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Synaptic structure of the lateral line lobe nucleus in mormyrid fish
Neuroscience Letters, 2 (1976) 127--131
127
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
SYNAPTIC STRUCTURE OF THE L A T E R A L LINE LOBE NUCLEUS IN MORMYRID FISH
T. SZABO and M. RAVAILLE Laboratoire de Physiologie Nerveuse, D$parternent de Neurophysiologie Sensorielle, CNRS, 91190 Gif sur Yvette (France)
(Received March 22nd, 1976) (Accepted March 24th, 1976)
SUMMARY Electron microscopical observations reveal a complex synaptic structure (Fig. 1) in the nucleus of the lateral line lobe (nLLL). Different types of axosomatic and axoaxonic synapses are demonstrated to be in contact with the large cells. The results furnish morphological evidence for electrotonic transmission (by way of club endings with gap junctions) at this level of the electrosensory pathway of mormyrid fish. A new ultrastructural finding is the existence of presynaptic gap junctions on the unmyelinated surface area of the club endings.
Recent electrophysiologicalinvestigations [1,2] have shown that the nucleus of the lateralline lobe (nLLL) displays an electroreceptor input, probably from the receptor type I, and represents the rhombencephalic relay of the rapid electrosensory pathway of mormyrid fish [2,6]. Anatomically this nucleus is located between the entrance of the lateralline nerves and the ganglion celllayer of the lateralline lobe. According to Maler [4], the posterior part of the n L L L receives fibres from the posterior lateralline nerve. The aim of the present paper is to give an ultrastructural account of this nucleus which is assumed to transmit electroreceptive impulses to the mesencephalon [2,7]. For this purpose the brains of three mormyrids were examined: G n a t h o n e m u s petersii, M o r m y r u s rurne and M a r c u s e n i u s sp. They were either immersed in 4% formalin or Bodian's solution or else perfused with glutaraldehyde/paraformaldehyde fixative. Intracardiac perfusion was performed with Ringer solution and consecutively with the aldehyde fixative during deep MS-222 anaesthesia and after injection of 0.5% NaNO3. The perfused brains were then immersed for 12 h in the perfusion solution and postfixed for 2 h with OSO4. For light microscopical observations the
128 brains were embedded either in paraffin or araldite. In the former case the brains were cut in transverse, sagittal and horizontal serial sections and stained alternatively according to Bodian's and Nissl's method. The semi-thin sections of the araldite embedded material were stained with toluidine blue. Ultra-thin sections were contrasted by Reynold's double stain method [ 5]. Paraffin sections revealed a non-homogeneous structure of the n L L L in all the species examined. The nucleus was found to be composed of different types of large and small neurones disposed rather loosely in a network of differently sized myelinated fibres. Observations of serial ultra-thin sections showed that the small cells are located only at the periphery of the nucleus whereas the large cells are located in its centre. A l l of the large cells (19 out of the 20 cells examined) have a spherical appearance with a diameter of about 20 pm, and t h e y do not display any dendritic processes (Fig. 2). However, the surface of their perikarya is not smooth but possesses spine-like extrusions. The cytoplasm contains a large number of mitochondria and a well-developed granular endoplasmic reticulum. Because of the large number of free ribosomes the cell has a dark appearance; in contrast, the peripheral cytoplasm close to the synaptic contacts is almost free of them. A slender axon develops from a rather discrete axon hillock; the initial segment of the axon is 1.5 times longer (30 ~m) than the diameter of the cell. The myelinated axon is of medium size (5 ~m). The surface of the large cells (Fig. 1) typically carries a great number o f very regularly distributed small 'boutons terminaux' (1 pm) and one or two large club endings (6--8 pm). The boutons terminaux are of at least two types (Fig. 2B): one type possesses a dark cytoplasm and contains mitochondria
7
Fig. 1. Schematic drawing of the synaptic structure of the large cells in the nLLL. ce, large club endings of myelinated preterminals; el, boutons with gap junctions; ch, boutons with chemical synapses. Insert: schematic representation of a transverse section through the mormyrid brain at the level of the lateral line lobe nucleus (nLLL). lob.c., lobus caudalis; lob.lat., lobus lateralis; the cerebellum is not represented.
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Fig. 2. A: club ending (ce) of a myelinated preterminal fibre (pf). Note the large contact area (arrows) between club ending (ce) and large neuron (In) of the nLLL. X 4500. B: two types of endings with dark (1) and clear (2) cytoplasm. X 40,000. C: gap junction (arrows) between club ending (ce) and a large neuron (ln). X 60,000. D: axoaxonic gap junction (arrows) between a bouton (bt) and a club ending (ce) of a large neurone in the nLLL. Note to the left of the gap the presence of a typical chemical axoaxonic junction (mixed synapse ?). × 70,000.
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and vesicles which are particularly densely packed at the synaptic contact; the other type is filled with many mitochondria and contains only a few vesicles which are concentrated at the membrane area making contact with the large cell. The boutons containing either clear or dense vesicles form typical chemical synapses with the postsynaptic membrane. Boutons with chemical synapses are also present at the axon hillock and all along the initial axon segment. The club endings originate from large preterminal fibres (6--7 ~m in diameter) which are myelinated up to the several micra-long neck of the club (Fig. 2A). The club contains many densely packed vesicles of different sizes. The contact area (approximately 10 pm in diameter) between club endings and the postsynaptic membrane is characterized by several gap junctions (Fig. 2C). Beside these axosomatic junctions several axoaxonic contacts were observed in the nLLL (Fig. 1). In particular, terminals with gap junctions (el) were found on the foot (unmyelinated portion) of the club ending (Fig, 2D). The terminals, in turn, bear boutons (ch) showing the characteristics of chemical synapses (Fig. 1). Finally, axoaxonic contacts, with some kind of tight junction, were also found on endings located at the initial segment. The present findings reveal a more complex synaptology of the large cells in the nLLL than one would expect for a simple relay. The gap junctions of the club endings are the morphological correlates of electrotonic transmission. Thus, it seems likely that the nLLL represents the rhombencephalic relay for electrosensory impulses rapidly conveyed to the mesencephalon [2], and the large cells in the nucleus probably correspond functionally to the pear-shaped second order neurones described in the fast conducting electrosensory pathway of gymnotids [6]. Although the contact area of the individual club endings with the cell membrane is relatively large (about 12% of the cell surface) the small boutons occupy the major part of this membrane. This fact suggests a complex and powerful postsynaptic action of different origins on the large neurones in this nucleus. In addition, the findings furnish morphological evidence for a possible presynaptic effect on the club endings, and arrangement which, to our knowledge, has never been demonstrated before. Finally, neither the form, nor the size or the other features of the large nLLL cells described by Maler [3] from Golgi preparations could be confirmed by our observations. REFERENCES 1 Bell, C.C. and Russell, C.J., Responses to the electric organ discharge and c o m m a n d signal in the mormyrid lateral line lobe. In 7th International Neurobiol. Meeting on Afferent and Intrinsic Organisation of Laminated Structures in the Brain, 1975, abstract. 2 Enger, P.S., Libouban, S. and Szabo, T., Fast conducting electrosensory pathway in the m o r m y r i d fish, Gnathonemus petersii. Neuroscience Letters, 2 (1976) 133--136. 3 Maler, L., The posterior lateral line lobe of a m o r m y r i d fish -- a Golgi study, J. comp. Neurol., 152 (1973) 181--197.
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4 Maler, L., Karten, H.J. and Bennett, M.V.L., The central connections of the posterior lateral line nerve of Gnathonemus petersii, J. comp. Neurol., 151 (1973) 57--66. 5 Reynolds, E.S., The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17 (1963) 208--212. 6 Szabo, T., Central processing of messages from tuberous electroreceptors in teleosts. In A. Fessard (Ed.), Electroreceptors and other Specialised Receptors in Lower Vertebrates, Handbook of Sensory Physiology, III/3, Springer, Berlin, 1974, pp. 95--124. 7 Szabo, T., Sakata, H. and Raville, M., An electrotonic coupled pathway in the central neuron system of some teleost fishl Gymnotidae and Mormyridae, Brain Res., 95 (1975) 459--474.
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© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
SYNAPTIC STRUCTURE OF THE L A T E R A L LINE LOBE NUCLEUS IN MORMYRID FISH
T. SZABO and M. RAVAILLE Laboratoire de Physiologie Nerveuse, D$parternent de Neurophysiologie Sensorielle, CNRS, 91190 Gif sur Yvette (France)
(Received March 22nd, 1976) (Accepted March 24th, 1976)
SUMMARY Electron microscopical observations reveal a complex synaptic structure (Fig. 1) in the nucleus of the lateral line lobe (nLLL). Different types of axosomatic and axoaxonic synapses are demonstrated to be in contact with the large cells. The results furnish morphological evidence for electrotonic transmission (by way of club endings with gap junctions) at this level of the electrosensory pathway of mormyrid fish. A new ultrastructural finding is the existence of presynaptic gap junctions on the unmyelinated surface area of the club endings.
Recent electrophysiologicalinvestigations [1,2] have shown that the nucleus of the lateralline lobe (nLLL) displays an electroreceptor input, probably from the receptor type I, and represents the rhombencephalic relay of the rapid electrosensory pathway of mormyrid fish [2,6]. Anatomically this nucleus is located between the entrance of the lateralline nerves and the ganglion celllayer of the lateralline lobe. According to Maler [4], the posterior part of the n L L L receives fibres from the posterior lateralline nerve. The aim of the present paper is to give an ultrastructural account of this nucleus which is assumed to transmit electroreceptive impulses to the mesencephalon [2,7]. For this purpose the brains of three mormyrids were examined: G n a t h o n e m u s petersii, M o r m y r u s rurne and M a r c u s e n i u s sp. They were either immersed in 4% formalin or Bodian's solution or else perfused with glutaraldehyde/paraformaldehyde fixative. Intracardiac perfusion was performed with Ringer solution and consecutively with the aldehyde fixative during deep MS-222 anaesthesia and after injection of 0.5% NaNO3. The perfused brains were then immersed for 12 h in the perfusion solution and postfixed for 2 h with OSO4. For light microscopical observations the
128 brains were embedded either in paraffin or araldite. In the former case the brains were cut in transverse, sagittal and horizontal serial sections and stained alternatively according to Bodian's and Nissl's method. The semi-thin sections of the araldite embedded material were stained with toluidine blue. Ultra-thin sections were contrasted by Reynold's double stain method [ 5]. Paraffin sections revealed a non-homogeneous structure of the n L L L in all the species examined. The nucleus was found to be composed of different types of large and small neurones disposed rather loosely in a network of differently sized myelinated fibres. Observations of serial ultra-thin sections showed that the small cells are located only at the periphery of the nucleus whereas the large cells are located in its centre. A l l of the large cells (19 out of the 20 cells examined) have a spherical appearance with a diameter of about 20 pm, and t h e y do not display any dendritic processes (Fig. 2). However, the surface of their perikarya is not smooth but possesses spine-like extrusions. The cytoplasm contains a large number of mitochondria and a well-developed granular endoplasmic reticulum. Because of the large number of free ribosomes the cell has a dark appearance; in contrast, the peripheral cytoplasm close to the synaptic contacts is almost free of them. A slender axon develops from a rather discrete axon hillock; the initial segment of the axon is 1.5 times longer (30 ~m) than the diameter of the cell. The myelinated axon is of medium size (5 ~m). The surface of the large cells (Fig. 1) typically carries a great number o f very regularly distributed small 'boutons terminaux' (1 pm) and one or two large club endings (6--8 pm). The boutons terminaux are of at least two types (Fig. 2B): one type possesses a dark cytoplasm and contains mitochondria
7
Fig. 1. Schematic drawing of the synaptic structure of the large cells in the nLLL. ce, large club endings of myelinated preterminals; el, boutons with gap junctions; ch, boutons with chemical synapses. Insert: schematic representation of a transverse section through the mormyrid brain at the level of the lateral line lobe nucleus (nLLL). lob.c., lobus caudalis; lob.lat., lobus lateralis; the cerebellum is not represented.
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Fig. 2. A: club ending (ce) of a myelinated preterminal fibre (pf). Note the large contact area (arrows) between club ending (ce) and large neuron (In) of the nLLL. X 4500. B: two types of endings with dark (1) and clear (2) cytoplasm. X 40,000. C: gap junction (arrows) between club ending (ce) and a large neuron (ln). X 60,000. D: axoaxonic gap junction (arrows) between a bouton (bt) and a club ending (ce) of a large neurone in the nLLL. Note to the left of the gap the presence of a typical chemical axoaxonic junction (mixed synapse ?). × 70,000.
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and vesicles which are particularly densely packed at the synaptic contact; the other type is filled with many mitochondria and contains only a few vesicles which are concentrated at the membrane area making contact with the large cell. The boutons containing either clear or dense vesicles form typical chemical synapses with the postsynaptic membrane. Boutons with chemical synapses are also present at the axon hillock and all along the initial axon segment. The club endings originate from large preterminal fibres (6--7 ~m in diameter) which are myelinated up to the several micra-long neck of the club (Fig. 2A). The club contains many densely packed vesicles of different sizes. The contact area (approximately 10 pm in diameter) between club endings and the postsynaptic membrane is characterized by several gap junctions (Fig. 2C). Beside these axosomatic junctions several axoaxonic contacts were observed in the nLLL (Fig. 1). In particular, terminals with gap junctions (el) were found on the foot (unmyelinated portion) of the club ending (Fig, 2D). The terminals, in turn, bear boutons (ch) showing the characteristics of chemical synapses (Fig. 1). Finally, axoaxonic contacts, with some kind of tight junction, were also found on endings located at the initial segment. The present findings reveal a more complex synaptology of the large cells in the nLLL than one would expect for a simple relay. The gap junctions of the club endings are the morphological correlates of electrotonic transmission. Thus, it seems likely that the nLLL represents the rhombencephalic relay for electrosensory impulses rapidly conveyed to the mesencephalon [2], and the large cells in the nucleus probably correspond functionally to the pear-shaped second order neurones described in the fast conducting electrosensory pathway of gymnotids [6]. Although the contact area of the individual club endings with the cell membrane is relatively large (about 12% of the cell surface) the small boutons occupy the major part of this membrane. This fact suggests a complex and powerful postsynaptic action of different origins on the large neurones in this nucleus. In addition, the findings furnish morphological evidence for a possible presynaptic effect on the club endings, and arrangement which, to our knowledge, has never been demonstrated before. Finally, neither the form, nor the size or the other features of the large nLLL cells described by Maler [3] from Golgi preparations could be confirmed by our observations. REFERENCES 1 Bell, C.C. and Russell, C.J., Responses to the electric organ discharge and c o m m a n d signal in the mormyrid lateral line lobe. In 7th International Neurobiol. Meeting on Afferent and Intrinsic Organisation of Laminated Structures in the Brain, 1975, abstract. 2 Enger, P.S., Libouban, S. and Szabo, T., Fast conducting electrosensory pathway in the m o r m y r i d fish, Gnathonemus petersii. Neuroscience Letters, 2 (1976) 133--136. 3 Maler, L., The posterior lateral line lobe of a m o r m y r i d fish -- a Golgi study, J. comp. Neurol., 152 (1973) 181--197.
131
4 Maler, L., Karten, H.J. and Bennett, M.V.L., The central connections of the posterior lateral line nerve of Gnathonemus petersii, J. comp. Neurol., 151 (1973) 57--66. 5 Reynolds, E.S., The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17 (1963) 208--212. 6 Szabo, T., Central processing of messages from tuberous electroreceptors in teleosts. In A. Fessard (Ed.), Electroreceptors and other Specialised Receptors in Lower Vertebrates, Handbook of Sensory Physiology, III/3, Springer, Berlin, 1974, pp. 95--124. 7 Szabo, T., Sakata, H. and Raville, M., An electrotonic coupled pathway in the central neuron system of some teleost fishl Gymnotidae and Mormyridae, Brain Res., 95 (1975) 459--474.