplasmic reticulum (E in Fig. 1). Most of the membrane are rough-surfaced, but sometimes smooth-surfaced ones are also observed. Regardless of its surface structure the membrane is about 70 A in thickness. The ribosomes on the surface of the endoplasmic reticulum are similar, with respect to the size, 200 • in diameter, and electron density, to Palade granules ( P A L A D E 1955). The same granules are also observed free in the cytoplasm. Generally speaking, the rough surfaced cytomembranes are well-differentiated in cells with no or only few neurosecretory granules. By contrast, when neurosecretory granules are abundant in the cytoplasm of Philosamia few eytomembranes are seen (Fig. 4). In Bombyx, cells with many secretory granules

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contain smooth-surfaced cndoplasmic reticulum which seems to be of the tubular type (E in Fig. 2). c) Golgi apparatus. A complex structure, consisting of piles of smooth double membranes and small vacuoles surrounding them, is frequently encountered in the cytoplasm (G in Figs. 3--5). This structure is referred to as the Golgi apparatus. I t much resembles the Golgi apparatus found in the neurosecretory cells in

Fig. 3.

N e u r o s e c r e t o r y cell of B o m b y x showing c o n s t r i c t e d m i t o c h o n d r i a a m o n g t h e e l e m e n t a r y granules. M Mitochondria; G Golgi l a m e n a e ; E G E l e m e n t a r y g r a n u l e ; N Nucleus. Magnification: 13300 •

the caudal spinal cord of Tinca vulgaris (SANo and KNOOP 1959). Each component of the double membranes is about 60 • in thickness and the space between the two components is about 90/~. The small vacuoles vary in size, and are filled with a homogeneous substance with less electron density than its membranous wall.

d) Neurosecretory granules. The most striking features often encountered in the neurosecretory cells are fine, spherical or ellipsoidal, electron-dense granules (EG in Figs. 2--5). These granules are relatively uniform in electron density, and each one is about 1000--3200 A in longer diameter (1800 A on the average) in Philosamia, while they measure about 600--3600 A (1200 A on the average) in Bombyx. From their form and dimension, these granules m a y be considered to be equivalent to the neurosecretory granules of the neurohypophysis (BAROMANN 1958; DUNCAN 1956; FUJITA 1957; GREEN 1955; HARTMANN 1958; PALAY

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1957), in the caudal neurosecretory system of the vertebrates (E~AMI and IMAI 1958; SAgO and KNooP 1959) and in the ncurosecretory terminal organs of the

Fig. 4. P e r i p h e r y of a n e u r o s e c r e t o r y cell of Philosamia p a c k e d w i t h e l e m e n t a r y granules. N Neuroglial cell nucleus; P ~ / P l a s m a m e m b r a n e of n e u r o s e c r e t o r y cell; C Canaliculi b e t w e e n t h e neuroglial processes; E G E l e m e n t a r y g r a n u l e ; GG G i a n t g r a n u l e ; G Golgi lamellae; C" Canaliculus p r o b a b l y t e r m i n a t i n g on the p l a s m a m e m b r a n e . Magnification: 17 150 •

invertebrates (KNowLES 1958; MEYER and PFLUGFELDER 1958; HODGE and CHAPMAN 1958). I t is uncertain whether they are the neurosecretory materials

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themselves or structures very intimately associated with them. The granules are called elementary granules according to FUJITA (1957), who coined this term in

Fig. 5. E l e m e n t a r y g r a n u l e s n e a r the Golgi lamellae of P h i l o s a m i a . G Golgi lamellae; E G E l e m e n t a r y g r a n u l e ; M ivIitoehondria. ( P a r t i a l m a g n i f i c a t i o n of Fig. 4). Magnification: 55200 •

his studies of the neurohypophysis of the dog. The granules occur in groups in the peripheral part of the cell or sometimes aggregated into giant granules (7500 A) as is seen in Fig. 4 (GG). It is highly probable that the giant granules correspond

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to the secretory granules staining with chrome-hematoxylin as seen under the light microscope. I t seems likely that the Golgi apparatus is related to the formation of the elementary granules, since these are frequently encountered within the Golgi zone. As has been reported by the present author (1960), in Philosamia a peculiar feature of mitochondria forming a rosary of ellipsoidal bodies occurs near the Golgi membranes (M in Fig. 5). Some distance away from the Golgi membrane, these ellipsoidal bodies appear to be constricted and separated from each other and to migrate among the electron-dense elementary granules described above. Some of these fragmented mitochondria lose their cristae. Gradation with respect to the disappearance of the cristae can be seen and the resulting bodies cannot be distinguished from the elementary granules with membranous walls. These observations seem to favour the view t h a t the elementary granules m a y originate from mitochondria which receive some material from the Golgi zone as has been suggested for zymogen granules of the mouse pancreas (CHALLIC]~and LAcY 1954). The membranous wall of the elementary granule would appear to be derived from the mitochondrial wall, although in Tinca vulgaris, the outer membrane of the neurosecretory elementary granule evidently originates from the Golgi lamella (SAgo and KNooP 1959). e) Other cytoplasmic inclusions. I n the cytoplasm, electron-dense bodies larger and with more irregular outline than the mitochondria are seen. These bodies are presumably of lipide nature, equivalent to the lipochondria or lipide bodies which have been seen in the neurons of the cockroach (HEss 1958). They take random disposition in the perikaryon (L in Fig. 1). Besides, in Bombyx there is a stupendous vacuole in the large neurosecretory cell. This vacuole is packed with a structureless substance under the electron microscope.

II. Sheath o/the brain in the area o/the pars intercerebralis According to HEss' terminology, the sheath of the brain will be divided into the neural lamella and perilemma: the neural lamella is the structureless outer layer, while the perilemma is the inner cellular layer. But, as seen in Fig. 6, the neural lamella (NL) in the area of the pars intercerebralis is not subdivided into four parts as in the cockroach ganglion described by HEss (1958). The perilemma is principally made up of glial cells, the processes of which imbricate extensively and wrap the nerve cells, neurosecretory cells and their axons. Between the neural lamella and the perilemma, there exists a space (S) which seems to contain coagulated hemolymph. Tracheoles (T) are often seen in it. This space would then be a blood sinus, and it is evidently connected with the canaliculi (C), t h a t take a zigzag course between the processes of the glial ceils, to be discussed later. 1. ~euroglia. The neuroglial ceils, in the perilemmal layer, have the closest connection with the neurosecretory cells in the pars intercerebralis. These glial cells have an irregularly shaped nucleus (N in Fig. 6), which contains in turn dense masses of chromatin. One or two, sometimes three nucleoli, are observed in the nucleus. The glial cytoplasm is rich in rough surfaced endoplasmic reticulum (E), mitochondria (M) and Golgi apparatus (G) as seen in Fig. 7. I t forms

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Fig. 6. S h e a t h of t h e b r a i n n e a r t h e p a r s intercerebralis of Philosamia. N L N e u r a l lamella; S Bloodsinus; N Neuroglial nucleus; M Mitochondria; C Cross section o5 eanaliculi n e a r neuroglial cell processes; T Tracheole. Magnification: 10800 •

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a thin layer around the nucleus and extends into many processes. These processes ramify extensively as reported by tt~ss (1958) in the cockroach ganglia. The ramified processes are extremely complicated. Lipide bodies (L in Fig. 7) can often be seen within these processes. Only in Bombyx, the glial cell has many

Fig. 7. Neuroglial cell of Philosamia. N Neuroglial cell nucleus; G Golgi lamellae; M Mitochondria; E E n d o p l a s m i c r c t i c u h m l ; L Lipide b o d y ; C Canaliculi b e t w e e n the i m b r i c a t e d neuroglial cell processes. Magnification: 21600 •

electron-dense bodies (PB in Fig. 2) measuring 300--700 m/~ in diameter. They take random disposition in the glial processes. From their form and dimension, they seem to coincide with the pigment granules seen in non-stained preparations under the light microscopic observation. The elongated mitochondria of the neuroglial cell are much more electrondense than those in the neurosecretory cell and the nerve cell. The prominent features in the perilemmal layer of this part of the brain are many canahculi of varying sizes (C in Figs. 6 and 7). Some parts of canaliculi enlarge into "sinus" containing the coagulation product of hemolymph. The canaliculi seem to be the same as the "glial lacunar system" reported by WIGGLESWORTH (1960) in cockroach ganglia.

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2. Relation between the neuroglial cell and the neurosecretory cell. The ramified and overlapping processes of neuroglial ceils enmesh the neurosecretory cells, as has been demonstrated by SANO (1958) in hypothalamic neurosecretory cells of the cat as seen under the light microscope. Sometimes, these glial processes

F i g . 8. C r o s s s e c t i o n of t h e c o r p u s c a r d i a c u m of Philosamia s h o w i n g n e r v e e n s h e a t h e d b y S c h w a n ~ cell. F N e r v e f i b e r ; N S c h w a n n cell n u c l e u s ; S c h S c h w a n n cell p r o c e s s e s . M a g n i f i c a t i o n : 8400 •

invaginate in the neurosecretory cells and in the nerve cells as reported by HESS (1958) in the cockroach-ganglia. This invagination m a y facilitate the transport of nutritive material from the glial cells to the neurons. Recently, WIGGLESWORTK (1960) made a similar statement regarding cockroach ganglia. On the other hand, the aforesaid canaliculi passing through the glial cell processes come in contact with, or sometimes m a y terminate on the plasma membrane of the neurosecretory cells (C' in Fig. 4) as well as of the nerve cells. These structures also might serve as the transfer of the nutritive substance from the blood to the ganglion cells, although WIOGLESWORTH (1960) has suggested their role as a pool of nutrients. At the same time, these structures m a y constitute a pathway of the neurosecretory products into the blood system.

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F i g . 9. E l e m e n t a r y g r a n u l e s w i t h i n n e r v e f i b e r i n t h e c o r p u s c a r d i a c u m o f Philosamia. E G E l e m e n t a r y g r a n u l e ; F N e r v e f i b e r ; T T r a e h e o l e ; Seh S c h w a r m cell p r o c e s s e s . M a g n i f i c a t i o n : 1 4 4 0 0 •

F i g . 10. Cell of t h e c o r p u s a l l a t u m of Philosamia. M M i t o c h o n d r i a ; N M N u c l e a r m e m b r a n e ; N N u c l e u s . M a g n i f i c a t i o n : 13 600 •

III. 1Veurosecretory pathway Light microscopic observation reveals that the axons of the medial neurosecretory cell group, after crossing, leave the brain to form the nervus corporis

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Fig. 11. N u c l e u s in c o r p u s a l l a t u m cell of B o m b y x . N M N u c l e a r m e m b r a n e ; Gr G r o u p s of s m a l l g r a n u l e s ; D U n k n o w n droplet. ) I a g n i f i c a t i o n : 7100 •

Fig. 12. N e r v e t e r m i n a l s c o n t a i n i n g e l e m e a t a r y granlfles a n d e m p t y vesicles in B o m b y x . E G E l e m e n t a r y g r a n u l e ; V E m p t y vesicle. M a g n i f i c a t i o n : 17550 x

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cardiaci I, while the fiber tract originating from the lateral group passes through the brain without crossing and leaves as nervus corporis cardiaci II. The latter joins with the former on the way to the corpus cardiacum. This nerve leaves the organ to innervate the corpus allatum. 1. Corpus cardiacum. On electron micrographs the nerve fibers in this organ are enveloped b y the imbricated processes of Schwann cells (Figs. 8 and 9), and the nerve fibers contain numerous mitochondria and sometimes neurosecretory elementary granules (EG in Fig. 9). No nerve endings could be identified with confidence in this organ. This m a y bear a relationship to the experimental result t h a t the organ functions, if at all, only as a minor storage center of neurosecretory material (ICHIKAWA and NISHIITSUTSUJI-Uwo 1959). 2. Corpus allatum. The corpus allatum is an oviform organ connected with the posterior part of the corpus cardiacum b y the nervus corporis allati. I t bifurcates into two main branches immediately after its entry into the corpus allatum; one runs along the peripheral part of this organ and the other terminates in the center of it. As is well known, the organ secretes the juvenile hormone; besides it acts as the storage center of the brain hormone. a) Cells o/the corpus allatum. The cells of the corpus allatum are so complicated in shape that it is difficult to trace their outline. They contain m a n y ribosomes and mitochondria of various sizes and shapes. Both structures are found scattered throughout the cytoplasm. The nuclear membrane is also rather complicated in outline (NM in Figs. 10 and 11). In the karyoplasm, small granules occur in groups of different sizes (Gr in Fig. 11). Furthermore, in Bombyx, peculiar, giant, less electron-dense droplets are seen (D in Fig. 11). Nothing can be said about the nature of these droplets, except that it is safe to say that they are not artifacts, because various fixatives were used to ascertain this point. I t is also a problem of the future to disclose a correlation between these droplets and the juvenile hormone. b) Nerve terminal el the neurosecretory cell. The neurosecretory nerve terminal of the intercerebralis-cardiacum-allatum tract seems to form a sac-like dilatation. As seen in Fig. 12, the terminals contain electron-dense granules, ranging between 600--4000 A in diameter (EG). I t is highly probable that these granules are identical with the elementary granules specified in the perlkaryon of the neurosecretory ceils. The terminals contain also m a n y e m p t y vesicles (V) measuring 900 4600 A in diameter, which m a y correspond to the e m p t y vesicles observed in the nerve terminals in the neurohypophysis of the cat (GREEN and VA~ BRE~MEN 1955) and of the rat (ttARTMANN 1958). They m a y also be equivalent to the granules of type 3 described by FUJITA (1957) in the neurohypophysis of the dog. Depending perhaps on the phase of secretory activity the proportion of the granules and the vesicles m a y vary, i.e., when the granules are more abundant, the vesicles are more scarce, and vice versa.

Discussion 1. Formation of the neurosecretory material. In the present investigation on the commercial silkworm and the Eri-silkworm, the most striking feature of the neurosecretory cells in the pars intercerebralis is the occurrence of fine, spherical or ellipsoidal, electron-dense granules measuring about 600--3600 A in diameter.

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They are referred to as the neurosecretory elementary granules according to FUJITA'S terminology coined on investigation of the vertebrate hypophysis (1957). The same structures have been detected within the nerve fibers in the neurohypophysis of various vertebrates (BARoMANN 1958; DTZNCAN 1956; G R ~ N and VAN BREE~EN 1955; I-IARTMANN 1958; PALAY 1957) and in the neurosecretory cells of the caudal spinal cord of vetrebrates (ENAMI and IMAI 1958; SANO and KNooP 1959), as well as in the nerve fibers in the corpus cardiacum of the stick insect (M]~YE~ and PFLUGFELDER 1958), in the post-commissure organ of the prawn (K~owLES 1958) and in the sinus gland of the land crab (HoDoE and CJZAPMA~ 1958). Based on light microscopic observation, KUROTSU and KONDO (1941) stated that the neurosecretory granules in the cells of the hypothalamus of Bu/o vulgaris japonieus are derived from the plastosome (mitochondria). This statement was confirmed by ITO and OIsrn (1950) with the same species. As to the insect, NAYA~ (1955) suggested that the neurosecretory granules of the pars intercerebralis of Iphita limbata are also derived from mitochondria. On the other hand, RO~IEV, STAHL and COTTE (1953) have stated that the Golgi apparatus of the cat may be responsible for the production of neurosecretory material. From the electron microscopic observation of the caudal neurosecretory system, ENAMI and IMAI (1958) have demonstrated that in Anguilla japonica the elementary granules are present in close association with Nissl substance, whereas SANo and KNooP (1959) have insisted that in Tinca vulgaris they are produced in the Golgi apparatus or its surroundings. The present electron microscopic study of the insect neurosecretory cells in the brain revealed the following points: 1. When the elementary granules in neurosecretory cells are rather scanty, there occurs, in general, a well-defined membranous endoplasmic reticulum with Palade granules; the latter granules also occur free in the cytoplasm. By contrast, when the elementary granules are numerous, the rough endoplasmic reticulum becomes scanty. 2. The elementary granules with membranous covering are frequently encountered in the Golgi zone. 3. In the Golgi zone, some of the mitochondria are divided into small fragments and lose gradually their cristae. It is impossible to distinguish such structureless mitoehondrial fragments from the elementary granules. From these observations, it may be surmised that the formation of the elementary granules is in close connection with the mitochondria in the Golgi zone, but that the rough endoplasmic reticulum appears also to be involved. The membranous wall of the elementary granules could be a derivative from the mitochondrial wall. 2. Transportation of the neuroseeretory material and its release. In 1944, B. and E. SC~ARRER discovered that in Leucophaea the neurosecretory granules migrate along the axons from the neurosecretory cells in the pars intercerebrahs of the brain to the corpus cardiaeum. This observation was subsequently confirmed by many workers in various insects. In lepidopterans, ARVEY, BOVNHIOL and GABE (1953) and KOBAYASI~ (1957) have observed that the neurosecretory granules in the pars intercercbrahs of Bombyx are transported along the axon to reach not only the corpora cardiaca but also the corpora allata. However, there is also some information indicating the direct release of the neurosecretory

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substance from the perikaryon (I~EK~ 1950, Ephestia, and WILLIAMS 1952, Cecropia). Dual mode of secretion from the neurosecretory cells seems probable in the lepidopterous insects. Our previous experimental data are favorable to the transportation theory (ICHIKAWA and NISgIITSUTSUJI-Uwo 1959, 1960; ICI~IKAWA and TAKAttASItI 1959), but a recent experiment showing t h a t brains with nervi corporis cardiaci severed on both sides, can release enough brain hormone to cause the imaginal differentiation of brainless diapausing pupae of Philosamia, speaks in favor of direct secretion from the perikaryon (NISttlITSUTSUJI-Uwo, unpublished data). The electron micrographs indicate that the neurosecretory cells in the pars intercerebralis are enmeshed by the neuroglial cell processes, among which there exist canaliculi communicating with a blood sinus. The canaliculi are in contact with the plasma membrane of the neurosecretory cell. Although elementary granules can not be seen within the canaliculi, these structures are apparently convenient for direct secretion of the secretory material from cell into blood system. On the other hand, other electron micrographs indicate that the elementary granules are transferred via axons to the corpora ailata, because the sac-like dilations of the nerve endings contain the same elementary granules and e m p t y vesicles as found in the perikaryon of the neurosecretory cells. The vesicle m a y be the cast of the secretory material, as has been claimed b y FVZITA (1957) and HA~TMANN (1958) regarding mammals.

Summary 1. Electron microscopic studies of the neurosecretory system in the lepidopterous insects, Bombyx mori and Philosamia cynthia ricini, were performed in the mature larvae. 2. The perikaryon of the neurosecretory cell in the pars interccrebralis contains characteristic granules in addition to the common features of the nerve cells. These granules are fine, spherical or eilipsoidal and relatively uniform in electron density. Each one is about 600--3600 A in diameter (1200/~ on the average) ia Bombyx and about 1000--3200 A (1800/k on the average) in Philosamia. These granules are referred to as the elementary granules. 3. When the neurosecretory cell is rather poor in elementary granules, there occur, as a rule, numerous well differentiated, rough surfaced cytomembranes. 4. The elementary granules are produced in close relation to the mitochondria in the Golgi zone. The endoplasmic reticulum with R N A granules seems also concerned with the formation of the neurosecretory material. 5. The neurosecretory cells are enveloped by processes of neuroglial ceils, and between the processes there exist m a n y branched canaliculi of varying sizes. Some of these canaliculi make contact with the plasma membrane of the neurosecretory cell. 6. The release of the neurosecretory material from the perikaryon m a y take place into these canaliculi and from there into the blood sinus. But there is also indication t h a t the elementary granules are transported via axon into the corpora ailata and stored in them. The nerve terminals in the corpus ailatum form bulbous dilatations in which elementary granules and e m p t y vesicles are abundant. In

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t h e l i g h t of o u r e x p e r i m e n t a l r e s u l t s , i t is l i k e l y t h a t t h e n e u r o s e c r e t o r y m a t e r i a l is d i s c h a r g e d f r o m t h e t e r m i n a l s i n t o t h e b o d y fluid, a l t h o u g h t h e a c t u a l disc h a r g e of t h e m a t e r i a l c o u l d n o t be d e m o n s t r a t e d in e l e c t r o n m i c r o g r a p h s . Literature

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