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Peripheral neurosecretory cells in the stick insect (Carausius morosus) and the blowfly larva (Phormia terrae-novae)
J. Insect Physiol., 1968, Vol. 14, @a. 1793 to 1801. Perg-
Press. printed in Grta
Britain
PERIPHERAL NEUROSECRETORY CELLS IN THE STICK INSECT (CARA.USIUS MOROSUS) AND THE BLOWFLY LARVA (PHORMIA TERRAE-NOVAE) L. H. FINLAYSON
and M. P. OSBORNE
Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham 15
Abstract-On each side of the abdominal segments of the stick insect there are at least ten multiterminal neurones lying on major nerves or on the tracheae that run with the nerves. The dendritic processes of the neurones run on and in the nerves. Sections stained with paraldehyde-fuchsin or examined by electron microscopy show typical neurosecretory granules in some of these neurones, in both cell body and cell processes. Superficial processes adjoin the haemocele and appear to be releasing their secretions into the blood. A group of about four cells situated on the nerve linking the ‘somatic’ and the ‘median’ nervous systems shows varying degrees of secretory activity. They may be contributing electrondense granules to the lateral neurohaemal organs and to diffuse neurohaemal tissue in the general region of the spiracle and/or releasing material directly into the haemol~ph. Electron-microscopic examination of a large neurone on the ventrolateral sheet of fat body reveals no conclusive evidence of neurosecretion. In the blowfly larva a neurosecretory neurone is located on each side of the body segments at the junction between the somatic and median nerves, INTRODUCTION
IN A discussion of the concept of neurosecretion BERN and KNOWLES(1966) point out the difficulty of establishing by light or electron microscopy whether a neurone or a neuronal process is neurosecretory. They exclude neurones that innervate exocrine glands, muscles, or other tissues, even though they or their processes contain secretory granules of a type found in neurosecreto~ neurones. Apart from the neurosecreto~ neurones of the central nervous system of vertebrates and invertebrates, the only other neurones that almost certainly fit this concept of neurosecretory are those in the corpus cardiacurn of insects (SCHARRER, 1963 ; SMITH and SMITH, 1966). A neurosecretory function has been postulated for a number of other cells, but electron microscopical investigation has revealed that they do not contain granules characteristic of neurosecretory cells (BERN, 1966). In the absence of evidence that a secretory neurone is engaged directly or indirectly in endocrine control (Bw and KNOWLES, 1966) there are two morphological criteria that are accepted as good evidence of neurosecretory function. One is the presence in the neurons of secretory inclusions (‘granules’, ‘vesicles’, ‘droplets’) measuring 100 to 300 rnp dia. and the other is the termination of the processes
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of the neurone in close proximity to a cavity containing blood or other fluid. Such terminations occur in discrete tissue, called neurohaemal tissue, as in the corpora cardiaca (SMITH and SMITH, 1966) and the segmental neurohaemal organs of phasmids (RAABE, 1965) or at individual endings, as in the abdominal nerves of Rhodnius (MADDRELL,1966), the heart of Peripluneta (JOHNSON, 1966), and the rectal papillae of Culliphora (GUPTA and BERRIDGE,1966). It has been generally assumed that neurohaemal endings belong to neurones whose cell bodies lie in the ganglia of the central nervous system or in the corpora cardiaca. In this paper wiI1 be described peripheral neurones that appear to have all the morphological attributes of neurosecretory cells as defined by BERN and KNOWLES(1966). Multite~inal neurones are found in the periphery of the insect body in various locations. Some are subepidermal and others are more deeply situated, with endings on a variety of tissues. Others are situated on the course of nerves and it is not known where their processes terminate (for review see FINLAYSON,1968). In the course of a study of the peripheral neurones of the stick insect it was found that certain neurones associated with several major nerves appeared to be neurosecretory. These neurones are described in this paper. The distribution of peripheral multiterminal neurones in the blowfly larva has already been described by OSBORNE (1963,1964). 0 ne of them has been investigated for neurosecretory activity. MATERIAL AND METHODS Larvae and adults of the stick insect (Curausius ntorosus) and final instar larvae of the black blowfly (Pbmia tevrae-novae) were used for this study. Vital staining of dissected animals was carried out with methylene blue in Ringer solution (NaCl, 6.31 g; KCI, 1.86; MgCl, 0.1 g; CaCl, O-2 g; NaH,PO,, 0.2 g; NaHCO,, 0.05 g; dextrose, 16 g; Hz0 to 1 1. Sections for light microscopy were cut at 3~ and stained with paraldehyde-fuchsin (CAMERONand STEELE, 1959) after fixation in Bouin with acetic acid content reduced to 2 per cent. For electron microscopy the stick insects and blowfly larvae were opened along the dorsal midline, the gut was removed, and the remaining tissues were flooded with 2.5% glutaraldehyde for 20 min. Tissues for sectioning were then transferred to 2% osmium tetroxide. Both the glutaraidehyde and the osmium tetroxide were buffered to pH 7.4 with phosphate buffer and made up to a total molarity of 0.45 M by the addition of sucrose. Dehydration in 70,90%, and absolute ethanol was followed by embedding in Epon (MANTON, 1964). Sections were double-stained in uranyl acetate and lead citrate. NEURONES OF THE STICK INSECT Topography Neurones associated with major nerves have been located by means of methylene blue staining and their distribution in the second abdominal segment of an adult is shown in Fig. 1. The cell bodies vary in position (Figs. 2,3) and it is
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diacult to be sure of the precise number of neurones or whether there is variation in number between segments. However, there is no doubt that there are about ten on each side of the major abdominal segments. Near the first nerve junctions, where the lateral nerve (nl,, MARQUHARDT, 1939) Ieaves the main segmental nerve there are several neurones whose relationship to the nerves is not clear. At least one of them sends its processes along the nerves (Fig. 5). In addition to neurone No. 8 (Fig. 1) a second neurone is sometimes to be seen on naSa, the nerve that runs by the spiracle. The cell bodies and the processes of these neurones are superficial in position. Frequently the cell body lies on the tracheae that runs beside the nerve and the processes ramify over tracheal and nerve branches (Figs. 3,6). The
FIG. 1. Stick insect. Diagram of innervation of one side of an abdominal segment. Neurones on the course of nerves are numbered 1 to 10; fb, fat body; fbn, fat body neurone; mn, median nerve; na,, naS, nata, nl,, nl,, np, npa, nerves labelled according to MARQUHARDT'S (1939) terminology; nh, neurohaemal tissue; sp, spiracle; ms, transverse nerve swelling (neurohaemal tissue).
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L. W. FINLAYSONAND M. P. OSBORNE
cell body may be detached, especially at nerve junctions (Fig, 5). The processes may run for long distances from the cell body or may terminate close to it (Figs, $6). Some endings have been observed to have a distinctive appearance in methylene blue preparations. They resemble irregular ink blots (Fig. 5) or a reticulum of fine fibrils (Fig. 8). Similar endings can be seen in multite~inal neurones on the epidermis in Crrrausius. In addition to the neurones on the nerves, one other is shown on Fig. 1. It is situated at the posterior margin of the ventrolateral sheet of fat body. Its possible functions are discussed below. N~~o~es OBthe link wve A nerve, designated na,, by MARQUHARDT (19391, links the main (somatic) nerve system of each segment with the lateral branch of the median (unpaired) nerve (Fig. 1). We shall refer to this nerve as the ‘link’ nerve for convenience and to emphasize its important ~atomic~ relationships~ There are, typically, four neurones on the trachea running alongside the nerve (Fig. 2). They vary considerably in position and one may lie at the junction of the link nerve and the anterior segmental nerve. In appearance, also, they show considerable variation, ranging from the typical multiterminal appearance with prominent processes to a condition in which the cell body is distended with secretory inclusions and the processes are fine and inco~picuous (Fig. 3). These cefls may stain heavily or only slightly with methylene blue. Sections stained with paraldehyde-fu~hsin revealed typical deeply stained neurosecretory inclusion in the cell processes but less deeply stained ones in the cell body. Electron microscopy, however, showed that the cell body also contains inclusions that are typical of neurosecreto~ cells (Figs. 10, 11). They are electron dense after treatment with osmium tetroxide, uranyl acetate, and lead citrate. As in the corpus cardiacurn of the same insect (SMOTE and SMITH, 1966) each secretory granule appears to be enveloped by a membrane of the Golgi complex (Fig. 11). On the vast majority of granules this membrane cannot be seen, perhaps because it has become closely applied to the surface of the secreted material, as suggested by SMITH and SMITH (1966). The endoplasmic reticulum is well developed in these cells and is densely coated with ribosomes (‘rough-surfaced’). Parts of the endoplasmic reticulum are frequently juxtaposed to the cell membrane (plasmalemma). Ribosomes are missing from the membrane surface applied to the pl~male~a (Fig. 11). This phenomenom may have no significance, but it is noteworthy that a large area of the plasmalemma is in very close apposition with the endopl~mi~ reticulum. The electron micrographs show how superficial are the neurosecretory cells of the link nerve. In transverse sections of the link nerve can be seen both superficial and deep processes containing neurosecreto~ granules (Fig. 12). The superficial processes have a puckered membrane with granules closely adhering to it (Figs. 12-14) and ~v~inations that may mark the points of release of the secretory material, the ‘omega profiles’ of SMITH and SMITH (1966). The deeper profiles show no such evidence of release of secretion (Fig. 12). They are wrapped
FIc. 2. Stick insect, second abdominal segment. Link nerve (nal in Fig. 1) with three neurones on the trachea and one at the junction with the main segmental nerve. Neurohaemal tissue (marked by arrow) at junction with transverse branch of median nerve. (Methylene blue.) Scale = 150/z. FIG. 3. Stick insect, third abdominal segment. Neurones at junction of link nerve (nal) and main segmental nerve, showing different degrees of distension with secretion. (Methylene blue.) Scale = 40/z. FIG. 4. Stick insect, second abdominal segment. Neurone (No. 10, Fig. 1) on nerve na2 with processes encircling trachea and returning to nerve. (Methylene blue.) Scale = 50/z. FIG. 5. Stick insect, first abdominal segment. Neurone No. 1 (Fig. 1) showing four processes; axon labelled (A). A short process terminates near the cell body and a long thick process has 'ink-blot' endings (I) on its branches. Fracture of nerve, trachea, and thick process marked by arrow. (Methylene blue.) Scale -50 ~.
FIc. 6. Stick insect, fourth abdominal segment. N e u r o n e near junction of na2 and na,a (Fig. 1). Note the short process terminating near the cell body (marked by arrow) similar to one in Fig. 5. (Methylene blue.) S c a l e - - 5 0 / z . FIG. 7. Stick insect, fourth abdominal segment. Fat body neurone; axon marked by arrow. (Methylene blue.) Scale -= 150/z. FIc. 8. Stick insect, fifth abdominal segment. N e u r o n e with process ending in a tangle of fine fibres (I). Link nerve (nal) with two neurones marked by arrow. (Methylene blue.) Scale = 100 p. FIc. 9. Stick insect abdominal segment. Neurohaemal tissue along course of nerves near spiracle. (Methylene blue.) Scale= 150/z.
FIG. 10. Survey picture of the cell body of a neurone on the link nerve of the stick insect. T h e neurone is bounded by a tenuous Schwann cell sheath (Sc) and a thin layer of basement membrane material (Bin). T h e cell contains numerous Golgi bodies (G) which are apparently producing secretory granules (arrows). H, haemolymph space. Scale = 2000 m/z.
FIC. 11. Stick insect. Detail of cytoplasmic contents of a link nerve neurone. The Golgi body (G) is sequestering granules as is evident from the continuity of Golgi membranes with the limiting membrane of the granule (straight arrow). Not all the granules have a limiting membrane (compare granules gl and g2). Profiles of rough-surfaced endoplasmic reticulum (Er) are evident and in some areas the reticulum is closely apposed to the plasma membrane (Pm) of the neurone. The apposed surface of the endoplasmic reticulum is free of the coating of ribosomes (curved arrow). Sc, Schwann cell. Scale = 500 m/z. The inset shows membrane continuity between a secretory granule and Golgi body (arrow). Scale = 500 m/z.
FIG. 12. Transverse section through a part of the link nerve of the stick insect. A group of nerve fibres (Nf) some of which contain granules is engulfed by wrappings of the Schwann cell (Sc). A large nerve process (top left) is packed with granules and has no Schwann cell sheath. This 'naked' process is separated from the haemolymph (H) by only a thin coating of basement membrane material (Bm). Note how markedly convoluted is the plasma membrane of the naked process (arrow) when compared with the smoother plasma membranes of the ensheathed nerve process. N, Schwann cell nucleus; T, trachea. Scale = 1000 mu.
FIG. 13. Stick insect. T w o nerve processes embedded in the basement membrane (Bm), surrounding the link nerve. One process ( N p l ) is 'naked' whereas the other (Np2) is partly invested by the Schwann cell (Sc). Both processes contain secretory granules, but Np2 is apparently subterminal since it contains neurotubules (straight arrows) which are very similar to the microtubules (curved arrows) in the nearby Schwann cell. Scale = 500 rap,. T h e inset shows a large naked nerve process on the link nerve. It is packed with secretory granules, some of which have an obvious limiting membrane (short arrows). T h e plasma membrane is deeply invaginated at some points (curved arrows) and in other regions where it is convoluted is in intimate contact with secretory granules (straight arrows). Note that only a thin layer of basement membrane material (Bm) is interposed between this nerve process and the haemolymph (H). Scale = 1000 m/z.
FIG. 14. Transverse section through part of the link nerve of the stick insect to show two ensheathed nerve processes (Npl, Np2) and a third, naked process (Np3). The latter is full of secretory granules whereas N p l contains only two such granules. Note the convolutions of the plasma membrane of Np3. Bm, basement membrane material; H, haemolymph; Sc, Schwann cell. Scale = 500 m/z.
FtG. 15. Stick insect. Section through part of the cell body of neurone No. 9 (Fig. 1) on nerve Na2. T h e cell contains free ribosomes, rough-surfaced endoplasmic reticulum (Er) and Golgi bodies (G). T h e Golgi bodies seem to be producing small dense granules (short arrows). However, one of the granules is larger (curved arrow) and resembles those (straight arrow) in the neighbouring naked-nerve process (Np). Sc, Schwann cell. Scale = 1000 m/z.
FIo. 16. Neurohaemal tissue of stick insect at the junction of the link nerve with the median nervous system. Some nerve fibres contain many secretory granules whilst others are relatively empty. Process N p l contains very. dense spherical granules of the type produced by the link nerve neurons. Elongated and spherical granules in the other nerve fibres have varying electron opacities. Bm, basement membrane; H, haemolymph; Sc, Schwann cell; T, tracheole. Scale = 1000 m/~.
FIG. 17. Parts of t h r e e n e r v e processes from stick insect n e u r o h a e m a l tissue at j u n c t i o n of link n e r v e w i t h m e d i a n n e r v o u s system. T h e process at u p p e r r i g h t contains spherical, d e n s e granules characteristic of those p r o d u c e d b y the link n e r v e n e u r o n e s . T h e o t h e r two processes have granules of lower electron density w h i c h show a n i n t e r n a l lamellate structure. Scale = 500 m/~. I n s e t shows detail of the i n t e r n a l s t r u c t u r e of the secretory granules. Scale = 250 m/z.
FIc. 18. Part of the neurone located on the fat body of the stick insect. The cell body contains many profiles of rough-surfaced endoplasmic reticulum (Er) and free ribosomes, symptomatic of protein production. The Golgi body (G) is producing small electron-dense droplets (straight arrows). Other organelles are mitochondria and microtubules (curved arrow). N, nucleus of neurone; Sc, Schwann cell. Scale = 1000 m/~. A cross-section of one of the dendrites of the fat body neurone is shown in the inset. Small electron dense droplets (arrows) resemble those associated with the Golgi body in the cell soma. Scale = 1000 m/~.
FIG. 19.
Blowfly larva. Part of cell body of neurone Mnl to show large numbers of electron-dense granules (arrows). Scale = 500 mp.
FIG. 20. Blowfly larva. Section across a dendrite of cell Mnl. This process is only partially enveloped by the Schwann cell (SC). The naked portion of the fibre has a puckered plasma membrane (straight arrow) close to which are clustered dense granules (curved arrows), suggesting that material is being liberated into the haemolymph (H) through the basement membrane (Bm). Scale = 1000 ml*.
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in Schwann (neurilemma) cell membranes; the superficial processes become completely divested of wrappings and are separated from the haemolymph only by a thin layer of connective tissue. Figs. 12 and 14 show superGal profiles without wrappings and deep profiles with wrappings; Fig. 13 shows two superficial processes, one of which is still partially enveloped by a Schwann cell and another which is completely naked. Both are embedded in the connective tissue sheath surrounding the nerve.
Nerve processes containing neurosecretory granules have been found in the vicinity of other neurones of the peripheral system. Sections of the nerve distal to neurons No. 8 on naza show a process containing many electron-dense granules similar to those described above. The other main nerve bearing multiterminal neurones is the one that runs to the posterior dorsal region (na,). Sections of the first neurone (Fig. 1, neurone No. 9) of nerve na, show typical neurosecretory granules in the processes in the vicinity of the cell (Fig. 15) but not so obviously in the cell body. The cell that was sectioned contained a few granules of the type found in the processes and larger numbers of smaller, electron-transparent vesicles (Fig. 15). There is an indication that some of these vesicles are filling up with electron-dense material and so may be forerunners of the granules seen in the nerve processes. This cell, if neurosecretory, could be considered to be in an inactive phase of the secretory cycle. NEUROHAEMAL
TISSUE
IN THE STICK
INSECT
Paired, segmentally arranged neurohaemal organs on the lateral branches of the median nerve have been described by RAABE(1965) in the stick insects CZ~t#~#~s and C~~~~, and BRADYand MODEL (1967) and RAA~Eand F&MADE(1967) have studied the fine structure of these tissues with the electron microscope. The swellings described by these authors are clearly visible in Carausius and they stain deeply with methylene blue. However, the use of the term ‘neurohaemal organ’ with the implication that the swellings are discrete organs for the release of neurosecretory material is somewhat misleading. We have found that clumps of neurosecretory fibres occur along the length of the lateral branch of the median nerve (the ‘transverse nerve’), at its junction with the link nerve and over a wide area of nerve branches in the vicinity of the spiracle and the transverse muscles of the inte~e~ental region (Figs. 1,9). The majority of the inclusions in the nerve fibres of conspicuous swellings on the transverse nerve (‘transverse nerve swellings’ of BRADYand MADDRELL,1967 ; ‘organes perisympatbiques of RAABEand RAMADE, 1967) are nearly electron transparent and in this respect they resemble the inclusions of the corpus cardiacurn of Carausius (SMITH and SMITH, 1966). Some fibres contain electron-dense granules and BRAJIYand MADDRELL(1967) describe how these fibres are more numerous in the distal region of the neurohaemal organ. We have found that the fibres in neurohaemal tissue at the junction of the link nerve (nal) and the transverse nerve contain a great majority of electron-dense
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L.H. FINLAYSON AND M.P.OSBORNE
granules (Fig. 16). The neurones of the link nerves are sometimes situated close to its junction with the lateral branch of the median nerve, but even when they are at a considerable distance from the junction their processes can be seen running along the link nerve and into the neurohaemal tissue. It is possible, therefore, that the fibres containing electron-dense granules originate in peripheral neurones, and those containing electron-transparent granules in the central nervous system. This would agree with the finding of BRADYand MADDRELL(1967) that fibres containing electron-dense granules are more numerous in the distal part of the ‘transverse nerve swelling’. Some sections of neurohaemal tissue show large numbers of elongated granules. As their cross-sections are approximately circular it is possible that all the granules in these regions are of the elongated type (Fig. 16). Elongated granules occur in the link nerve but are not so numerous as in the neurohaemal tissue. Details of the ultrastructure of granules can also be seen in sections of neurohaemal tissue. They appear to consist of tightly packed tubules oriented parallel to the long axis of the elongated granules (Fig. 17). THE FAT BODY NEURONE This large neurone lies at the posterior end of the ventro-lateral sheet of fat body (Figs. 1, 7). The cell body is free from the fat body, as are the main dendritic branches, but the fine processes are firmly attached to the fat body. Fig. 18 inset shows a cross-section of a single process, well sheathed in Schwann cell wrappings and firmly embedded in the fat body. These processes, as seen in methylene blue preparations, terminate in ‘ink-blot’ endings. Electron microscopy has so far not revealed any convincing evidence of neurosecretion although the presence of many conspicuous neurotubules, well-developed Golgi bodies, rough surfaced E.R., and assorted vesicles in the cell body are symptomatic of an actively secreting cell (Fig. 18). Occasionally granules of various sizes can be seen in a dendrite (Fig. 18). Action potentials can be picked up from this neurone, but it does not appear to have a marked or consistent response to mechanical stimulation. A detailed study of the cell and its activity is in progress. It is probably homologous with the neurone described in the ventral diaph~~ of the locust by GUTHRIE(1962). A PERIPHERAL NEUROSECRETORY CELL IN THE BLOWFLY LARVA In the blowfly larva OSBORNE(1963) described, among others, a multiterminal neurone in the transverse branch of the median (unpaired) nerve (cell Mnl). In a later paper (OSBORNE,1964) he described more precisely the topography of this neurone. It has a process in the lateral branch of the median nerve; presumably this is the axon. A second process runs along the edge of a transverse muscle of the intersegmental region and ends in branches on the stigmatic cord. A third process runs along the link nerve and enters a motor nerve that runs to the ventral muscles. Sections of this neurone examined under the electron microscope reveal typical electron-dense neurosecreto~ granules in the cell body and in the process that
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runs along the edge of the muscle (Figs. 19,ZO). The granules are smaller than those of the stick insect; they measure about 100 w dia. DISCUSSION
The evidence presented in this paper cannot by itself establish that the peripheral neurones studied are neurosecretory in the full meaning of the term as defined by BERN and KNOWLES(1966) because we have no information about the physiological functioning of the cells. The definition of BERNand KNOWLES(1966) excludes all neurones, however actively engaged in secretion, that do not themselves produce hormone or stimulate other glandular cells to produce hormone. Neurosecretory neurones are, by this definition, part of the endocrine system. DE ROBERTIS(1964), on the other hand, emphasizes the basic secretory activity of all neurones and would categorize the endocrine function as but one manifestation of this activity. His concept is of the ‘unitary’ nature of secretion by neurones. It is true, as BERNand KNOWLIS (1966) point out, that the endocrine function of neurosecretion has been emphasized since its discovery, but now the concept has been extended to cover those secretory neurones that form a link in the endocrine chain but do not themselves act as endocrine glands. It is difficult to see the distinction between this type of ‘chemical addressing’ (HORRIDGE, 1961) and that which presumably takes place in other tissues (JOHNSON, 1966; C&PTA and BERRIDGE, 1966) in which there are nerve endings containing secretory granules simiiar in structure and appearance to the ‘neurose~reto~’ granules of BERN and KNOWLES (1966). The cells described in this paper almost certainly release their secretions into the blood and there seems little doubt that they are ‘neurosecretory’ even by the narrowest of definitions. The neurosecretory material is transported from the cell body to a number of dendritic terminations. This is unusual, most neurosecretory material travels along a single process, probably the axon in morphological terms. In insects no sensory neurones have been identified within the central nervous system and it is reasonable to assume that the neurosecretory cells in the central nervous system are modified motor cells and that the processes that run to the neurohaemoral tissues are ‘axons’. The sensory neurones of insects are all peripheral and send their axons into the central nervous system. It seems, therefore, that the peripheral neurosecreto~ cells are sensory in origin because they resemble anatomically the ‘ordinary’ multiterminal sensory neurones. Because their products must be liberated peripherally the secretory terminations are on the dendrites. The question arises whether the whole neurone is functionally reversed and receives ‘information’ from the central nervous system via its axon to regulate its secretory activities or whether the release of secretion is controlled by a blood-borne factor. In the latter case secretory activity may be accompanied by spikes travelling along the axon to the central nervous system, feeding back the information that material is being secreted into the haemolymph. The origin of the secretory granules in the Golgi complex of the peripheral neurones is also typical of neurosecretion (BERNand KNOWLES,1966).
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OSBORNE
The mechanism of release of neurosecretory material from neurones is not known for certain, but there is good evidence that the limiting membrane of the granule fuses with the cell membrane (plasmalemma) and that the release of the granule leaves a characteristic indentation in the cell membrane, called an ‘omega profile’ by SMITH and SRIITH (1966). The limiting membrane of the terminations of the peripheral neurosecretory neurones has a puckered appearance compared with the smoother outline of the processes that are ensheathed in Schwann cell wrappings. There are also indentations resembling SMXTHand SMITH’S (1966) ‘omega profiles’ and in many sections granules appear, to be adhering to the cell membrane as if about to be discharged. Our evidence supports the hypothesis that the granules are discharged by the fusion of cell membrane and granule membrane followed by rupture at the region of fusion. BOWERSand JOHNSON(1966) d es&be peripheral secretory neurones in the lateral and median nerves of the aphid Myxuspersicae, but the granules did not stain with paraldehyd~fuc~in or ~hrom~haematoxylin-phloxin. It is possible that the neurosecretory material described by JOHNSON(1966) in the heart of Periplaneta may originate in the heart neurones (ALEXANDROWICZ, 1926). There is also the possibility that multiterminal neurones in other locations in the insect body (FINLAYSON,1968) may be secretory in function. WHITEN (1963a, b, 1964) has postulated secretory activity in cells on the nerves of blowfly larvae but she states that the extensions of the cells are ‘not narrowed into typical dendrite or axon’, Other descriptions of possible secretory cells by Whitten could in some cases refer to neurones but it is sometimes difficult to be sure which cells she is describing. WHITTEN’S (1963a) account of secretory cells in the median (unpaired) nervous system of the blowfly larva has already been criticized by OSBORNE(1964). The presence in both stick insect and blowfly larva of secretory neurones on link nerves suggests that they are probably universally present in insects. The neurosecretory cells and neurohaemal tissue described in this paper may be part of an extensive system which includes the transverse nerve swellings of the median system. The corpora cardiaca may represent this system in a much condensed form in the region of maximum condensation of segments, the head. Ac~~~ledg~~t~-~e are indebted to the Rockefeller Foundation for providing the electron microscope and to Mr. R. THORNHILLand Miss J. NE~BITfor technical advice and assistance. REFERENCES ALEXANDROWICZ J. S. (1926) The innervation of the heart of the cockroach (Periptnneta orientalis). J. amp. Neural 41,291-309. BERN H. A. (1966) On the pmductio~ of hormones by neurones and the role of neurosecretion in neuroendocrine mechanisms. Symp. Sot. exp. Biol. 20,325-344. BERN H. A. and KNOWLBS F. G. W. (1966) Neurosecretion. En Neuroendocrinology (Ed. by MARTINIL. and GANONCW. F.) 1, 139-186. Academic Press, New York. Bownns B. and JOHNSON B. (1966) An electron microscope study of the corpora cardiaca and secretory neurones in the aphid, Myzus persicae (Sulz.). Gen. camp. E:ndocr. 6, 213-230.
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BRADY J. and MADI)KKLLS. H. P. (1967) Neurohaemal organ8 in the medial nervous system of insects. 2. .ZeUjors&. 76, 389-404. CAMKRONM. L. and STEELE J. E. (1959) Simplified aldebyd~~c~ staining of neurosecretory cells. St& Teehnol. 34,265-266. DK RonxKYrs E. D. P. (1964) ~~t~hy~o~o# of Sy~ses and ??ewosecretion. Pergamon Fress, Oxford. RNLAYS~N L. H. (1968) Froprioceptors in the invertebrates. Xn I~~tebr~te Receptors, Symp. zoo& SW. Lond. 23,217-249. CUFTA I3, 2, and BERRILXE M. J. (1966) Fine structural organization of the rectum in the blowfly, C~~~iphor~ erythroc~~~ (Meig.) with special reference to connective tissue, tracheae and neurosecretory iteration in the rectal papillae. 3. ~~~, 120, 23-82. GUTHRIB D, M. (1962) Control of the ventral diaphragm in an insect. Nuture, Land. 196, 1010-1012. HORRIDGE C. A. (1961) The organization of the primitive central nervous system as suggested by examples of inhibition and the structure of the neuropile. In Nervow Inhibition (Ed. by FLOKKYE.), Pergamon Press, Oxford. JOHNSONB, (1966) Fine structure of the lateral cardiac nerves of the cockroach, Periplanetu amwicana (t.). J. Insect Physiol. 12,645-653. MAD~KKLLS. H. P. (1966) The site of release of the diuretic hormone in Rhodnius-a new neurohaemal system in insects. 3. exp. Biol. 45, 499-508. MAKTON I. (1964) The possible significance of some details of flagellar bases in plants. J. R, n&. Sot. 82, 279-285. MARQURARDT I?. (1939) Beitrsge zur Anatomie der Muskulatur und der peripheren Nerven von Cawaths (Dixsppus) ~OYOSUSBr. Zool.JtS. (Anat.) 6663-128. OSB~~ M. P. (1963) The sensory neurones and sensilla in the abdomen and thorax of the blowfly larva. Quart. r_ m&r. Sk 104, 227-241. OSBORNSXM. P. (1964) The structure of the unpaired Ventral nerves in the blowfly larva. @xat.t.J. n&m. is. N-s, 325-329. RAABE M. (1965) Recherches sur la neuros&r&ion dans la chaine nerveuse ventrale du phasme ~~~~~ e~~u~~~~~: Les Cpaississements des nerfs transverses, organes de s~~i~~tio~ probablem~t neurohemale. C. R. Acad. Z&i., Paris 261,424U-4243. RAAKKM. and RA~IADEF. (1967) Observations sur l’ultrastructure des organes perisympathiques des phasmides. C. R. Acud. Sci., Paris 2&#,77-80. SCHARRERB. (1963) Neurosecrerion-XIII. The ultrastrncture of the corpus cardiacum of the insect Leucophaea maderae. 2. Zellforsch. m&r. Anat. 60, 761-796. SMITH U. and SMITH D. S. (1966) Observations on the secretory processes in the corpus cardiacum of the stick insect, Curausius morosus. J. Cell Sci. 1, 59-66. WHITTEN J. M. (1963a) The dorsal nerves of cyclorrhaphan larvae: giant cells in which secretory channels appear at the onset of puparium farmation. Quart. J. micr. Sci. 104, 217-225. WHITTEN J. M. (1963b) Observations on the cyclorrhaphan larvae peripheral nervous system: muscle and tracheal receptor organs and independent type II neurons associated with tbe lateral segmental nerves. Ann. ent. Sot. Am. 56,755-763. WHXTTENJ. M. (1964) Connective tissue membranes and their apparent role in transporting neurosectory and other secretory products in insects. Gen. camp. Endocr. 4, 176-192.
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PERIPHERAL NEUROSECRETORY CELLS IN THE STICK INSECT (CARA.USIUS MOROSUS) AND THE BLOWFLY LARVA (PHORMIA TERRAE-NOVAE) L. H. FINLAYSON
and M. P. OSBORNE
Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham 15
Abstract-On each side of the abdominal segments of the stick insect there are at least ten multiterminal neurones lying on major nerves or on the tracheae that run with the nerves. The dendritic processes of the neurones run on and in the nerves. Sections stained with paraldehyde-fuchsin or examined by electron microscopy show typical neurosecretory granules in some of these neurones, in both cell body and cell processes. Superficial processes adjoin the haemocele and appear to be releasing their secretions into the blood. A group of about four cells situated on the nerve linking the ‘somatic’ and the ‘median’ nervous systems shows varying degrees of secretory activity. They may be contributing electrondense granules to the lateral neurohaemal organs and to diffuse neurohaemal tissue in the general region of the spiracle and/or releasing material directly into the haemol~ph. Electron-microscopic examination of a large neurone on the ventrolateral sheet of fat body reveals no conclusive evidence of neurosecretion. In the blowfly larva a neurosecretory neurone is located on each side of the body segments at the junction between the somatic and median nerves, INTRODUCTION
IN A discussion of the concept of neurosecretion BERN and KNOWLES(1966) point out the difficulty of establishing by light or electron microscopy whether a neurone or a neuronal process is neurosecretory. They exclude neurones that innervate exocrine glands, muscles, or other tissues, even though they or their processes contain secretory granules of a type found in neurosecreto~ neurones. Apart from the neurosecreto~ neurones of the central nervous system of vertebrates and invertebrates, the only other neurones that almost certainly fit this concept of neurosecretory are those in the corpus cardiacurn of insects (SCHARRER, 1963 ; SMITH and SMITH, 1966). A neurosecretory function has been postulated for a number of other cells, but electron microscopical investigation has revealed that they do not contain granules characteristic of neurosecretory cells (BERN, 1966). In the absence of evidence that a secretory neurone is engaged directly or indirectly in endocrine control (Bw and KNOWLES, 1966) there are two morphological criteria that are accepted as good evidence of neurosecretory function. One is the presence in the neurons of secretory inclusions (‘granules’, ‘vesicles’, ‘droplets’) measuring 100 to 300 rnp dia. and the other is the termination of the processes
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of the neurone in close proximity to a cavity containing blood or other fluid. Such terminations occur in discrete tissue, called neurohaemal tissue, as in the corpora cardiaca (SMITH and SMITH, 1966) and the segmental neurohaemal organs of phasmids (RAABE, 1965) or at individual endings, as in the abdominal nerves of Rhodnius (MADDRELL,1966), the heart of Peripluneta (JOHNSON, 1966), and the rectal papillae of Culliphora (GUPTA and BERRIDGE,1966). It has been generally assumed that neurohaemal endings belong to neurones whose cell bodies lie in the ganglia of the central nervous system or in the corpora cardiaca. In this paper wiI1 be described peripheral neurones that appear to have all the morphological attributes of neurosecretory cells as defined by BERN and KNOWLES(1966). Multite~inal neurones are found in the periphery of the insect body in various locations. Some are subepidermal and others are more deeply situated, with endings on a variety of tissues. Others are situated on the course of nerves and it is not known where their processes terminate (for review see FINLAYSON,1968). In the course of a study of the peripheral neurones of the stick insect it was found that certain neurones associated with several major nerves appeared to be neurosecretory. These neurones are described in this paper. The distribution of peripheral multiterminal neurones in the blowfly larva has already been described by OSBORNE (1963,1964). 0 ne of them has been investigated for neurosecretory activity. MATERIAL AND METHODS Larvae and adults of the stick insect (Curausius ntorosus) and final instar larvae of the black blowfly (Pbmia tevrae-novae) were used for this study. Vital staining of dissected animals was carried out with methylene blue in Ringer solution (NaCl, 6.31 g; KCI, 1.86; MgCl, 0.1 g; CaCl, O-2 g; NaH,PO,, 0.2 g; NaHCO,, 0.05 g; dextrose, 16 g; Hz0 to 1 1. Sections for light microscopy were cut at 3~ and stained with paraldehyde-fuchsin (CAMERONand STEELE, 1959) after fixation in Bouin with acetic acid content reduced to 2 per cent. For electron microscopy the stick insects and blowfly larvae were opened along the dorsal midline, the gut was removed, and the remaining tissues were flooded with 2.5% glutaraldehyde for 20 min. Tissues for sectioning were then transferred to 2% osmium tetroxide. Both the glutaraidehyde and the osmium tetroxide were buffered to pH 7.4 with phosphate buffer and made up to a total molarity of 0.45 M by the addition of sucrose. Dehydration in 70,90%, and absolute ethanol was followed by embedding in Epon (MANTON, 1964). Sections were double-stained in uranyl acetate and lead citrate. NEURONES OF THE STICK INSECT Topography Neurones associated with major nerves have been located by means of methylene blue staining and their distribution in the second abdominal segment of an adult is shown in Fig. 1. The cell bodies vary in position (Figs. 2,3) and it is
~~OSEC~ORY
CELLS IN STICK INBOX
BLOWFLYLARVA
1795
diacult to be sure of the precise number of neurones or whether there is variation in number between segments. However, there is no doubt that there are about ten on each side of the major abdominal segments. Near the first nerve junctions, where the lateral nerve (nl,, MARQUHARDT, 1939) Ieaves the main segmental nerve there are several neurones whose relationship to the nerves is not clear. At least one of them sends its processes along the nerves (Fig. 5). In addition to neurone No. 8 (Fig. 1) a second neurone is sometimes to be seen on naSa, the nerve that runs by the spiracle. The cell bodies and the processes of these neurones are superficial in position. Frequently the cell body lies on the tracheae that runs beside the nerve and the processes ramify over tracheal and nerve branches (Figs. 3,6). The
FIG. 1. Stick insect. Diagram of innervation of one side of an abdominal segment. Neurones on the course of nerves are numbered 1 to 10; fb, fat body; fbn, fat body neurone; mn, median nerve; na,, naS, nata, nl,, nl,, np, npa, nerves labelled according to MARQUHARDT'S (1939) terminology; nh, neurohaemal tissue; sp, spiracle; ms, transverse nerve swelling (neurohaemal tissue).
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L. W. FINLAYSONAND M. P. OSBORNE
cell body may be detached, especially at nerve junctions (Fig, 5). The processes may run for long distances from the cell body or may terminate close to it (Figs, $6). Some endings have been observed to have a distinctive appearance in methylene blue preparations. They resemble irregular ink blots (Fig. 5) or a reticulum of fine fibrils (Fig. 8). Similar endings can be seen in multite~inal neurones on the epidermis in Crrrausius. In addition to the neurones on the nerves, one other is shown on Fig. 1. It is situated at the posterior margin of the ventrolateral sheet of fat body. Its possible functions are discussed below. N~~o~es OBthe link wve A nerve, designated na,, by MARQUHARDT (19391, links the main (somatic) nerve system of each segment with the lateral branch of the median (unpaired) nerve (Fig. 1). We shall refer to this nerve as the ‘link’ nerve for convenience and to emphasize its important ~atomic~ relationships~ There are, typically, four neurones on the trachea running alongside the nerve (Fig. 2). They vary considerably in position and one may lie at the junction of the link nerve and the anterior segmental nerve. In appearance, also, they show considerable variation, ranging from the typical multiterminal appearance with prominent processes to a condition in which the cell body is distended with secretory inclusions and the processes are fine and inco~picuous (Fig. 3). These cefls may stain heavily or only slightly with methylene blue. Sections stained with paraldehyde-fu~hsin revealed typical deeply stained neurosecretory inclusion in the cell processes but less deeply stained ones in the cell body. Electron microscopy, however, showed that the cell body also contains inclusions that are typical of neurosecreto~ cells (Figs. 10, 11). They are electron dense after treatment with osmium tetroxide, uranyl acetate, and lead citrate. As in the corpus cardiacurn of the same insect (SMOTE and SMITH, 1966) each secretory granule appears to be enveloped by a membrane of the Golgi complex (Fig. 11). On the vast majority of granules this membrane cannot be seen, perhaps because it has become closely applied to the surface of the secreted material, as suggested by SMITH and SMITH (1966). The endoplasmic reticulum is well developed in these cells and is densely coated with ribosomes (‘rough-surfaced’). Parts of the endoplasmic reticulum are frequently juxtaposed to the cell membrane (plasmalemma). Ribosomes are missing from the membrane surface applied to the pl~male~a (Fig. 11). This phenomenom may have no significance, but it is noteworthy that a large area of the plasmalemma is in very close apposition with the endopl~mi~ reticulum. The electron micrographs show how superficial are the neurosecretory cells of the link nerve. In transverse sections of the link nerve can be seen both superficial and deep processes containing neurosecreto~ granules (Fig. 12). The superficial processes have a puckered membrane with granules closely adhering to it (Figs. 12-14) and ~v~inations that may mark the points of release of the secretory material, the ‘omega profiles’ of SMITH and SMITH (1966). The deeper profiles show no such evidence of release of secretion (Fig. 12). They are wrapped
FIc. 2. Stick insect, second abdominal segment. Link nerve (nal in Fig. 1) with three neurones on the trachea and one at the junction with the main segmental nerve. Neurohaemal tissue (marked by arrow) at junction with transverse branch of median nerve. (Methylene blue.) Scale = 150/z. FIG. 3. Stick insect, third abdominal segment. Neurones at junction of link nerve (nal) and main segmental nerve, showing different degrees of distension with secretion. (Methylene blue.) Scale = 40/z. FIG. 4. Stick insect, second abdominal segment. Neurone (No. 10, Fig. 1) on nerve na2 with processes encircling trachea and returning to nerve. (Methylene blue.) Scale = 50/z. FIG. 5. Stick insect, first abdominal segment. Neurone No. 1 (Fig. 1) showing four processes; axon labelled (A). A short process terminates near the cell body and a long thick process has 'ink-blot' endings (I) on its branches. Fracture of nerve, trachea, and thick process marked by arrow. (Methylene blue.) Scale -50 ~.
FIc. 6. Stick insect, fourth abdominal segment. N e u r o n e near junction of na2 and na,a (Fig. 1). Note the short process terminating near the cell body (marked by arrow) similar to one in Fig. 5. (Methylene blue.) S c a l e - - 5 0 / z . FIG. 7. Stick insect, fourth abdominal segment. Fat body neurone; axon marked by arrow. (Methylene blue.) Scale -= 150/z. FIc. 8. Stick insect, fifth abdominal segment. N e u r o n e with process ending in a tangle of fine fibres (I). Link nerve (nal) with two neurones marked by arrow. (Methylene blue.) Scale = 100 p. FIc. 9. Stick insect abdominal segment. Neurohaemal tissue along course of nerves near spiracle. (Methylene blue.) Scale= 150/z.
FIG. 10. Survey picture of the cell body of a neurone on the link nerve of the stick insect. T h e neurone is bounded by a tenuous Schwann cell sheath (Sc) and a thin layer of basement membrane material (Bin). T h e cell contains numerous Golgi bodies (G) which are apparently producing secretory granules (arrows). H, haemolymph space. Scale = 2000 m/z.
FIC. 11. Stick insect. Detail of cytoplasmic contents of a link nerve neurone. The Golgi body (G) is sequestering granules as is evident from the continuity of Golgi membranes with the limiting membrane of the granule (straight arrow). Not all the granules have a limiting membrane (compare granules gl and g2). Profiles of rough-surfaced endoplasmic reticulum (Er) are evident and in some areas the reticulum is closely apposed to the plasma membrane (Pm) of the neurone. The apposed surface of the endoplasmic reticulum is free of the coating of ribosomes (curved arrow). Sc, Schwann cell. Scale = 500 m/z. The inset shows membrane continuity between a secretory granule and Golgi body (arrow). Scale = 500 m/z.
FIG. 12. Transverse section through a part of the link nerve of the stick insect. A group of nerve fibres (Nf) some of which contain granules is engulfed by wrappings of the Schwann cell (Sc). A large nerve process (top left) is packed with granules and has no Schwann cell sheath. This 'naked' process is separated from the haemolymph (H) by only a thin coating of basement membrane material (Bm). Note how markedly convoluted is the plasma membrane of the naked process (arrow) when compared with the smoother plasma membranes of the ensheathed nerve process. N, Schwann cell nucleus; T, trachea. Scale = 1000 mu.
FIG. 13. Stick insect. T w o nerve processes embedded in the basement membrane (Bm), surrounding the link nerve. One process ( N p l ) is 'naked' whereas the other (Np2) is partly invested by the Schwann cell (Sc). Both processes contain secretory granules, but Np2 is apparently subterminal since it contains neurotubules (straight arrows) which are very similar to the microtubules (curved arrows) in the nearby Schwann cell. Scale = 500 rap,. T h e inset shows a large naked nerve process on the link nerve. It is packed with secretory granules, some of which have an obvious limiting membrane (short arrows). T h e plasma membrane is deeply invaginated at some points (curved arrows) and in other regions where it is convoluted is in intimate contact with secretory granules (straight arrows). Note that only a thin layer of basement membrane material (Bm) is interposed between this nerve process and the haemolymph (H). Scale = 1000 m/z.
FIG. 14. Transverse section through part of the link nerve of the stick insect to show two ensheathed nerve processes (Npl, Np2) and a third, naked process (Np3). The latter is full of secretory granules whereas N p l contains only two such granules. Note the convolutions of the plasma membrane of Np3. Bm, basement membrane material; H, haemolymph; Sc, Schwann cell. Scale = 500 m/z.
FtG. 15. Stick insect. Section through part of the cell body of neurone No. 9 (Fig. 1) on nerve Na2. T h e cell contains free ribosomes, rough-surfaced endoplasmic reticulum (Er) and Golgi bodies (G). T h e Golgi bodies seem to be producing small dense granules (short arrows). However, one of the granules is larger (curved arrow) and resembles those (straight arrow) in the neighbouring naked-nerve process (Np). Sc, Schwann cell. Scale = 1000 m/z.
FIo. 16. Neurohaemal tissue of stick insect at the junction of the link nerve with the median nervous system. Some nerve fibres contain many secretory granules whilst others are relatively empty. Process N p l contains very. dense spherical granules of the type produced by the link nerve neurons. Elongated and spherical granules in the other nerve fibres have varying electron opacities. Bm, basement membrane; H, haemolymph; Sc, Schwann cell; T, tracheole. Scale = 1000 m/~.
FIG. 17. Parts of t h r e e n e r v e processes from stick insect n e u r o h a e m a l tissue at j u n c t i o n of link n e r v e w i t h m e d i a n n e r v o u s system. T h e process at u p p e r r i g h t contains spherical, d e n s e granules characteristic of those p r o d u c e d b y the link n e r v e n e u r o n e s . T h e o t h e r two processes have granules of lower electron density w h i c h show a n i n t e r n a l lamellate structure. Scale = 500 m/~. I n s e t shows detail of the i n t e r n a l s t r u c t u r e of the secretory granules. Scale = 250 m/z.
FIc. 18. Part of the neurone located on the fat body of the stick insect. The cell body contains many profiles of rough-surfaced endoplasmic reticulum (Er) and free ribosomes, symptomatic of protein production. The Golgi body (G) is producing small electron-dense droplets (straight arrows). Other organelles are mitochondria and microtubules (curved arrow). N, nucleus of neurone; Sc, Schwann cell. Scale = 1000 m/~. A cross-section of one of the dendrites of the fat body neurone is shown in the inset. Small electron dense droplets (arrows) resemble those associated with the Golgi body in the cell soma. Scale = 1000 m/~.
FIG. 19.
Blowfly larva. Part of cell body of neurone Mnl to show large numbers of electron-dense granules (arrows). Scale = 500 mp.
FIG. 20. Blowfly larva. Section across a dendrite of cell Mnl. This process is only partially enveloped by the Schwann cell (SC). The naked portion of the fibre has a puckered plasma membrane (straight arrow) close to which are clustered dense granules (curved arrows), suggesting that material is being liberated into the haemolymph (H) through the basement membrane (Bm). Scale = 1000 ml*.
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in Schwann (neurilemma) cell membranes; the superficial processes become completely divested of wrappings and are separated from the haemolymph only by a thin layer of connective tissue. Figs. 12 and 14 show superGal profiles without wrappings and deep profiles with wrappings; Fig. 13 shows two superficial processes, one of which is still partially enveloped by a Schwann cell and another which is completely naked. Both are embedded in the connective tissue sheath surrounding the nerve.
Nerve processes containing neurosecretory granules have been found in the vicinity of other neurones of the peripheral system. Sections of the nerve distal to neurons No. 8 on naza show a process containing many electron-dense granules similar to those described above. The other main nerve bearing multiterminal neurones is the one that runs to the posterior dorsal region (na,). Sections of the first neurone (Fig. 1, neurone No. 9) of nerve na, show typical neurosecretory granules in the processes in the vicinity of the cell (Fig. 15) but not so obviously in the cell body. The cell that was sectioned contained a few granules of the type found in the processes and larger numbers of smaller, electron-transparent vesicles (Fig. 15). There is an indication that some of these vesicles are filling up with electron-dense material and so may be forerunners of the granules seen in the nerve processes. This cell, if neurosecretory, could be considered to be in an inactive phase of the secretory cycle. NEUROHAEMAL
TISSUE
IN THE STICK
INSECT
Paired, segmentally arranged neurohaemal organs on the lateral branches of the median nerve have been described by RAABE(1965) in the stick insects CZ~t#~#~s and C~~~~, and BRADYand MODEL (1967) and RAA~Eand F&MADE(1967) have studied the fine structure of these tissues with the electron microscope. The swellings described by these authors are clearly visible in Carausius and they stain deeply with methylene blue. However, the use of the term ‘neurohaemal organ’ with the implication that the swellings are discrete organs for the release of neurosecretory material is somewhat misleading. We have found that clumps of neurosecretory fibres occur along the length of the lateral branch of the median nerve (the ‘transverse nerve’), at its junction with the link nerve and over a wide area of nerve branches in the vicinity of the spiracle and the transverse muscles of the inte~e~ental region (Figs. 1,9). The majority of the inclusions in the nerve fibres of conspicuous swellings on the transverse nerve (‘transverse nerve swellings’ of BRADYand MADDRELL,1967 ; ‘organes perisympatbiques of RAABEand RAMADE, 1967) are nearly electron transparent and in this respect they resemble the inclusions of the corpus cardiacurn of Carausius (SMITH and SMITH, 1966). Some fibres contain electron-dense granules and BRAJIYand MADDRELL(1967) describe how these fibres are more numerous in the distal region of the neurohaemal organ. We have found that the fibres in neurohaemal tissue at the junction of the link nerve (nal) and the transverse nerve contain a great majority of electron-dense
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L.H. FINLAYSON AND M.P.OSBORNE
granules (Fig. 16). The neurones of the link nerves are sometimes situated close to its junction with the lateral branch of the median nerve, but even when they are at a considerable distance from the junction their processes can be seen running along the link nerve and into the neurohaemal tissue. It is possible, therefore, that the fibres containing electron-dense granules originate in peripheral neurones, and those containing electron-transparent granules in the central nervous system. This would agree with the finding of BRADYand MADDRELL(1967) that fibres containing electron-dense granules are more numerous in the distal part of the ‘transverse nerve swelling’. Some sections of neurohaemal tissue show large numbers of elongated granules. As their cross-sections are approximately circular it is possible that all the granules in these regions are of the elongated type (Fig. 16). Elongated granules occur in the link nerve but are not so numerous as in the neurohaemal tissue. Details of the ultrastructure of granules can also be seen in sections of neurohaemal tissue. They appear to consist of tightly packed tubules oriented parallel to the long axis of the elongated granules (Fig. 17). THE FAT BODY NEURONE This large neurone lies at the posterior end of the ventro-lateral sheet of fat body (Figs. 1, 7). The cell body is free from the fat body, as are the main dendritic branches, but the fine processes are firmly attached to the fat body. Fig. 18 inset shows a cross-section of a single process, well sheathed in Schwann cell wrappings and firmly embedded in the fat body. These processes, as seen in methylene blue preparations, terminate in ‘ink-blot’ endings. Electron microscopy has so far not revealed any convincing evidence of neurosecretion although the presence of many conspicuous neurotubules, well-developed Golgi bodies, rough surfaced E.R., and assorted vesicles in the cell body are symptomatic of an actively secreting cell (Fig. 18). Occasionally granules of various sizes can be seen in a dendrite (Fig. 18). Action potentials can be picked up from this neurone, but it does not appear to have a marked or consistent response to mechanical stimulation. A detailed study of the cell and its activity is in progress. It is probably homologous with the neurone described in the ventral diaph~~ of the locust by GUTHRIE(1962). A PERIPHERAL NEUROSECRETORY CELL IN THE BLOWFLY LARVA In the blowfly larva OSBORNE(1963) described, among others, a multiterminal neurone in the transverse branch of the median (unpaired) nerve (cell Mnl). In a later paper (OSBORNE,1964) he described more precisely the topography of this neurone. It has a process in the lateral branch of the median nerve; presumably this is the axon. A second process runs along the edge of a transverse muscle of the intersegmental region and ends in branches on the stigmatic cord. A third process runs along the link nerve and enters a motor nerve that runs to the ventral muscles. Sections of this neurone examined under the electron microscope reveal typical electron-dense neurosecreto~ granules in the cell body and in the process that
~~EC~ORY
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1799
runs along the edge of the muscle (Figs. 19,ZO). The granules are smaller than those of the stick insect; they measure about 100 w dia. DISCUSSION
The evidence presented in this paper cannot by itself establish that the peripheral neurones studied are neurosecretory in the full meaning of the term as defined by BERN and KNOWLES(1966) because we have no information about the physiological functioning of the cells. The definition of BERNand KNOWLES(1966) excludes all neurones, however actively engaged in secretion, that do not themselves produce hormone or stimulate other glandular cells to produce hormone. Neurosecretory neurones are, by this definition, part of the endocrine system. DE ROBERTIS(1964), on the other hand, emphasizes the basic secretory activity of all neurones and would categorize the endocrine function as but one manifestation of this activity. His concept is of the ‘unitary’ nature of secretion by neurones. It is true, as BERNand KNOWLIS (1966) point out, that the endocrine function of neurosecretion has been emphasized since its discovery, but now the concept has been extended to cover those secretory neurones that form a link in the endocrine chain but do not themselves act as endocrine glands. It is difficult to see the distinction between this type of ‘chemical addressing’ (HORRIDGE, 1961) and that which presumably takes place in other tissues (JOHNSON, 1966; C&PTA and BERRIDGE, 1966) in which there are nerve endings containing secretory granules simiiar in structure and appearance to the ‘neurose~reto~’ granules of BERN and KNOWLES (1966). The cells described in this paper almost certainly release their secretions into the blood and there seems little doubt that they are ‘neurosecretory’ even by the narrowest of definitions. The neurosecretory material is transported from the cell body to a number of dendritic terminations. This is unusual, most neurosecretory material travels along a single process, probably the axon in morphological terms. In insects no sensory neurones have been identified within the central nervous system and it is reasonable to assume that the neurosecretory cells in the central nervous system are modified motor cells and that the processes that run to the neurohaemoral tissues are ‘axons’. The sensory neurones of insects are all peripheral and send their axons into the central nervous system. It seems, therefore, that the peripheral neurosecreto~ cells are sensory in origin because they resemble anatomically the ‘ordinary’ multiterminal sensory neurones. Because their products must be liberated peripherally the secretory terminations are on the dendrites. The question arises whether the whole neurone is functionally reversed and receives ‘information’ from the central nervous system via its axon to regulate its secretory activities or whether the release of secretion is controlled by a blood-borne factor. In the latter case secretory activity may be accompanied by spikes travelling along the axon to the central nervous system, feeding back the information that material is being secreted into the haemolymph. The origin of the secretory granules in the Golgi complex of the peripheral neurones is also typical of neurosecretion (BERNand KNOWLES,1966).
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L. H. FINLAYS~N ANDM. P.
OSBORNE
The mechanism of release of neurosecretory material from neurones is not known for certain, but there is good evidence that the limiting membrane of the granule fuses with the cell membrane (plasmalemma) and that the release of the granule leaves a characteristic indentation in the cell membrane, called an ‘omega profile’ by SMITH and SRIITH (1966). The limiting membrane of the terminations of the peripheral neurosecretory neurones has a puckered appearance compared with the smoother outline of the processes that are ensheathed in Schwann cell wrappings. There are also indentations resembling SMXTHand SMITH’S (1966) ‘omega profiles’ and in many sections granules appear, to be adhering to the cell membrane as if about to be discharged. Our evidence supports the hypothesis that the granules are discharged by the fusion of cell membrane and granule membrane followed by rupture at the region of fusion. BOWERSand JOHNSON(1966) d es&be peripheral secretory neurones in the lateral and median nerves of the aphid Myxuspersicae, but the granules did not stain with paraldehyd~fuc~in or ~hrom~haematoxylin-phloxin. It is possible that the neurosecretory material described by JOHNSON(1966) in the heart of Periplaneta may originate in the heart neurones (ALEXANDROWICZ, 1926). There is also the possibility that multiterminal neurones in other locations in the insect body (FINLAYSON,1968) may be secretory in function. WHITEN (1963a, b, 1964) has postulated secretory activity in cells on the nerves of blowfly larvae but she states that the extensions of the cells are ‘not narrowed into typical dendrite or axon’, Other descriptions of possible secretory cells by Whitten could in some cases refer to neurones but it is sometimes difficult to be sure which cells she is describing. WHITTEN’S (1963a) account of secretory cells in the median (unpaired) nervous system of the blowfly larva has already been criticized by OSBORNE(1964). The presence in both stick insect and blowfly larva of secretory neurones on link nerves suggests that they are probably universally present in insects. The neurosecretory cells and neurohaemal tissue described in this paper may be part of an extensive system which includes the transverse nerve swellings of the median system. The corpora cardiaca may represent this system in a much condensed form in the region of maximum condensation of segments, the head. Ac~~~ledg~~t~-~e are indebted to the Rockefeller Foundation for providing the electron microscope and to Mr. R. THORNHILLand Miss J. NE~BITfor technical advice and assistance. REFERENCES ALEXANDROWICZ J. S. (1926) The innervation of the heart of the cockroach (Periptnneta orientalis). J. amp. Neural 41,291-309. BERN H. A. (1966) On the pmductio~ of hormones by neurones and the role of neurosecretion in neuroendocrine mechanisms. Symp. Sot. exp. Biol. 20,325-344. BERN H. A. and KNOWLBS F. G. W. (1966) Neurosecretion. En Neuroendocrinology (Ed. by MARTINIL. and GANONCW. F.) 1, 139-186. Academic Press, New York. Bownns B. and JOHNSON B. (1966) An electron microscope study of the corpora cardiaca and secretory neurones in the aphid, Myzus persicae (Sulz.). Gen. camp. E:ndocr. 6, 213-230.
NKIJROSECKKTORY CELLS IN STICK INSKC’rANDBLOWFLY LARVA
1801
BRADY J. and MADI)KKLLS. H. P. (1967) Neurohaemal organ8 in the medial nervous system of insects. 2. .ZeUjors&. 76, 389-404. CAMKRONM. L. and STEELE J. E. (1959) Simplified aldebyd~~c~ staining of neurosecretory cells. St& Teehnol. 34,265-266. DK RonxKYrs E. D. P. (1964) ~~t~hy~o~o# of Sy~ses and ??ewosecretion. Pergamon Fress, Oxford. RNLAYS~N L. H. (1968) Froprioceptors in the invertebrates. Xn I~~tebr~te Receptors, Symp. zoo& SW. Lond. 23,217-249. CUFTA I3, 2, and BERRILXE M. J. (1966) Fine structural organization of the rectum in the blowfly, C~~~iphor~ erythroc~~~ (Meig.) with special reference to connective tissue, tracheae and neurosecretory iteration in the rectal papillae. 3. ~~~, 120, 23-82. GUTHRIB D, M. (1962) Control of the ventral diaphragm in an insect. Nuture, Land. 196, 1010-1012. HORRIDGE C. A. (1961) The organization of the primitive central nervous system as suggested by examples of inhibition and the structure of the neuropile. In Nervow Inhibition (Ed. by FLOKKYE.), Pergamon Press, Oxford. JOHNSONB, (1966) Fine structure of the lateral cardiac nerves of the cockroach, Periplanetu amwicana (t.). J. Insect Physiol. 12,645-653. MAD~KKLLS. H. P. (1966) The site of release of the diuretic hormone in Rhodnius-a new neurohaemal system in insects. 3. exp. Biol. 45, 499-508. MAKTON I. (1964) The possible significance of some details of flagellar bases in plants. J. R, n&. Sot. 82, 279-285. MARQURARDT I?. (1939) Beitrsge zur Anatomie der Muskulatur und der peripheren Nerven von Cawaths (Dixsppus) ~OYOSUSBr. Zool.JtS. (Anat.) 6663-128. OSB~~ M. P. (1963) The sensory neurones and sensilla in the abdomen and thorax of the blowfly larva. Quart. r_ m&r. Sk 104, 227-241. OSBORNSXM. P. (1964) The structure of the unpaired Ventral nerves in the blowfly larva. @xat.t.J. n&m. is. N-s, 325-329. RAABE M. (1965) Recherches sur la neuros&r&ion dans la chaine nerveuse ventrale du phasme ~~~~~ e~~u~~~~~: Les Cpaississements des nerfs transverses, organes de s~~i~~tio~ probablem~t neurohemale. C. R. Acad. Z&i., Paris 261,424U-4243. RAAKKM. and RA~IADEF. (1967) Observations sur l’ultrastructure des organes perisympathiques des phasmides. C. R. Acud. Sci., Paris 2&#,77-80. SCHARRERB. (1963) Neurosecrerion-XIII. The ultrastrncture of the corpus cardiacum of the insect Leucophaea maderae. 2. Zellforsch. m&r. Anat. 60, 761-796. SMITH U. and SMITH D. S. (1966) Observations on the secretory processes in the corpus cardiacum of the stick insect, Curausius morosus. J. Cell Sci. 1, 59-66. WHITTEN J. M. (1963a) The dorsal nerves of cyclorrhaphan larvae: giant cells in which secretory channels appear at the onset of puparium farmation. Quart. J. micr. Sci. 104, 217-225. WHITTEN J. M. (1963b) Observations on the cyclorrhaphan larvae peripheral nervous system: muscle and tracheal receptor organs and independent type II neurons associated with tbe lateral segmental nerves. Ann. ent. Sot. Am. 56,755-763. WHXTTENJ. M. (1964) Connective tissue membranes and their apparent role in transporting neurosectory and other secretory products in insects. Gen. camp. Endocr. 4, 176-192.