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Symmetrical axo-axonic synapses in the axon cap of the goldfish Mauthner cell
SHORT COMMUNICATIONS
255
Symmetrical axo-axonic synapses in the axon cap of the goldfish Mauthner cell Axo-axonic synapses between the terminals containing the synaptic vesicles on both sides have been frequently reported in the central nervous system of the vertebrates6, 20. In each case, the direction of transmission could usually be judged by the accumulation of the vesicles on either side of the synaptic membranes. On the other hand, in some invertebrates v,lI bidirectional synapses with symmetrical accumulation of the vesicles on both sides of the synaptic membranes were observed between the parallel-arranged or crossing giant nerve fibers. The present communication shows similar symmetrical and more differentiated presynaptic-to-presynaptic synapses found in the axon cap region of the Mauthner cell of the goldfish medulla. An electron micrograph of the axon cap was previously published by Robertson et al. ag, but it was low in magnification and insufficient to analyze the finer structure of the synapses. Common goldfish (Carassius auratus), 10-15 cm in length, treated according to the method of Robertson et alfl 9, were perfused through the aorta with a mixture of I ~ paraformaldehyde and 1.25 ~ glutaraldehyde in phosphate buffer (pH 7.4) containing CaCI~ (ref. 14). Tissue was removed and, after postfixation in 2 ~,, osmium tetroxide, was dehydrated in ethanol and embedded in Epon. The position of the axon cap in the embedded block was ascertained by tracing the large myelinated axon of the Mauthner cell in the successive thick sections for light microscopy. Thin sections were double-stained with uranyl acetate and lead acetatelL It is known from the light microscopic studies~,a, is that the 'axon cap' is a peculiar synaptic apparatus which spherically surrounds the axon hillock and the initial axon segment of the Mauthner cell, in association with the external boundary of the 'cap cells', and into which numerous nerve fibers enter and lose their myelin sheaths at the boundary layer; also, 'cap dendrites' from the cell soma penetrate into this region, Specifically, Bodian 3 found that the central core of the axon cap immediately investing the initial axon segment is of a different nature than that of the peripheral portion of the axon cap. The central core consists of a dense conglomeration of extremely fine terminal branches of the 'spiral fibers' which come to the axon cap, enveloping the Mauthner axon from the supracommissural bundles of the fasciculus longitudinalis medialis. In electron micrographs (Fig. 1), the central core (CEC) is readily recognized as a compact aggregation of small clear nervous profiles (A) around the initial axon segment (INS). These profiles represent the intricate terminal branches of the spiral fibers. The terminal branches contain numerous vesicles and a few mitochondria. The vesicles, 30-60 nm in diameter, are spherical and clear in the center. Some large vesicles, about 80 nm in diameter, include a dense core in the center. Neurofilaments, microtubules and tubular structures with irregular swellings are also contained within the terminals. Slender processes (P), often forming bundles, penetrate into the mass of the terminal branches. The cytoplasm is filled with numerous longitudinally arranged filaments and microtubules. Between them, free ribosomes and cisternae Brain Research, 23 (1970) 255-258
256
SHORI (OMMUNI('AIlONS
Fig. I. -File central core (CEC) of lhe axon cap surrounding the initial axon segmenl (INS) of goMfish M a u t h n e r cell. Arrows indicate symmetrical axo-axonic synapses between axon terminals (A) containing vesicles and m i t o : h o n d r i a . Three adjacent ierminals (A,, Ae, A:0 are serially connected by lwo symmetrical synapses. Bundles of slender cap cell processes (P) are lilied with n u m e r o u s tilaments, microtubules and cisternae with ribosomes. Fascicles of microlubules (F) and a dense undercoaied a x o l e m m a (U) are seen in the cross section of the INS. 21,000. h~seri, Symmelrical apposition of two presynaptic acli~e zones x~itl~ clusters of vesicles anti wilh 4 opposite pairs of dense projections (arrows), 60,000.
Brain Research, 23 (1970) 255-258
SHORT COMMUNICATIONS
257
with their associated ribosomes are dispersed. These are the elongated processes extending from the cap cells enclosing the axon cap. The initial axon segment (INS) of the Mauthner cell, passing through the center of the axon cap, contains numerous evenly dispersed neurofilaments and fascicles of the microtubules which are connected in series by short arms (F), and the axoplasm is surrounded by a plasma membrane whose axoplasmic surface is coated by a thin layer of dense material (U). These characteristic structures have been also observed in the initial axon segment of the multipolar neurons in frog 1° and rat brains la-16. The symmetrical axo-axonic synapses are frequently found in the central core of the axon cap (arrows), and serial occurrence of the synapses is often observed between the several adjacent terminals (A1, A2, Aa). In the synaptic region, each cluster of the vesicles contained within the two adjacent terminals is symmetrically aggregated toward both cytoplasmic surfaces of the apposed membranes. The aggregation of the vesicles forms a typical presynaptic active zone on some small region of the apposed membranes. The synaptic cleft is about 10 nm wide, and the active zones facing each other usually have a width of 0.2-0.6/~m. In this synapse two similar presynaptic active zones are always directly opposite. This symmetrical synapse is different from the reciprocal synapse of the bulbus olfactorius8,9,15, where two presynaptic active zones with an accumulation of vesicles are not directly opposite but appear separately in two neighboring places of the apposed membranes. Small dense bodies project into a cluster of the vesicles from the active zones of the symmetrical synapses (arrows in insert of Fig. 1). It is often clearly observed in the sections cut perpendicular to the synaptic membrane that 2-4 of the dense bodies are arranged on an active zone with an interval of 100-150 nm from center to center, and they are often situated opposite each other on both sides of the synaptic cleft. At these places the synaptic cleft appears denser than elsewhere. These dense bodies found in the uranyl- and lead-stained materials are very similar to the 'presynaptic dense projections', which are arranged in a hexagonal pattern on the presynaptic membrane of the mammalian synapses stained with PTAS, 6 or bismuth1,17. The presynaptic-to-presynaptic symmetrical synapses are exclusively found between the spiral fiber terminals in the central core of the axon cap and never observed in the peripheral region of the axon cap where the common axo-dendritic synapses between the afferent nerve fibers and the cap dendrites are usual (not shown). Electrophysiologically, Furukawa and Furshpan 4 found two types of postsynaptic inhibition which act upon the Mauthner cell in association with the activity of the cell axon collaterals. One is the common chemical type of inhibition, in which changes in the permeability of the postsynaptic membrane are brought about, probably, by a transmitter substance. The other is the special electrical type of inhibition in which the excitability of the Mauthner cell is lowered by the extracellular positive potential referred to as the 'extrinsic hyperpolarizing potential' (EHP), in the vicinity of the initial axon segment of the Mauthner cell. It may be reasonably supposed, therefore, that the symmetrical axo-axonic synapses shown in this study, exclusively in the central core of the axon cap, participate
Brain Research, 23 (1970) 255-258
258
SHORT COMMUNICATIONS
in the production of the EHP around the initial axon segment, although the definite mechanism can not be explained as yet. The helpful advice and encouragement of Prof. Heiya Yamada and Prof. Sanford L. Palay are gratefully acknowledged. Department of Anatomy, Tokyo Medical and Dental University, School of Medicine, Bunkyo-Ku, Tokyo (Japan)
K
KOHNO
1 AKERT, K., MOOR, H., PFENN1NGER, K., AND SANDRI, C., Contributions of new impregnation methods and freeze etching to the problems of synaptic fine structure. In K. AKERT AND P. G. WASER (Eds.), Mechanisms of Synaptie Transmission, Progress in Brain Research, Vol, 31, Elsevier, Amsterdam, 1969, pp. 223-240. 2 BARTELMEZ,G. W., Mauthner cell and the nucleus motorius tegmenti, J. eomp. Neurot., 25 (I 915) 87-128. 3 BODIAN, D., The structure of the vertebrate synapse. A study of the axon endings on Mauthner's cell and neighbouring centers in the goldfish, J. comp. NeuroL, 68 (1938) 117-145. 4 FURUKAWA, T., AND FURSm'AN, E. J., Two inhibitory mechanisms in the Mauthner neurons of goldfish, J. Neurophysiol., 26 (1963) 140-176. 5 GRAY, E. G., Electron microscopy of presynaptic organelles of the spinal cord, J. Anat. (Lond.), 97 (1963) 101-106. 6 GRAY, E. G., Electron microscopy of excitatory and inhibitory synapses: a brief review. In K. AKERT AND P. G. WASER (Eds.), Mechanisms of Synaptic Transmission, Progress in Brahl Research. Vol. 31, Elsevier, Amsterdam, 1969, pp. 141-155. 7 HAMA, K., Some observations on the fine structure of the giant fibers of the crayfishes (Cambarus virilus and Cambarus clarkii) with special reference to the submicroscopic organization of the synapses, Anat. Rec., 141 (1961) 275-294. 8 HINDS, J. W., Reciprocal and serial dendro-dendritic synapses in the glomerular layer of the rat olfactory bulb, Brain Reseaeh, 17 (1970) 530-534. 9 HIRATA, Y., Some observations on the fine structure of the synapses in the olfactory bulb of the mouse, with particular reference to the atypical synaptic configurations, Arch. histol, jap., 24 (1964) 293-302. 10 KOHNO, K., Neurotubules contained within the dendrite and axon of Purkinje cell of frog, Bull. Tokyo Med. Dent. Univ., 11 (1964) 411~142. 11 MARTIN, R., The structural organization of the intracerebral giant fiber system of cephalopods. The chiasma of the first order giant axons, Z. Zellforseh., 97 (t969) 50-68. 12 MILLONIG, G., A modified procedure for lead staining of thin sections, J. biophys, biochem. Cytol.. 11 (1961) 736-739. 13 PALAY, So L., The structural basis for neural action. In M. A. B. BRAZIER (Ed.), Brain Fum'tion, Vol. 2, RNA and Brain Function; Memory and Learning, Univ. of Calif. Press, Berkeley, 1964, pp. 69-108. 14 PALAY, S. L., SOTELO, C., PETERS, A., AND ORKAND, P. M., The axon hillock and the initial segment, J. Cell Biol., 38 (1968) 193-201. 15 PETERS, A., PALAV, S. L., AND WEaSTER, H. DE F., The Fine Structure of the Nervous ~vstem: Tire Cells and Their Processes, Hoeber, New York, 1970, p. 55 and p. 158. 16 PETERS,A., PROSKAUER,C. C., AND KAISERMAN-ABRAMOr,I. R., The small pyramidal neuron of the rat cerebral cortex: the axon hillock and initial segment, J. Cell Biol., 39 (1968) 604-6i9. 17 PFENN1NGER, K., SANDRI, C., AKERT, K., AND EUGSTER, C. H., Contribution to the problem of structural organization of the presynaptic area, Brain Research, 12 (1969) 10-18. 18 RETZLAFF, E., Neurohistological basis for the functioning of paired half centers, J. comp. Neurol., 101 (1954) 407-447. 19 ROaERTSON, J. D., BODENHEIMER, T. S., AND STAGE, D. E., The ultrastructure of Mauthner celt synapses and nodes in goldfish brains, J. Cell Biol., 19 (1963) 159-199. 20 SZENTA,GOTr~AI,J., Synaptic structure and the concept of presynaptic inhibition. In C. yon EtJLER, S. SKOGLtrND, AND U. S6DERBERG (Eds.), Structure and Function of Inhibitory Neuronal Mechanisms, Pergamon, Oxford, 1968, pp. 15-31. (Accepted July 14th, 1970)
Brain Research, 23 (1970) 255-258
255
Symmetrical axo-axonic synapses in the axon cap of the goldfish Mauthner cell Axo-axonic synapses between the terminals containing the synaptic vesicles on both sides have been frequently reported in the central nervous system of the vertebrates6, 20. In each case, the direction of transmission could usually be judged by the accumulation of the vesicles on either side of the synaptic membranes. On the other hand, in some invertebrates v,lI bidirectional synapses with symmetrical accumulation of the vesicles on both sides of the synaptic membranes were observed between the parallel-arranged or crossing giant nerve fibers. The present communication shows similar symmetrical and more differentiated presynaptic-to-presynaptic synapses found in the axon cap region of the Mauthner cell of the goldfish medulla. An electron micrograph of the axon cap was previously published by Robertson et al. ag, but it was low in magnification and insufficient to analyze the finer structure of the synapses. Common goldfish (Carassius auratus), 10-15 cm in length, treated according to the method of Robertson et alfl 9, were perfused through the aorta with a mixture of I ~ paraformaldehyde and 1.25 ~ glutaraldehyde in phosphate buffer (pH 7.4) containing CaCI~ (ref. 14). Tissue was removed and, after postfixation in 2 ~,, osmium tetroxide, was dehydrated in ethanol and embedded in Epon. The position of the axon cap in the embedded block was ascertained by tracing the large myelinated axon of the Mauthner cell in the successive thick sections for light microscopy. Thin sections were double-stained with uranyl acetate and lead acetatelL It is known from the light microscopic studies~,a, is that the 'axon cap' is a peculiar synaptic apparatus which spherically surrounds the axon hillock and the initial axon segment of the Mauthner cell, in association with the external boundary of the 'cap cells', and into which numerous nerve fibers enter and lose their myelin sheaths at the boundary layer; also, 'cap dendrites' from the cell soma penetrate into this region, Specifically, Bodian 3 found that the central core of the axon cap immediately investing the initial axon segment is of a different nature than that of the peripheral portion of the axon cap. The central core consists of a dense conglomeration of extremely fine terminal branches of the 'spiral fibers' which come to the axon cap, enveloping the Mauthner axon from the supracommissural bundles of the fasciculus longitudinalis medialis. In electron micrographs (Fig. 1), the central core (CEC) is readily recognized as a compact aggregation of small clear nervous profiles (A) around the initial axon segment (INS). These profiles represent the intricate terminal branches of the spiral fibers. The terminal branches contain numerous vesicles and a few mitochondria. The vesicles, 30-60 nm in diameter, are spherical and clear in the center. Some large vesicles, about 80 nm in diameter, include a dense core in the center. Neurofilaments, microtubules and tubular structures with irregular swellings are also contained within the terminals. Slender processes (P), often forming bundles, penetrate into the mass of the terminal branches. The cytoplasm is filled with numerous longitudinally arranged filaments and microtubules. Between them, free ribosomes and cisternae Brain Research, 23 (1970) 255-258
256
SHORI (OMMUNI('AIlONS
Fig. I. -File central core (CEC) of lhe axon cap surrounding the initial axon segmenl (INS) of goMfish M a u t h n e r cell. Arrows indicate symmetrical axo-axonic synapses between axon terminals (A) containing vesicles and m i t o : h o n d r i a . Three adjacent ierminals (A,, Ae, A:0 are serially connected by lwo symmetrical synapses. Bundles of slender cap cell processes (P) are lilied with n u m e r o u s tilaments, microtubules and cisternae with ribosomes. Fascicles of microlubules (F) and a dense undercoaied a x o l e m m a (U) are seen in the cross section of the INS. 21,000. h~seri, Symmelrical apposition of two presynaptic acli~e zones x~itl~ clusters of vesicles anti wilh 4 opposite pairs of dense projections (arrows), 60,000.
Brain Research, 23 (1970) 255-258
SHORT COMMUNICATIONS
257
with their associated ribosomes are dispersed. These are the elongated processes extending from the cap cells enclosing the axon cap. The initial axon segment (INS) of the Mauthner cell, passing through the center of the axon cap, contains numerous evenly dispersed neurofilaments and fascicles of the microtubules which are connected in series by short arms (F), and the axoplasm is surrounded by a plasma membrane whose axoplasmic surface is coated by a thin layer of dense material (U). These characteristic structures have been also observed in the initial axon segment of the multipolar neurons in frog 1° and rat brains la-16. The symmetrical axo-axonic synapses are frequently found in the central core of the axon cap (arrows), and serial occurrence of the synapses is often observed between the several adjacent terminals (A1, A2, Aa). In the synaptic region, each cluster of the vesicles contained within the two adjacent terminals is symmetrically aggregated toward both cytoplasmic surfaces of the apposed membranes. The aggregation of the vesicles forms a typical presynaptic active zone on some small region of the apposed membranes. The synaptic cleft is about 10 nm wide, and the active zones facing each other usually have a width of 0.2-0.6/~m. In this synapse two similar presynaptic active zones are always directly opposite. This symmetrical synapse is different from the reciprocal synapse of the bulbus olfactorius8,9,15, where two presynaptic active zones with an accumulation of vesicles are not directly opposite but appear separately in two neighboring places of the apposed membranes. Small dense bodies project into a cluster of the vesicles from the active zones of the symmetrical synapses (arrows in insert of Fig. 1). It is often clearly observed in the sections cut perpendicular to the synaptic membrane that 2-4 of the dense bodies are arranged on an active zone with an interval of 100-150 nm from center to center, and they are often situated opposite each other on both sides of the synaptic cleft. At these places the synaptic cleft appears denser than elsewhere. These dense bodies found in the uranyl- and lead-stained materials are very similar to the 'presynaptic dense projections', which are arranged in a hexagonal pattern on the presynaptic membrane of the mammalian synapses stained with PTAS, 6 or bismuth1,17. The presynaptic-to-presynaptic symmetrical synapses are exclusively found between the spiral fiber terminals in the central core of the axon cap and never observed in the peripheral region of the axon cap where the common axo-dendritic synapses between the afferent nerve fibers and the cap dendrites are usual (not shown). Electrophysiologically, Furukawa and Furshpan 4 found two types of postsynaptic inhibition which act upon the Mauthner cell in association with the activity of the cell axon collaterals. One is the common chemical type of inhibition, in which changes in the permeability of the postsynaptic membrane are brought about, probably, by a transmitter substance. The other is the special electrical type of inhibition in which the excitability of the Mauthner cell is lowered by the extracellular positive potential referred to as the 'extrinsic hyperpolarizing potential' (EHP), in the vicinity of the initial axon segment of the Mauthner cell. It may be reasonably supposed, therefore, that the symmetrical axo-axonic synapses shown in this study, exclusively in the central core of the axon cap, participate
Brain Research, 23 (1970) 255-258
258
SHORT COMMUNICATIONS
in the production of the EHP around the initial axon segment, although the definite mechanism can not be explained as yet. The helpful advice and encouragement of Prof. Heiya Yamada and Prof. Sanford L. Palay are gratefully acknowledged. Department of Anatomy, Tokyo Medical and Dental University, School of Medicine, Bunkyo-Ku, Tokyo (Japan)
K
KOHNO
1 AKERT, K., MOOR, H., PFENN1NGER, K., AND SANDRI, C., Contributions of new impregnation methods and freeze etching to the problems of synaptic fine structure. In K. AKERT AND P. G. WASER (Eds.), Mechanisms of Synaptie Transmission, Progress in Brain Research, Vol, 31, Elsevier, Amsterdam, 1969, pp. 223-240. 2 BARTELMEZ,G. W., Mauthner cell and the nucleus motorius tegmenti, J. eomp. Neurot., 25 (I 915) 87-128. 3 BODIAN, D., The structure of the vertebrate synapse. A study of the axon endings on Mauthner's cell and neighbouring centers in the goldfish, J. comp. NeuroL, 68 (1938) 117-145. 4 FURUKAWA, T., AND FURSm'AN, E. J., Two inhibitory mechanisms in the Mauthner neurons of goldfish, J. Neurophysiol., 26 (1963) 140-176. 5 GRAY, E. G., Electron microscopy of presynaptic organelles of the spinal cord, J. Anat. (Lond.), 97 (1963) 101-106. 6 GRAY, E. G., Electron microscopy of excitatory and inhibitory synapses: a brief review. In K. AKERT AND P. G. WASER (Eds.), Mechanisms of Synaptic Transmission, Progress in Brahl Research. Vol. 31, Elsevier, Amsterdam, 1969, pp. 141-155. 7 HAMA, K., Some observations on the fine structure of the giant fibers of the crayfishes (Cambarus virilus and Cambarus clarkii) with special reference to the submicroscopic organization of the synapses, Anat. Rec., 141 (1961) 275-294. 8 HINDS, J. W., Reciprocal and serial dendro-dendritic synapses in the glomerular layer of the rat olfactory bulb, Brain Reseaeh, 17 (1970) 530-534. 9 HIRATA, Y., Some observations on the fine structure of the synapses in the olfactory bulb of the mouse, with particular reference to the atypical synaptic configurations, Arch. histol, jap., 24 (1964) 293-302. 10 KOHNO, K., Neurotubules contained within the dendrite and axon of Purkinje cell of frog, Bull. Tokyo Med. Dent. Univ., 11 (1964) 411~142. 11 MARTIN, R., The structural organization of the intracerebral giant fiber system of cephalopods. The chiasma of the first order giant axons, Z. Zellforseh., 97 (t969) 50-68. 12 MILLONIG, G., A modified procedure for lead staining of thin sections, J. biophys, biochem. Cytol.. 11 (1961) 736-739. 13 PALAY, So L., The structural basis for neural action. In M. A. B. BRAZIER (Ed.), Brain Fum'tion, Vol. 2, RNA and Brain Function; Memory and Learning, Univ. of Calif. Press, Berkeley, 1964, pp. 69-108. 14 PALAY, S. L., SOTELO, C., PETERS, A., AND ORKAND, P. M., The axon hillock and the initial segment, J. Cell Biol., 38 (1968) 193-201. 15 PETERS, A., PALAV, S. L., AND WEaSTER, H. DE F., The Fine Structure of the Nervous ~vstem: Tire Cells and Their Processes, Hoeber, New York, 1970, p. 55 and p. 158. 16 PETERS,A., PROSKAUER,C. C., AND KAISERMAN-ABRAMOr,I. R., The small pyramidal neuron of the rat cerebral cortex: the axon hillock and initial segment, J. Cell Biol., 39 (1968) 604-6i9. 17 PFENN1NGER, K., SANDRI, C., AKERT, K., AND EUGSTER, C. H., Contribution to the problem of structural organization of the presynaptic area, Brain Research, 12 (1969) 10-18. 18 RETZLAFF, E., Neurohistological basis for the functioning of paired half centers, J. comp. Neurol., 101 (1954) 407-447. 19 ROaERTSON, J. D., BODENHEIMER, T. S., AND STAGE, D. E., The ultrastructure of Mauthner celt synapses and nodes in goldfish brains, J. Cell Biol., 19 (1963) 159-199. 20 SZENTA,GOTr~AI,J., Synaptic structure and the concept of presynaptic inhibition. In C. yon EtJLER, S. SKOGLtrND, AND U. S6DERBERG (Eds.), Structure and Function of Inhibitory Neuronal Mechanisms, Pergamon, Oxford, 1968, pp. 15-31. (Accepted July 14th, 1970)
Brain Research, 23 (1970) 255-258