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Ultrastructural analysis of the ocellar tract of the wasps, Paravespula Vulgaris L. and P. Germanica L. (Hymenoptera : Vespidae)
Int. J. InsectMorphoL&Embryol.. Vol. 12, No. 5/6, pp. 313 to 320, 1983. Printed in Great Britain.
0020- 7322/83$3.00+ .00 © 1983PergamonPressLtd.
ULTRASTRUCTURAL ANALYSIS OF THE OCELLAR TRACT OF T H E W A S P S , P A R A V E S P U L A V U L G A R I S L. A N D P. G E R M A N I C A L. ( H Y M E N O P T E R A • VESPIDAE) KARL KRAL Department of Zoology, University of Graz, A-8010 Graz, Austria
(Accepted 25 May 1983) A b s t r a c t - - T h e oceUar tract of the wasp conveys visual information from the 3 ocelli to the protocerebrum, and consists of 26 thick axons (15 - 30 gm) of large I st-order interneurones and a larger number of thin axons of small interneurones. In the present study, the ocellar tract of the wasps, Paravespula vulgaris and P. germanica (Hymenoptera : Vespidae) was investigated at the ultrastructural level. The axons of the large I st-order interneurones are separated from one another by glial cells, but on entering the posterior slopes of the protocerebrum the glial cells disappear so that the axons come very close to each other and form junctions. Slightly farther, they arborize into dendritic branches, which are also interconnected by such junctions. In addition, lateral chemical synapses of the bar-shaped type also exist a m o n g the dendritic branches. These synapses connect interneurones of the individual lateral nerves and of the median ocellar nerve, but synaptic contacts could also be observed between the lateral nerves and the median nerve. It was also demonstrated that there are profiles with clear vesicles and large dense-cored vesicles, possibly neurosecretory granules, in the ocellar tract that form synapses with other neural elements. Index descriptors (in addition to those in title): Ocellar tract, electron microscopic examination, neuronal interaction.
INTRODUCTION
THE OCELLAR tract of adult insects contains large afferent lst-order interneurones and smaller afferent and efferent interneurones, which connect the ocelli with the brain (dragonfly: Dowling and Chappell, 1972; locust: Goodman, 1976; Goodman and Williams, 1976; cabbage looper moth: Pappas and Eaton, 1977; Eaton and Pappas, 1978; Honey bee: Pan and Goodman, 1977; Guy et al., 1979; wasp: Kral, 1982). The fact that the giant fibres (up to 30 ~tm in diameter) of the ocellar tract are ideally suited for intracellular recording and marking allows those parts of the pathways, which are responsible for the generation, transfer and output of visual signals, to be studied in much detail. Recent electrophysiological investigations of migratory locusts (Simmons, 1982) and behavioural experiments with honey bees (Kral and Heran, 1983) have shown that integration of incoming visual messages is completed in the ocellar tract. It was with this in mind that the present study was undertaken to examine the ocellar tract of vespid wasps at the ultrastructural level. MATERIALS
AND
METHODS
Adult wasps (P. vulgaris and P. germanica) were collected from nests in the Graz area. Brains were fixed for 3 hr in p h o s p h a t e - b u f f e r e d 6.25°70 glutaraldehyde (pH 7.3; 4°C), post-fixed in 10?00sO, (pH 7.3; 4°C) and embedded in Epon 812. Semi-thin sections from brain were cut to localize the region to be investigated in the ocellar tract. Thereafter, trimming was done with razor blades. At the beginning and end of each series of ultrathin sections (interval of 3 - 5 ~tm) 1 ~tm-thick control sections were cut, stained with toluidine blue and 313
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examined with the light microscope. Ultra-thin sections were stained with lead citrate and examined with a Zeiss EM IOA electron microscope, which was supplied by the Stiftung Volkswagenwerk.
RESULTS
Adult wasps (P. vulgaris and P. germanica) possess one median ocellus and 2 lateral ocelli. The photoreceptor cells form synaptic contacts with the dendrites of a small number of large 1st-order interneurones (L neurones) and a larger group of small interneurones (Kral, 1979). The pathway of the ocellar tract in the brain of the wasp has been described previously (Kra.l, 1982). The output region of the ocellar tract (illustrated in Fig. 1) was not investigated in the present study, since conventional electron microscopy does not allow the identification of individual elements within the complex neuropile of the posterior slopes. Some preliminary observations indicate, however, that chemical synapses also exist between ocellar fibre terminals and central neurones.
ocellus
dendrite
. :~
dendr PS Flo. 1. Reconstruction o f 2 parallel large afferent lst-order ocellar interneurones of P. germanica, based on photographs of Procion yellow filled oceUar nerves (Kral, 1982). Sites of lateral synapses are marked by curved arrows. Direction of information flow is indicated by arrows. PS = posterior slope neurOpile.
Ultrastructural Analysis of the Ocellar Tract of Wasps
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The electron microscopic examination of the ocellar tract showed that the axons of the large lst-order interneurones and most of the axons of the small interneurones contain a clear cytoplasm with microtubules aligned along the axonal axis. The axons form terminal arborizations within the CNS. Except in the terminal arborizations, the cytoplasm contains only a few small mitochondria, whose inner membrane is slightly folded. The axons of the large interneurones are completely separated from each other by glial cells (Fig. 2), whereas the thin axons, which are mainly found in the outer part of the tract, are mostly naked. Some of the thin axons contain structures that may be neurosecretory granules of up to 160 nm diameter. These axons can be in synaptic contact with other neural elements (Fig. 3).
FIG. 2. Cross section through median ocellar nerve of P. vulgaris, showing axons of 2 large lst-order interneurones (LA). Note that the axons are isolated from each other by glia (G). Numerous vacuoles, lacunae and widened spaces of endoplasmic reticulum (arrows) are found in glial layer. Scale = 2.0 gm.
Within the glia, there are widened spaces of the endoplasmic reticulum and large vacuoles that are often filled with electron-dense material (Figs. 2,3). Large magnification showed that this material is bound to fine membranes that bridge the lumen. The vacuoles are often in contact with the cytoplasm of the axons. Tracheoles were also observed in the glia. On entering the neuropile of the posterior slopes of the protocerebrum the axons of the large 1st-order interneurones form dendritic branches. The glia disappears and the interaxonal spaces become narrow. Axons a n d / o r dendritic branches come close to each other and form tight junctions (Fig. 4). Fibres often are deeply invaginated into their neighbouring fibres. Small aggregates of vesicle-like structures, ellipsoidal and up to 80 nm in diameter, can appear on one side of these finger-like appositions.
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FIG. 3. Cross section through perineural cell layer of lateral ocellar nerve of P. vulgaris. Shown is a small axon (SA), which m a y contain neurosecretory granules (thick arrow), being likely presynaptic to other neural elements within nerve (arrowhead). Note electron-dense material lying in lacunae of glia (thin arrow). LA = axon of large lst-order interneurone; G = glia. Scale = 1.0 I.tm.
FIG. 4. Cross section through median ocellar nerve of P. germanica. Shown are close appositional sites (arrows) between axons of large I st-order interneurones (LA). Note finger-like invaginations and membrane-associated vesicle-like organelles. Scale = 1.0 p,m. FIG. 5. Cross section taken from lateral ocellar nerve of P. vulgaris, showing an axon of a large lstorder interneurone (LA) being both pre- and postsynaptic to other fibres within nerve (arrows). Large axon gives off short collaterals (arrowhead). A presynaptic element (star) contains vesicles. Scale = 1.0 v,m.
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In the middle of the terminal arborizations of the large 1st-order interneurones, lateral chemical synapses between neighbouring branches were observed (Figs. 5,6). The synapses connect adjacent lst-order interneurones of the ocellar nerves and it looks as if all ocellar nerve fibres synapse with each other. However, with the methods employed, it was difficult to sort out whether the synapses are between neurones of the same a n d / o r different ocellar nerves, but synaptic contacts between the nerve of the median ocellus and the lateral nerves could be observed. The interneuronal synapses are situated close to each other, the distance being often only a few micrometers. The presynaptic part of the synapses contains an electron-dense body, which in uninterrupted series of sections was seen to be bar-shaped. The bars of the presynaptic membrane protrude as far as 0.5 ~tm into the lumen of the fibre terminal. The observed shape of the bars depends on the plane of section. In transverse cuts, the bars appeared round or oval, 5 0 - 120 nm in diameter (Fig. 6). Such presynaptic figures were also seen in the distal terminals of the ocellar nerves within the subretinal neuropile (Kral, 1979). Clear spherical vesicles with an average diameter of 40 nm are arrayed around the presynaptic bars. The width of the synaptic cleft measures approximately 20 nm. Electron-dense material often occurs in the cleft. On the postsynaptic side there is mostly an increase in the electron density of the plasma membrane. Electron opaque substances were frequently seen beneath the postsynaptic m e m b r a n e (Fig. 6).
FIG. 6. Transverse section through lateral ocellar nerve of P. vulgaris. One large lst-oraer interneurone dendritic branch (LD) is presynaptic to dendritic branches of adjacent large lst-order interneurones (arrowed). Note different views of presynaptic bars. Scale = 1.0 ~tm. Chemical synapses between putative neurosecretory cells and other neural elements were also observed (Fig. 3). The putative neurosecretory axons contain large dense-cored (up to 160 nm) and small clear spherical vesicles, but only the small vesicles, 2 0 - 30 nm in diameter, cluster at synaptic complexes, suggesting that these small vesicles may contain
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FIG. 7. Cross section through ocellar nerve of P. vulgaris, within brain showing pre- and postsynaptic contacts (arrows) between a dendritic branch of a large lst-order interneurone (LD) and a small profile (star) containing round clear and round dense-cored vesicles. Scale = 2.0 tam.
the neurotransmitter. The large dense-cored vesicles occur in regions distant from the synapse. Within the arborization area of the large lst-order interneurones small profiles were observed, which also contain 2 populations of vesicles: spherical dense-cored vesicles of varying size ( 5 0 - 1 6 0 nm) and smaller spherical clear vesicles, 3 0 - 4 0 nm in diameter. These profiles and lst-order ocellar interneurones seem to have reciprocal synaptic connections (Fig. 7). DISCUSSION
The present ultrastructural analysis of the ocellar tract of the wasp demonstrated that the axons of the large lst-order interneurones are completely isolated from one another in their course from the ocelli to their arborizations within the central nervous system. In the area where the axons start to arborize, the gila disappear or at least become very thin, which enables adjacent axons and adjacent dendritic branches to form close appositions between one another. The membrane appositions possibly form junctions, the assumption being that at these appositions the normal intercellular gap of about 2 0 - 40 nm narrows to about 3 rim. The vesicle-like organelles at each side of the contacts may represent cation reservoirs (L. Schneider, personal communication). Up to now, electrotonic junctions in the ocellar system had been demonstrated only in the cabbage looper moth (Eaton and Pappas, 1977). These electrotonic synapses in the moth connect different photoreceptor cells, photoreceptor cells and lst-order interneurones and different lst-order interneurones. In contrast, in the wasp such junctions were only found within the ocellar tract.
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Besides these junctions, lateral chemical bar-type synapses among lst-order interneurones were frequently observed in the wasp. It could clearly be shown that interneurones of the lateral ocellus nerves as well as those of the median ocellus nerve form such lateral synapses. Contacts between the lateral nerves and the median nerve were also observed, but it was not possible to clearly demonstrate chemical synapses between the interneurones of the 2 lateral nerves, although it is quite likely that the interneurones of all ocellar nerves synapse with one another. What is the functional significance of the junctions and the lateral chemical synapses within the proximal ocellar tract of the wasp? Recent intracellular recordings from locusts (Simmons, 1982) have shown that ocellar lst-order interneurones make both excitatory and inhibitory connections with one another. These results, together with ethological investigations (locust: Wilson, 1978; Taylor, 1981a,b; dragonfly: Stange, 1981), suggest that excitatory synapses may sharpen responsiveness in weak illumination conditions, and that during flight, inhibitory synapses may enhance the detection of rapid movement of the horizon, as it is required during roll or pitch. Furthermore, ethological experiments with freely running honey bees have shown that interaction of all afferent ocellar interneurones plays a major role in phototelotactic orientation (Frisch et al., 1982; Kral and Heran, 1983). Taken together, all these findings indicate that the ocellar tract is far from being a simple pathway for conveying visual signals. Electrophysiological (Simmons, 1982), ethological (Stange, 1981; Taylor, 1981a,b; Kral and Heran, 1983) and morphological (Goodman et ai., 1977; this paper) studies support the view that the ocellar nerves are interacting with each other in a complex manner, thus filtering or otherwise integrating ocellar information. The axons of the small interneurones are mainly situated at the outer part of the ocellar tract; some of them leave the tract at the protocerebral bridge, others more proximal to the CNS. It is well known that the small interneurones of the ocellar system innervate different regions of the brain (Goodman and Williams, 1976; Pan and Goodman, 1977; Eaton and Pappas, 1978). A number of thin axons within the ocellar tract contain electron-dense granules, 50 - 160 nm in diameter. Size and shape of the granules speak in favour of these ceils being putative neurosecretory cells. The term "neurosecretory cells" is used to denote nerve cells that are capable of synthesizing, transporting and releasing complex substances into the haemolymph. Physiological studies are needed to elucidate the functional significance of the ocellar neurosecretory cells and their synaptic contact with other neural elements of the ocellar tract. Within the arborization area of the large 1st-order interneurones, there are also thin profiles that contain clear synaptic vesicles and large dense-cored vesicles. Although the dense-cored vesicles are of the same dimensions as the above described neurosecretory granules, they are of a quite different appearance. It is likely that these profiles belong to central neurones. Worth noting is their reciprocal contact with 1st-order interneurones. Presynaptic terminals with dense-cored vesicles have also been described in the brains of other insects (Tolbert and Hildebrand, 1981). Both clear and dense-cored vesicles are simultaneously found in synapses of the vertebrate CNS and both types of vesicles seem to contain aminergic transmitter substances (see, for example, Andres and v.Dtiring, 1976). The detailed analysis of the output/input region of the ocellar tract within the posterior slopes of the protocerebrum requires special electron microscopic methods. It is hoped that future studies will achieve this goal and contribute to the understanding of the significance of the ocellar tract in various insect species.
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Acknowledgements--I thank Professor W. Burkl (Department of Histology, University of Graz) and Professor L. Schneider (Department of Zoology, University of Wttrzburg) for the invaluable assistance that they have offered in my work. I also thank Mrs. Astrid Gauster for technical help. I am very grateful to Dr. Peter Holzer for critical review of the manuscript. Supported by Grant P 4832 from the Osterr. Fonds zur F0rderung d. wiss. Forschung, Vienna. REFERENCES ANDRES, K. H. and M. v.Dt)RING. 1976. The synapse, pp. 3 - 4 5 . In W. H. GISPEN (ed.) Molecular and Functional Neurobiology, Elsevier, Amsterdam, Oxford, New York. DOWLING, J. E. and R. L. CHAPPELL. 1972. Neural organization of the median ocellus of the dragonfly. II. Synaptic structure. J. Gen. Physiol. 60:148-65. EATON, J. L. and L. G. PAPPAS. 1977. Synaptic organisation of the cabbage looper moth ocellus. Cell Tissue Res. 183:291 - 97. EATON, J. L. and L. G. PAPPAS. 1978. Small ocellar interneurons in the brain of the cabbage looper moth Trichoplusia ni (Hubner) (Lepidoptera : Noctuidae). Int. J. Insect Morphol. Embryol. 7: 337-45. FRISCH,B., K. KRAL and H. HERAN. 1982. Das phototaktische Verhalten der Honigbiene nach Eingriffen in das optische System. Verb. Dtsch. Zool. Ges, 1982: 305. GOODMAN, C. S. 1976. Anatomy of the ocellar interneurons of acridid grasshoppers. I. The large interneurons. Cell Tissue Res. 175:183 -202. GOODMAN, C. S. and J. L. D. WILLIAMS. 1976. Anatomy of the ocellar interneurons of acridid grasshoppers. II. The small interneurons. Cell Tissue Res. 175:203 - 26. GOODMAN, L. J., P. G. MOBaS and R. G. GuY. 1977. Information processing along the course of a visual interneuron. Experientia 33:748 - 50. Guy, R. G., L. J. GOODMANand P. G. Moaas. 1979. Visual interneurons in the bee brain: Synaptic organisation and transmission by graded potentials. J. Comp. Physiol. 134:253 - 64. KRAL, K. 1979. Neuronal connections in the ocellus of the wasp (Paravespula vulgaris L.). Cell Tissue Res. 203: 161-71. KRAL, K. 1982. Large ocellar interneurones (L neurones) in the brain of the wasps, Paravespula vulgaris L. and P. germanica L. (Hymenoptera : Vespidae). Int. J. Insect Morphol. Embryol. 11:307 - 18. KRAL, K. and H. HERAN. 1983. Phototelotaktische Wendereaktionen der Honigbiene (Apis mellifica carnica POLLM.) nach Eingriffen in das Ocellensystem. Zool. Jahrb. Physiol. 87: 127- 140. MADDRELL,S. H. P. 1974. Neurosecretion, pp. 307 -357. In J. E. TREHERNE(ed.) Insect Neurobiology, NorthHolland, Amsterdam, Oxford. PAN, K. C. and L. J. GOODMAN.1977. Ocellar projections within the central nervous system of the worker honey bee, Apis mellifera. Cell Tissue Res. 176:505 - 27. PAPPAS, L. G. and J. L. EATON. 1977. Large ocellar interneurons in the brain of the cabbage looper moth, Trichoplusia ni (Lepidoptera). Zoomorphologie 87:237 - 46. SIMMONS,P. J. 1982. Transmission mediated with and without spikes at connexions between large second-order neurones of locust ocelli. J. Comp. PhysioL 147:401 - 14. STANGE,G. 1981. The ocellar component of flight equilibrium control in dragonflies. J. Comp. Physiol. 141: 335 - 47. TAYLOR, C. P. 1981a. Contribution of compound eyes and ocelli to steering of locusts in flight. I. Behavioural analysis. J. Exp. Biol. 93:1 - 18. TAYLOR, C. P. 1981b. Contribution of compound eyes and ocelli to steering of locusts in flight. II. Timing changes in flight motor units. J. Exp. Biol. 93: 19- 31. TOLBERT, L. P. and J. G. HILDEBRAND. 1981. Organization and synaptic ultrastructure of glomeruli in the antennal lobes of the moth Manduca sexta: a study using thin sections and freeze-fracture. Proc. R. Soc. Lond. B 213: 279- 301. WILSON, M. 1978. The functional organisation of locust ocelli. J. Comp. Physiol. 124: 297- 316.
0020- 7322/83$3.00+ .00 © 1983PergamonPressLtd.
ULTRASTRUCTURAL ANALYSIS OF THE OCELLAR TRACT OF T H E W A S P S , P A R A V E S P U L A V U L G A R I S L. A N D P. G E R M A N I C A L. ( H Y M E N O P T E R A • VESPIDAE) KARL KRAL Department of Zoology, University of Graz, A-8010 Graz, Austria
(Accepted 25 May 1983) A b s t r a c t - - T h e oceUar tract of the wasp conveys visual information from the 3 ocelli to the protocerebrum, and consists of 26 thick axons (15 - 30 gm) of large I st-order interneurones and a larger number of thin axons of small interneurones. In the present study, the ocellar tract of the wasps, Paravespula vulgaris and P. germanica (Hymenoptera : Vespidae) was investigated at the ultrastructural level. The axons of the large I st-order interneurones are separated from one another by glial cells, but on entering the posterior slopes of the protocerebrum the glial cells disappear so that the axons come very close to each other and form junctions. Slightly farther, they arborize into dendritic branches, which are also interconnected by such junctions. In addition, lateral chemical synapses of the bar-shaped type also exist a m o n g the dendritic branches. These synapses connect interneurones of the individual lateral nerves and of the median ocellar nerve, but synaptic contacts could also be observed between the lateral nerves and the median nerve. It was also demonstrated that there are profiles with clear vesicles and large dense-cored vesicles, possibly neurosecretory granules, in the ocellar tract that form synapses with other neural elements. Index descriptors (in addition to those in title): Ocellar tract, electron microscopic examination, neuronal interaction.
INTRODUCTION
THE OCELLAR tract of adult insects contains large afferent lst-order interneurones and smaller afferent and efferent interneurones, which connect the ocelli with the brain (dragonfly: Dowling and Chappell, 1972; locust: Goodman, 1976; Goodman and Williams, 1976; cabbage looper moth: Pappas and Eaton, 1977; Eaton and Pappas, 1978; Honey bee: Pan and Goodman, 1977; Guy et al., 1979; wasp: Kral, 1982). The fact that the giant fibres (up to 30 ~tm in diameter) of the ocellar tract are ideally suited for intracellular recording and marking allows those parts of the pathways, which are responsible for the generation, transfer and output of visual signals, to be studied in much detail. Recent electrophysiological investigations of migratory locusts (Simmons, 1982) and behavioural experiments with honey bees (Kral and Heran, 1983) have shown that integration of incoming visual messages is completed in the ocellar tract. It was with this in mind that the present study was undertaken to examine the ocellar tract of vespid wasps at the ultrastructural level. MATERIALS
AND
METHODS
Adult wasps (P. vulgaris and P. germanica) were collected from nests in the Graz area. Brains were fixed for 3 hr in p h o s p h a t e - b u f f e r e d 6.25°70 glutaraldehyde (pH 7.3; 4°C), post-fixed in 10?00sO, (pH 7.3; 4°C) and embedded in Epon 812. Semi-thin sections from brain were cut to localize the region to be investigated in the ocellar tract. Thereafter, trimming was done with razor blades. At the beginning and end of each series of ultrathin sections (interval of 3 - 5 ~tm) 1 ~tm-thick control sections were cut, stained with toluidine blue and 313
KARL KRAL
314
examined with the light microscope. Ultra-thin sections were stained with lead citrate and examined with a Zeiss EM IOA electron microscope, which was supplied by the Stiftung Volkswagenwerk.
RESULTS
Adult wasps (P. vulgaris and P. germanica) possess one median ocellus and 2 lateral ocelli. The photoreceptor cells form synaptic contacts with the dendrites of a small number of large 1st-order interneurones (L neurones) and a larger group of small interneurones (Kral, 1979). The pathway of the ocellar tract in the brain of the wasp has been described previously (Kra.l, 1982). The output region of the ocellar tract (illustrated in Fig. 1) was not investigated in the present study, since conventional electron microscopy does not allow the identification of individual elements within the complex neuropile of the posterior slopes. Some preliminary observations indicate, however, that chemical synapses also exist between ocellar fibre terminals and central neurones.
ocellus
dendrite
. :~
dendr PS Flo. 1. Reconstruction o f 2 parallel large afferent lst-order ocellar interneurones of P. germanica, based on photographs of Procion yellow filled oceUar nerves (Kral, 1982). Sites of lateral synapses are marked by curved arrows. Direction of information flow is indicated by arrows. PS = posterior slope neurOpile.
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The electron microscopic examination of the ocellar tract showed that the axons of the large lst-order interneurones and most of the axons of the small interneurones contain a clear cytoplasm with microtubules aligned along the axonal axis. The axons form terminal arborizations within the CNS. Except in the terminal arborizations, the cytoplasm contains only a few small mitochondria, whose inner membrane is slightly folded. The axons of the large interneurones are completely separated from each other by glial cells (Fig. 2), whereas the thin axons, which are mainly found in the outer part of the tract, are mostly naked. Some of the thin axons contain structures that may be neurosecretory granules of up to 160 nm diameter. These axons can be in synaptic contact with other neural elements (Fig. 3).
FIG. 2. Cross section through median ocellar nerve of P. vulgaris, showing axons of 2 large lst-order interneurones (LA). Note that the axons are isolated from each other by glia (G). Numerous vacuoles, lacunae and widened spaces of endoplasmic reticulum (arrows) are found in glial layer. Scale = 2.0 gm.
Within the glia, there are widened spaces of the endoplasmic reticulum and large vacuoles that are often filled with electron-dense material (Figs. 2,3). Large magnification showed that this material is bound to fine membranes that bridge the lumen. The vacuoles are often in contact with the cytoplasm of the axons. Tracheoles were also observed in the glia. On entering the neuropile of the posterior slopes of the protocerebrum the axons of the large 1st-order interneurones form dendritic branches. The glia disappears and the interaxonal spaces become narrow. Axons a n d / o r dendritic branches come close to each other and form tight junctions (Fig. 4). Fibres often are deeply invaginated into their neighbouring fibres. Small aggregates of vesicle-like structures, ellipsoidal and up to 80 nm in diameter, can appear on one side of these finger-like appositions.
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FIG. 3. Cross section through perineural cell layer of lateral ocellar nerve of P. vulgaris. Shown is a small axon (SA), which m a y contain neurosecretory granules (thick arrow), being likely presynaptic to other neural elements within nerve (arrowhead). Note electron-dense material lying in lacunae of glia (thin arrow). LA = axon of large lst-order interneurone; G = glia. Scale = 1.0 I.tm.
FIG. 4. Cross section through median ocellar nerve of P. germanica. Shown are close appositional sites (arrows) between axons of large I st-order interneurones (LA). Note finger-like invaginations and membrane-associated vesicle-like organelles. Scale = 1.0 p,m. FIG. 5. Cross section taken from lateral ocellar nerve of P. vulgaris, showing an axon of a large lstorder interneurone (LA) being both pre- and postsynaptic to other fibres within nerve (arrows). Large axon gives off short collaterals (arrowhead). A presynaptic element (star) contains vesicles. Scale = 1.0 v,m.
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In the middle of the terminal arborizations of the large 1st-order interneurones, lateral chemical synapses between neighbouring branches were observed (Figs. 5,6). The synapses connect adjacent lst-order interneurones of the ocellar nerves and it looks as if all ocellar nerve fibres synapse with each other. However, with the methods employed, it was difficult to sort out whether the synapses are between neurones of the same a n d / o r different ocellar nerves, but synaptic contacts between the nerve of the median ocellus and the lateral nerves could be observed. The interneuronal synapses are situated close to each other, the distance being often only a few micrometers. The presynaptic part of the synapses contains an electron-dense body, which in uninterrupted series of sections was seen to be bar-shaped. The bars of the presynaptic membrane protrude as far as 0.5 ~tm into the lumen of the fibre terminal. The observed shape of the bars depends on the plane of section. In transverse cuts, the bars appeared round or oval, 5 0 - 120 nm in diameter (Fig. 6). Such presynaptic figures were also seen in the distal terminals of the ocellar nerves within the subretinal neuropile (Kral, 1979). Clear spherical vesicles with an average diameter of 40 nm are arrayed around the presynaptic bars. The width of the synaptic cleft measures approximately 20 nm. Electron-dense material often occurs in the cleft. On the postsynaptic side there is mostly an increase in the electron density of the plasma membrane. Electron opaque substances were frequently seen beneath the postsynaptic m e m b r a n e (Fig. 6).
FIG. 6. Transverse section through lateral ocellar nerve of P. vulgaris. One large lst-oraer interneurone dendritic branch (LD) is presynaptic to dendritic branches of adjacent large lst-order interneurones (arrowed). Note different views of presynaptic bars. Scale = 1.0 ~tm. Chemical synapses between putative neurosecretory cells and other neural elements were also observed (Fig. 3). The putative neurosecretory axons contain large dense-cored (up to 160 nm) and small clear spherical vesicles, but only the small vesicles, 2 0 - 30 nm in diameter, cluster at synaptic complexes, suggesting that these small vesicles may contain
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FIG. 7. Cross section through ocellar nerve of P. vulgaris, within brain showing pre- and postsynaptic contacts (arrows) between a dendritic branch of a large lst-order interneurone (LD) and a small profile (star) containing round clear and round dense-cored vesicles. Scale = 2.0 tam.
the neurotransmitter. The large dense-cored vesicles occur in regions distant from the synapse. Within the arborization area of the large lst-order interneurones small profiles were observed, which also contain 2 populations of vesicles: spherical dense-cored vesicles of varying size ( 5 0 - 1 6 0 nm) and smaller spherical clear vesicles, 3 0 - 4 0 nm in diameter. These profiles and lst-order ocellar interneurones seem to have reciprocal synaptic connections (Fig. 7). DISCUSSION
The present ultrastructural analysis of the ocellar tract of the wasp demonstrated that the axons of the large lst-order interneurones are completely isolated from one another in their course from the ocelli to their arborizations within the central nervous system. In the area where the axons start to arborize, the gila disappear or at least become very thin, which enables adjacent axons and adjacent dendritic branches to form close appositions between one another. The membrane appositions possibly form junctions, the assumption being that at these appositions the normal intercellular gap of about 2 0 - 40 nm narrows to about 3 rim. The vesicle-like organelles at each side of the contacts may represent cation reservoirs (L. Schneider, personal communication). Up to now, electrotonic junctions in the ocellar system had been demonstrated only in the cabbage looper moth (Eaton and Pappas, 1977). These electrotonic synapses in the moth connect different photoreceptor cells, photoreceptor cells and lst-order interneurones and different lst-order interneurones. In contrast, in the wasp such junctions were only found within the ocellar tract.
Ultrastructural Analysisof the Ocellar Tract of Wasps
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Besides these junctions, lateral chemical bar-type synapses among lst-order interneurones were frequently observed in the wasp. It could clearly be shown that interneurones of the lateral ocellus nerves as well as those of the median ocellus nerve form such lateral synapses. Contacts between the lateral nerves and the median nerve were also observed, but it was not possible to clearly demonstrate chemical synapses between the interneurones of the 2 lateral nerves, although it is quite likely that the interneurones of all ocellar nerves synapse with one another. What is the functional significance of the junctions and the lateral chemical synapses within the proximal ocellar tract of the wasp? Recent intracellular recordings from locusts (Simmons, 1982) have shown that ocellar lst-order interneurones make both excitatory and inhibitory connections with one another. These results, together with ethological investigations (locust: Wilson, 1978; Taylor, 1981a,b; dragonfly: Stange, 1981), suggest that excitatory synapses may sharpen responsiveness in weak illumination conditions, and that during flight, inhibitory synapses may enhance the detection of rapid movement of the horizon, as it is required during roll or pitch. Furthermore, ethological experiments with freely running honey bees have shown that interaction of all afferent ocellar interneurones plays a major role in phototelotactic orientation (Frisch et al., 1982; Kral and Heran, 1983). Taken together, all these findings indicate that the ocellar tract is far from being a simple pathway for conveying visual signals. Electrophysiological (Simmons, 1982), ethological (Stange, 1981; Taylor, 1981a,b; Kral and Heran, 1983) and morphological (Goodman et ai., 1977; this paper) studies support the view that the ocellar nerves are interacting with each other in a complex manner, thus filtering or otherwise integrating ocellar information. The axons of the small interneurones are mainly situated at the outer part of the ocellar tract; some of them leave the tract at the protocerebral bridge, others more proximal to the CNS. It is well known that the small interneurones of the ocellar system innervate different regions of the brain (Goodman and Williams, 1976; Pan and Goodman, 1977; Eaton and Pappas, 1978). A number of thin axons within the ocellar tract contain electron-dense granules, 50 - 160 nm in diameter. Size and shape of the granules speak in favour of these ceils being putative neurosecretory cells. The term "neurosecretory cells" is used to denote nerve cells that are capable of synthesizing, transporting and releasing complex substances into the haemolymph. Physiological studies are needed to elucidate the functional significance of the ocellar neurosecretory cells and their synaptic contact with other neural elements of the ocellar tract. Within the arborization area of the large 1st-order interneurones, there are also thin profiles that contain clear synaptic vesicles and large dense-cored vesicles. Although the dense-cored vesicles are of the same dimensions as the above described neurosecretory granules, they are of a quite different appearance. It is likely that these profiles belong to central neurones. Worth noting is their reciprocal contact with 1st-order interneurones. Presynaptic terminals with dense-cored vesicles have also been described in the brains of other insects (Tolbert and Hildebrand, 1981). Both clear and dense-cored vesicles are simultaneously found in synapses of the vertebrate CNS and both types of vesicles seem to contain aminergic transmitter substances (see, for example, Andres and v.Dtiring, 1976). The detailed analysis of the output/input region of the ocellar tract within the posterior slopes of the protocerebrum requires special electron microscopic methods. It is hoped that future studies will achieve this goal and contribute to the understanding of the significance of the ocellar tract in various insect species.
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Acknowledgements--I thank Professor W. Burkl (Department of Histology, University of Graz) and Professor L. Schneider (Department of Zoology, University of Wttrzburg) for the invaluable assistance that they have offered in my work. I also thank Mrs. Astrid Gauster for technical help. I am very grateful to Dr. Peter Holzer for critical review of the manuscript. Supported by Grant P 4832 from the Osterr. Fonds zur F0rderung d. wiss. Forschung, Vienna. REFERENCES ANDRES, K. H. and M. v.Dt)RING. 1976. The synapse, pp. 3 - 4 5 . In W. H. GISPEN (ed.) Molecular and Functional Neurobiology, Elsevier, Amsterdam, Oxford, New York. DOWLING, J. E. and R. L. CHAPPELL. 1972. Neural organization of the median ocellus of the dragonfly. II. Synaptic structure. J. Gen. Physiol. 60:148-65. EATON, J. L. and L. G. PAPPAS. 1977. Synaptic organisation of the cabbage looper moth ocellus. Cell Tissue Res. 183:291 - 97. EATON, J. L. and L. G. PAPPAS. 1978. Small ocellar interneurons in the brain of the cabbage looper moth Trichoplusia ni (Hubner) (Lepidoptera : Noctuidae). Int. J. Insect Morphol. Embryol. 7: 337-45. FRISCH,B., K. KRAL and H. HERAN. 1982. Das phototaktische Verhalten der Honigbiene nach Eingriffen in das optische System. Verb. Dtsch. Zool. Ges, 1982: 305. GOODMAN, C. S. 1976. Anatomy of the ocellar interneurons of acridid grasshoppers. I. The large interneurons. Cell Tissue Res. 175:183 -202. GOODMAN, C. S. and J. L. D. WILLIAMS. 1976. Anatomy of the ocellar interneurons of acridid grasshoppers. II. The small interneurons. Cell Tissue Res. 175:203 - 26. GOODMAN, L. J., P. G. MOBaS and R. G. GuY. 1977. Information processing along the course of a visual interneuron. Experientia 33:748 - 50. Guy, R. G., L. J. GOODMANand P. G. Moaas. 1979. Visual interneurons in the bee brain: Synaptic organisation and transmission by graded potentials. J. Comp. Physiol. 134:253 - 64. KRAL, K. 1979. Neuronal connections in the ocellus of the wasp (Paravespula vulgaris L.). Cell Tissue Res. 203: 161-71. KRAL, K. 1982. Large ocellar interneurones (L neurones) in the brain of the wasps, Paravespula vulgaris L. and P. germanica L. (Hymenoptera : Vespidae). Int. J. Insect Morphol. Embryol. 11:307 - 18. KRAL, K. and H. HERAN. 1983. Phototelotaktische Wendereaktionen der Honigbiene (Apis mellifica carnica POLLM.) nach Eingriffen in das Ocellensystem. Zool. Jahrb. Physiol. 87: 127- 140. MADDRELL,S. H. P. 1974. Neurosecretion, pp. 307 -357. In J. E. TREHERNE(ed.) Insect Neurobiology, NorthHolland, Amsterdam, Oxford. PAN, K. C. and L. J. GOODMAN.1977. Ocellar projections within the central nervous system of the worker honey bee, Apis mellifera. Cell Tissue Res. 176:505 - 27. PAPPAS, L. G. and J. L. EATON. 1977. Large ocellar interneurons in the brain of the cabbage looper moth, Trichoplusia ni (Lepidoptera). Zoomorphologie 87:237 - 46. SIMMONS,P. J. 1982. Transmission mediated with and without spikes at connexions between large second-order neurones of locust ocelli. J. Comp. PhysioL 147:401 - 14. STANGE,G. 1981. The ocellar component of flight equilibrium control in dragonflies. J. Comp. Physiol. 141: 335 - 47. TAYLOR, C. P. 1981a. Contribution of compound eyes and ocelli to steering of locusts in flight. I. Behavioural analysis. J. Exp. Biol. 93:1 - 18. TAYLOR, C. P. 1981b. Contribution of compound eyes and ocelli to steering of locusts in flight. II. Timing changes in flight motor units. J. Exp. Biol. 93: 19- 31. TOLBERT, L. P. and J. G. HILDEBRAND. 1981. Organization and synaptic ultrastructure of glomeruli in the antennal lobes of the moth Manduca sexta: a study using thin sections and freeze-fracture. Proc. R. Soc. Lond. B 213: 279- 301. WILSON, M. 1978. The functional organisation of locust ocelli. J. Comp. Physiol. 124: 297- 316.