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Synaptic organization in the pacemaker nucleus of a medium-frequency weakly electric fish,Eigenmannia sp
Brain Research, 237 (1982) 267-281
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Elsevier Biomedical Press
Research Reports
S Y N A P T I C O R G A N I Z A T I O N I N T H E P A C E M A K E R N U C L E U S OF A M E D I U M - F R E Q U E N C Y W E A K L Y E L E C T R I C FISH, E I G E N M A N N I A SP.
K. ELEKES* and T. SZABO Laboratoire de Physiologie Nerveuse, Departement de Neurophysiologie Sensorielle, CNRS, 91190 Gif sur Yvette (France) and Biological Research Institute of the Hungarian Academy o f Sciences, Tihany (Hungary)
(Accepted September 17th, 1981) Key words: synaptic contacts - - pacemaker nucleus - - weakly electric fish - - electro n microscopy
SUMMARY The medullary c o m m a n d nucleus (MCN) of the medium-frequency weakly electric fish, Eigenmannia sp., contains two types of neurones, namely large and small cells, which are embedded in a neuropile of large and small myelinated fibers. Using serial semi-thin and ultra-thin sectioning, combined with H R P labelling it has been established that both cell types possess rich dendritic arborization and large myelinated axons. Only the axons of the large cells leave the nucleus and these constitute the unique output of the MCN. Axon branching has been observed only in the axons of small cells and their collaterals show an exclusively intranuclear course. Two types of synaptic terminals have been found on large as well as on small cells: (1) large club endings forming both gap (electrotonic)junctions and polarized chemical synapses, which often appear at the same junction constituting morphologically mixed synapses; and (2) small bouton-like terminals forming exclusively chemical synaptic contacts. N o differences between the two neuron types could be detected with respect to the arrangement of the synaptic contacts: club endings and small bouton-like terminals synapse on dendritic processes as well as on perikarya, while the unmyelinated initial segments were always found to be free of synaptic contacts. Large and small cells were found to be simultaneously connected by the same club ending or small bouton-like terminal: in the case of club endings by means of gap junctions and chemical synapses, whereas in the case of boutons by chemical synapses only. Club endings sometimes form gap junctions with each other. The possible role of these * Present address: The Biological Research Institute of the Hungarian Academy of Sciences, H 8237 Tihany, Hungary. 00~6-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
268 unusual synaptic connections in local synchronization is suggested. Club endings originate from the large axons of small cells, while small bouton-like terminals originate from the fine myelinated fibers of extranuclear origin. In Eigenmannia, small cells, being connected to large cells as well as to each other by axo-somatic and axodendritic synapses, can be considered as the pacemaker cells of the MCN whereas large cells are relay cells. Small bouton-like terminals may convey exogeneous impulses towards the MCN exerting modulatory effects at both pacemaker and relay cell levels. The greater variety of ultrastructural correlates established in the MCN of Eigenmannia, in comparison with Sternarchus 5 (see also ref. 16), suggests increased modulation possibilities in the former fish's EOD behaviour.
INTRODUCTION The electric organ in all species of weakly electric fish discharges electric impulses of which the frequency is characteristic for different species, in many species, and in particular in gymnotids, the rhythmic discharge depends on a specific nucleus, the medullary command nucleus (MCN), situated in the rhombencephalic reticular formation. Several investigationsl0,1~, 1~ showed that the spinal electromotor neurons innervating the electric organ are directly excited with the MCN. In agreement with earlier data concerning the morphological organization of the MCN in two gymnotid species, Sternarchus and Eigenmannia r2, it has recently been shown by H R P injection into the spinal cord that only large (relay) cells of the MCN send their axon to the spinal electromotor neurons6,1L Furthermore, it has also been found following semithin serial sectioning in Sternarchus albifrons 5 that large (relay) cells failed to send off collaterals within the nucleus, while the axons of small (pacemaker) cells, which exhibit rich branching patterns, never leave the MCN. The ultrastructure of the MCN was first investigated in gymnotids by Bennett et al. 1 in a medium-frequency pulse species Steatogenys sp. and in a medium-frequency wave species Sternopygus sp. Club endings and small boutons of unidentified origin were localized on large (relay) as well as small (pacemaker) cells. In Sternopygus, electrotonic (gap)junctions between pacemaker cell dendrites were also seen. Recently detailed microscopic investigations were performed on the MCN of the high frequency Sternarchus albifronsS, 1~. Both small (pacemaker) and large (relay) cells are richly provided with different kinds of synaptic terminals: club endings with electrotonic junctions and small boutons with polarised chemical synapses. Elekes and Szabo 5 have furnished additional data as to the origin of these two types of synaptic terminals: large club endings originate from thick myelinated axon collaterals of the pacemaker cells, while small bouton-like terminals take their origin from thin myelinated fibers entering the nucleus from neighboring medullary areas. On the basis of these findings, the synaptic connections between the neuronal components of Sternarchus MCN have been established. The aim of the present work was to perform a detailed ultrastructural analysis on the MCN ofEigenmannia sp., a medium-frequency wave species gymnotid. Such an analysis would make it possible to compare the synaptology of the MCN in two
269 weakly electric fish with medium (Eigenmannia) and high frequency (Sternarchus) EOD discharges. This comparison is of particular interest since Eigenmanniaexhibits a more complex electrical behaviour than Sternarchus during social encountersa,S, la. MATERIALSAND METHODS Adult specimens, 8-12 cm long, of the weakly electric fish Eigenmanniasp. were used. After anesthesia with tricaine methanesulfonate (MS 222, Sandoz, 50 rag/l), the animals were perfused through the heart with a solution containing 2 ~ glutaraldehyde and 2 ~ formaldehyde buffered with 0.1 M phosphate at pH 7.2, for 45-60 min. The brains were thereafter immersed in the same solution overnight at 4 °C. A small part containing the MCN was dissected out of the medulla, washed in phosphate blaffer for 1 h and post-fixed in 2 ~ OsO4 buffered with 0.1 M phosphate for 2 h at 4 °C. After dehydration, tissue samples were embedded in araldite and cut in serial semi-thin and ultra-thin sections on an LKB III ultrotome. Semi-thin sections were stained with 1 ~o toluidine blue while ultra-thin sections were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Siemens Elmiskop 102. For HRP labelling, a 50~o solution of the enzyme was injected through a micropipette by pressure into the caudal part of the spinal cord. After a survival time of 10-14 days the animals were perfused and fixed as described above. For light microscopical investigations, the brains were cut on a Vibratome. The sections were treated with 3,3'-DAB for 20 min and the HRP reaction product was developed with H202. The mounted sections were lightly counterstained by cresyl violet. For electron microscopy, 60-/~m sections, cut on Vibratome and treated with DAB and H202, were postfixed with 2 ~ OsO4 for 60 min, then dehydrated and embedded as described above. Ultra-thin sections either without or following double staining with uranyl acetate and lead citrate were observed in a TESLA BS 500 electron microscope. RESULTS The MCN of the weakly electric fish, Eigenmanniasp., is situated in the reticular formation of the medulla just below the Mauthner cell axons. In semi-thin transverse sections it shows a heart-like form; it is separated from the neighbouring medullary area by large bundles of myelinated axons showing intense toluidine blue staining (Fig. 1). Ventrally, this bordering layer is constituted of extremely thick myelin sheathed axons, while dorsally the limit is formed by thin myelinated fibers (Fig. 1). The nucleus itself consists of a network of large and thin myelinated fibers in which two types of nerve cells are embedded: large cells with a diameter of 65-70/~na and small cells with a diameter of 30-35/,m (Fig. 1). The two cell types are more or less intermingled, but rather more large cells were found in the lateral part of the nucleus. Capillaries are present throughout in the MCN forming a dense vascularization. Large cells, with centrally located spherical nuclei, possess an initial segment about 20/zm long. The myelinated axon never gives off collaterals either within the
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Fig. 1. Semi-thin section from a MCN of Eigenmannia. Large (arrows) and small cells (double arrows) are embedded in a neuropile composed by myelinated axons. Region of the bordering layer of myelinated fibers through which large cell axons leave the nucleus is marked by arrowheads; see also Fig. 3. Scale: 50/~m.
271 nucleus or outside the nucleus in the neighboring medullary area. Concordantly, after H R P injection we failed to observe labelled axons sending off collaterals either in semithin or ultra-thin sections. However, HRP-labelled myelinated axon profiles were found in the ventral bordering layer of the M C N mentioned above (Fig. 3). These correspond to the large cell axons which course towards the spinal electromotor neurons. Ten to 14 days after spinal injection, only large cells were found to be labelled in the M C N (see ref. 6). While small cells were always free of reaction product (Fig. 4). H R P labelling also revealed a large number of dendritic arborization of the large cells, appearing in the light microscope as a fine network throughout the nucleus. Electron microscopical analysis reveals that the initial axon segments of the large cells is practically free of synaptic terminals but covered by glia processes (Fig. 2). In contrast, the cell body of the large cells is partly covered with club endings and small bouton-like terminals, partly with a multi-layered sheath of glial processes. On large cells, club endings and bouton-like terminals were also seen on primary dendrites as well as at some distance from the cell body (Figs. 5 and 6). HRP-labelled small dendritic processes made synaptic contact mainly with bouton-like terminals; club endings were less frequently seen at this level (Figs. 5 and 6). A considerable part of the dendritic surface is covered by glial processes (Fig. 6). Besides H R P labelling, identification of large cell dendrites was possible on the basis of glycogen content: large cells appear practically free of glycogen granules whereas small cells have always a large amount of glycogen (Figs. 10a, 12 and 17). Apart from their smaller size, the morphology of the small cells differs from the large ones in that that the initial segments of their axons are about twice as long (40 #m) and that their axons send off several collaterals, not far from the beginning of the myelin sheath (Fig. 7). The small cells have several short primary dendrites branching almost immediately at their origin (Fig. 9). The ultrastructural aspects of the small cells differ from those of the large ceils. A large amount of complex glycogen granules is found in their perikarya, mainly concentrated in the peripheral cytoplasm (Figs. 9 and 12). Consequently, glycogen granules are also seen in the dendritic processes, as well as in the axoplasm of the initial segment where they were uniformly distributed. In contrast, glycogen granules Fig. 2. Electron micrograph of the initial segment (IS) of a large cell axon. No synaptic terminals, but densely packed glial processes can be seen on its surface. C, capillary. Scale: 2/zm. Fig. 3. HRP-labelled (arrows) large cell axon (A) surrounded by thickly myelinated axons separating the MCN from the neighboring medullary areas. Unstained section. Scale: 2/~m. Fig. 4. Light microscopic view of large cells labelled retrogradely by HRP injection into the spinal cord. One cell only weakly labelled (arrow). Note the rich dendritic arborization of the two intensely labelled cells. Dark-field micrograph. Scale: 30/tm. Fig. 5. A club ending (CE) forms chemical synapses (arrowheads) on a HRP-labelled dendritic process of a large cell (RD). Simple glycogengranules and rER elements (asterisk) are present in the dendrite. Scale: 0.5/~m. Fig. 6. HRP-labelled dendritic process (RD) with 3 small bouton-like terminals (asterisk) forming chemical synapses. Note the presence of simple glycogen granules in the dendrite. Arrowhead, rER elements. G, glia processes. Scale: 0.5 ktm.
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Fig. 7. Light micrographs of a small cell seen in serial semi-thin sections, a: note the long initial axon segment (IS). Arrow points to the axon profile which appears as a typical collateral of the myelinated axon in Fig. 7b. b: arrow points to the branching point of the myelinated axon. P, perikaryon; C, capillary. Scale: 10/~rn. Fig. 8. Low power electron micrograph of the initial segment (IS) of a small cell axon. No terminals are present on the IS membrane which is completely covered by glial processes. Arrows indicate glycogen in the myelinated part of the axon (A). C, capillary. Scale: 2 ibm. Inset: high-power electron microscopic detail of the surface of small cell initial segment (IS). Note medium electron-dense material in the intercellular cleft and high electron-dense material attached to the inner surface of the 1S membrane (arrowheads). Scale: 0.1 /tm.
273 in the myelinated axoplasm were often clustered just below the myelin sheath (Fig. 8). The distribution o f synaptic terminals on the different parts o f the small cells, i.e. perikaryon, initial axon segment and dendrites was f o u n d to be similar to that observed on the large cells. The long initial axon segment is completely free o f synaptic terminals and connections and it is covered by a multilayered glial sheath. At lowpower magnification a strikingly increased electron density o f the initial axon m e m b r a n e is seen. At higher magnification a finely granulated electron-dense material was f o u n d in the intracellular cleft between the axolemma and the glial m e m b r a n e ; a layer o f dense material o f irregular thickness and o f granulated structure is attached to the inner surface o f the initial axon segment m e m b r a n e (Fig. 8 and inset). The perikaryon o f the small cells bears several bouton-like terminals and a few club endings f r o m thick myelinated axons. However, a large part o f the cell b o d y is covered with multilayered glial sheath. In contrast, the dendritic processes receive
Fig. 9. Three club endings (arrows) synapse on a bifurcating primary dendrite (D) of a small cell (PC). One of the club endings (filled asterisk) simultaneously connects the small cell dendrite (D) and a large cell dendritic process (open asterisk). C, capillary. Scale: 2/~m. Fig. 10. Synaptic terminals on small cell dendrites, a: large club ending (CE) and bouton-like terminals (arrows) on a small cell dendrite (D). Several large granular vesicles are present in neighboring bouton (asterisk). M, mitochondria. Scale: 0.5 #m. b: enlarged detail of Fig. 10a showing two gap junctions (arrows) of the club ending and a polarized chemical synapse of a bouton (arrowheads) on the dendrite (D). Several large granular vesicles are present in the neighbouring bouton (asterisk). M, mitochondria. Scale: 0.2/~m. Inset: high-power magnification of the gap junction shown in Fig. 10b. Scale: 0.05/~m.
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Fig. 11. Unusual nodal synaptic ending of a large bifurcating myelinated axon (A): a thin axoplasmic preterminal protrusion (arrow) contacts with a typical terminal (filled asterisk) filled with mitochondria. The terminal forms a gap junction (arrowhead) with a small cell dendrite (D). Open asterisk: glial processes. Scale: 1 itm. Fig. 12. A small (PC) and a large (RC) cell are connected by two en passant terminals (asterisks) of the same large myelinated axon (A). N, nucleus; C, capillary. Scale: 2/J,m. Upper inset: in the course of serial sectioning, one of the terminals (open asterisk) appears as a typical club ending forming a gap junction (arrow) with the large cell (RC). Scale: 1/~m. Bottom inset: high-power magnification of the gap junction indicated by arrow in the upper inset. Scale: 0.05/zm.
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Fig. 13. A club ending (CE) simultaneously connecting a large cell dendrite (open asterisk) with a chemical synapse (arrow) and a small cell perikaryon (filled asterisk) with a gap junction (double arrow). Note the spine-like postsynaptic profile (arrowhead) of the large cell dendrite at the chemical synapse. G, glial processes. Scale: 0.5 #m. Inset: high-power magnification of the gap junction indicated by double arrow in Fig. 13. Scale: 0.05/~m. Fig. 14. Electron micrograph of a club ending (CE) forming a mixed synapse on a large cell dendrite. The polarized chemical synapse is indicated by arrow, the gap junction by double arrow and a desmosome-like attachment by an arrowhead. Scale: 0.2 #m. Fig. 15. Gap junction (between arrows) between two club endings (CE). M, mitochondria. Scale: 0.25 /tm. Figs. 16 and 17. Ultrastructure and origin of small bouton-like terminals. Fig. 16. Fine myelinated fiber (A) synapses with a bouton-like terminal perikaryon (asterisk). C, capillary. Scale: 2 #m. Fig. 17. Small bouton-like terminal (asterisk) connect simultaneously by synapses the dendrite of a large cell (RD) and that of a small (PD) cell. Note granules (arrowheads) in the small cell dendrite and its absence in the mitochondria; G, glial processes. Scale: 0.25/~m.
(arrow) on a small cell means of two chemical the presence of glycogen large cell dendrite. M,
276 many synaptic endings: primary dendrites are covered by club endings and small bouton-like terminals (Fig. 9); smaller dendritic processes form synaptic contacts with several bouton-like terminals and few club endings (Figs. 10, 11 and 17).
Ultrastructure of synaptic terminals and synaptic connections Club endings occurring on both large and small cells take their origin from large myelinated axons (Figs. 9, 10, 11, 12 and 14). They form partly en passant terminals (Figs. 11 and 12). A new form ofen passant terminals was found (Fig. 11) consisting ol a thin (about 0.5/tm in diameter) axoplasmic 'neck' protruding from a node of Ranvier which, after 1-2/ma course, forms a typical club ending filled with mitochondria and surrounded by glial processes. Club endings were mainly characterized by two cellular compartments, namely mitochondria and small (40-50 /~m) clear synaptic vesicles (Figs. 5, 13 and 14). These latter were mostly found clustered in the lateral parts of the terminals. Most of the club endings also contained neurotubules and a few glycogen granules. Club endings with HRP labelling were never found. Club endings formed 3 typical junctional complexes both with large and small cells: gap (electrotonic) junctions, polarized chemical synapses and desmosome-like attachments (Figs. 5, 10, 13, 14 and 15). Polarized chemical synapses in a club ending could be distinguished easily from desmosome-like attachments. The former are characterized by an asymmetric clustering of clear synaptic vesicles, typical postsynaptic paramembranous dense apposition and presynaptic dense projections, whereas the latter show exclusively symmetrical apposition of dense material (Fig. 14). Gap junctions and polarized chemical synapses occur close to each other, having the same postsynaptic membrane and thus form typical mixed synapses (Fig. 14). Both types of contacts were also found in the same plane of section of a club ending, but making synaptic connections with different postsynaptic elements (Fig. 13). Such club endings were seen to synapse on a small cell dendrite with a gap junction and simultaneously with a polarized chemical junction on a large cell dendrite (Figs. 9 and 13). Similar connections were also observed between small and large cell perikarya. Furthermore, small and large cell dendrites or perikarya can also be connected by different club endings or en passant terminals of the same large myelinated axon (Fig. 12). This is the first time that an electrotonic (gap)junction between club endings has been described (Fig. 15), in which either one or both endings simultaneously contact typical postsynaptic element(s), i.e. dendritic profile(s). Small bouton-like terminals forming synaptic contacts both on large and small cells originate from thin myelinated axons entering the nucleus from neighboring medullary areas (Fig. 16). Thin myelinated axons show an electron transparent axoplasm containing only few mitochondria (even in the preterminal segment) and glycogen granules. Several boutons ending on large or small cells have been seen to originate from the same thin myelinated fiber forming terminals or en passant contacts. The boutons are small, with a diameter of about 0.5-1 #m ; besides many small clear spheroid-shaped vesicles of 40-60 nm in diameter, they also contain a few small
277 mitochondria and glycogen granules (Fig. 17). The clear vesicles represent a homogeneous population; a few large (85 nm) granular vesicles were sometimes observed in these boutons (Fig. 17) or in the preterminal myelinated segment of the thin fiber, although in a few cases, the boutons also contained many large, granular vesicles (Fig. 10b). Flattened, clear vesicles were never seen in boutons. Small bouton-like terminals of thin myelinated fibers form only chemical synapses (Figs. 7, 10 and 17). They never synapse on each other but may connect postsynaptic profiles of different origin (Fig. 17). DISCUSSION Our present findings concerning the MCN of Eigenmannia, a wave species, weakly electric fish with medium-frequency EOD, revealed that its general organization is essentially similar to that of Sternarchus albifrons, another wave species electric fish with high-frequency EOD 5. In both, two different neuron types constitute the MCN; these are embedded in a neuropile constituted by large and thin myelinated fibers. Large and small neurons as well as large myelinated fibers have earlier been described in the MCN of several weakly electric fish species1,12; however, the presence of thin myelinated fibers of extranuclear origin was described only recently by Elekes and Szabo 5 in Sternarchus. From the present data, the latter also appear to be a basic neuronal component of the MCN in Eigenmannia, representing the only afferent connections of the nucleus. Concerning the neuronal morphology within the MCN of Eigenmannia, large cells differ from the small ones in that that their axon leaves the nucleus without giving off collaterals, whereas small cell axons exhibit branching patterns; the branches have an exclusively intranuclear course. That only large cell axons project to the spinal cord was also proved by retrograde HRP labelling: following spinal injection of HRP only large cells were labelled. A similar conclusion has been drawn in several other gymnotid species, using Bodian impregnation methods 12, and recently HRP labelling6, as. Dendritic arborization of the large cells were also revealed by HRP labelling as well as by electron microscopic analysis. Thus, in Eigenmannia both neuronal cell types possess a rather large dendritic tree, in contrast to Sternarchus in which only small cells were found to have long arborized dendrites whereas large cells have only short spinelike dendritic processesL The more extensive dendritic development of large cells in Eigenmannia may partly explain the differences between Eigenmannia and Sternarchus concerning the arrangement ofsynaptic terminals on large cells. In Eigenmannia, club endings are also present on dendritic processes, while in Sternarchus, they are concentrated on the perikaryon5. In contrast, we failed to find club endings on the initial segment of large cell axons in Eigenmannia, whereas in Sternarchus the initial segments were completely and exclusively covered by them. This suggests that the coupling between pacemaker and relay cells may be much tighter in Sternarchus, which has a discharge frequency of 1000 c/s than in Eigenmannia with only 300 c/s. Elekes and Szabo 5 proposed that this particular synaptic arrangement perhaps represents the morphological correlate of
278 the reinforcing function of pacemaker impulses in view of the exact timing of large cell activity in the case of high frequency discharge. This explanation also holds for low frequency fish such as Hypopomus in which the initial segment of the large cells is also free from club endings 4. The arrangement of synaptic contacts on the small cells is different in Sternarchus and Eigenmannia in spite of a relatively rich dendritic arborization in both species: club endings and small bouton-like terminals occur on dendrites in Eigenmannia, whereas only small boutons are present on these in
Sternarchus 5. In Eigenmannia, large club endings form gap (electrotonic) junctions and chemical synapses on both large and small cells; they often occur together constituting so-called mixed synapses. Such synaptic contacts were also observed in the M C N of the medium-frequency wave species Sternopygus and in the variable-frequency pulse species Steaogenys as well as in different parts of the electrosensory and electromotor pathway of gymnotid and mormyrid CNS 1,2,9,1a,14. In contrast, we, as well as Tokunaga et al. 15, have failed to find mixed synapses in Sternarchus MCN, where only gap junctions occur on both large and small cellsL Whether these mixed synapses in Eigenmannia represent the morphological correlates of a functionally dual transmission cannot be stated without precise electrophysiological experiments. Recently, the existence of dual transmission at mixed synapses in goldfish Mauthner cell has been proven by cobalt injections at the postsynaptic levelL Another arrangement of
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Fig. 18. Schematic representation of synaptic connections and arrangements in the M C N of the weakly electric fish Eigenmannia sp. P, small pacemaker cell; R, large relay cell.
279 synaptic contacts found in certain club endings in the MCN of Eigenmannia may reflect a form of dual transmission at mixed synapses; the gap junction and polarized chemical synapses of the same club ending can connect different postsynaptic profiles suggesting a different function for the two forms of the neuronal connection of the club ending. The club endings must originate from the small pacemaker cells (Fig. 18), since no large myelinated axons enter the MCN and only the axons of small cells were found to send off collaterals; axons of large cells leave the nucleus without branching. Therefore, one has to conclude that all club endings synapsing both on large and small cells belong to the small cells. Additional indirect evidence may be mentioned in this connection: following retrograde HRP labelling of large cells, labelled club endings were never found, and HRP reaction product was present only in large myelinated fibers leaving the nucleus. Thus, it appears that similarly to the MCN of the highfrequency fish Sternarchus5, small cells in Eigenmannia are connected by their own terminals to each other as well as to the large cells. Supporting the physiological data of Szabo and Enger TM small cells of Eigenmannia MCN can be considered as pacemaker cells responsible for the activation of the large cells, in the same way as has also been described for other gymnotid species1,5. Large cells are relay cells connecting Eigenmannia MCN to the spinal electromotor neurons. Ellis and Szabo 6 recently gave light microscopical evidence of the dendrodendritic connections of large cells. At ultrastructural levels we failed to find any dendro-dendritic connections in Eigenmannia MCN, even in HRP-labelled material. Nor have dendro-dendritic contacts been established in Sternarchus5, though they exist between small cells in Sternopygus1, another medium-frequency wave species. In spite of the absence of electrotonic coupling between MCN neurons, we found club endings of small cells in Eigenmannia which were coupled to each other by gap junctions. Such coupled club endings interconnect postsynaptic elements. Gap junctions between club endings in the MCN or in other parts of the electrosensory--electromotor pathway of different weakly electric fish have not been described until now 1,z,5,9,13-15. Coming from similar origin, these connections may serve for the synchronization of presynaptic activities, or, if from different origins, they may represent morphological correlates for presynaptic modulation. Small bouton-like terminals have their origin in thin myelinated fibers. These fibers are the only afferents to the MCN of Eigenmannia; they mediate afferent information only chemically, but both at pacemaker and relay cell level. In Sternarchus, Tokunaga et al. 15 and Elekes and Szabo 5 ascribed a modulatory role upon the MCN activity to terminals of these thin fibers. Small bouton-like terminals were never seen to synapse with each other, but the same bouton was found to connect pacemaker and relay cell dendrites suggesting that simultaneous modulation at both cellular levels may take place in Eigenmannia. In conclusion (Fig. 18) in the medium-frequency weakly electric fish Eigenmannia small pacemaker cell axons connect with the relay cells by club endings; in addition, the pacemaker cells are interconnected with each other by axo-somatic, axodendritic and axo-axonic connections. The MCN receives extranuclear afferences by
280 means o f thin m y e l i n a t e d fibers o f u n k n o w n origin*. Thus, the synaptic organisation o f the c o n s t a n t m e d i u m - f r e q u e n c y weakly electric fish Eigenmannia sp. is essentially similar to t h a t o f the high-frequency weakly electric fish SternarchusZ, 1.~. However, differences between the two species were f o u n d in certain details, first o f all in the neuronal m o r p h o l o g y and a r r a n g e m e n t a n d also in the ultrastructural aspects o f the synaptic terminals. It can be stated that Eigenmannia M C N possesses a m o r e complex structure. These differences m a y c o r r e s p o n d to the observations that Eigenmannia displays a richer repertoire o f E O D activity m o d u l a t i o n 4. Indeed, Sternarchus exhibits only two types o f m o d u l a t i o n , n a m e l y a j a m m i n g avoidance response with exclusively increasing frequency a n d transient changes (increasing) in E O D rate; b o t h can only be e v o k e d by electrical stimuli3, s, (Behrend, personal c o m m u n i c a t i o n ; Scheich, personal c o m m u n i c a t i o n ) . In contrast, Eigenmannia can p r o d u c e 4 types o f E O D m o d u l a t i o n , n a m e l y j a m m i n g avoidance response with increasing a n d decreasing frequency, transient changes o f E O D rateS, 16 ; Eigenmannia can also stop its E O D ( K i r s c h b a u m , personal c o m m u n i c a t i o n ; Westby, personal c o m m u n i c a t i o n ) . All these m o d u l a t i o n s can be e v o k e d by electrical stimuli s. ACK NOWLEDGEM ENTS This w o r k was p a r t l y s u p p o r t e d by the F o n d a t i o n de la Recherche M e d i c a l e Franqaise and the E u r o p e a n T r a i n i n g P r o g r a m in Brain a n d Behaviour Research. * Note added in proof: afferent fibers to the MCN have been recently traced m Eigenmannia; according to Heiligenberg et al. (Brah7 Research, 211 (1981) 418-423) they originate from the mesencephalic torus semicircularis,
REFERENCES 1 Bennett, M. V. L., Pappas, G. D., Gimenez, M. and Nakajima, Y., Physiology and ultrastructure of electrotonic junction. IV, Medullary electromotor nuclei in Gymnotid fish. J. Neurophysiol., 30 (1967) 236-300. 2 Bennett, M. V. L., Sandri, C. and Akert, K., Neuronal gap junctions and morphologically mixed synapses in the spinal cord of a teleost, Sternarchus albifrons (Gymnotidei), Brain Research, 143 (1978) 43-60. 3 Bullock, T. H., Species differences in effect of electroreceptor input on electric organ pacemakers and other aspects of behavior in electric fish, Brain Behav. Evol., 2 (1969) 85--ll 8. 4 Elekes, K. and Szabo, T., Comparative synaptology of the pacemaker nucleus in the brain of weakly electric fish (Gymnotidae). In Szabo, T. and Czeh, G., (Eds.), Advances in Physiological Sciences, Vol. XXXI, Sensory Physiology of Aquatic Lower Vertebrates, Oxford and New York, Pergamon Press, pp. 107-127. 5 Elekes, K. and Szabo, T., Synaptology of the command (pacemaker) nucleus in the brain of the weakly electric fish Sternarchus (Apteronotus) albifrons, Neuroscience, 6 (1981) 443~460. 6 Ellis, D. and Szabo, T., Identification of different cell types in the command (pacemaker) nucleus of several gymnotiform species by retrograde transport of horseradish peroxidase, Neuroscience, 5 (1980) 1917-1929. 7 Faber, D. S., Kaars, C. and Zottoli, S. J., Dual transmission at morphologically mixed synapses: evidence from postsynaptic cobalt injections, Neuroscience, 5 (1980) 433-440. 8 Hopkins, C. D., Electric communication. In Sebeok (Ed.), How Animals Communicate, Indiana University Press, 1977, pp. 263-289.
281 9 Sotelo, C., Rethelyi, M. and Szabo, T., Morphological correlates of electrotonic coupling in the magnoceUular mesencephalic nucleus of the weakly electric fish Gymnotus carapo, J. Neurocytol., 4 (1975) 587-607. 10 Szabo, T., Organisation particuliere de la commande nerveuse centrale de la decharge chez un poisson electrique Gymnarchus niloticus, C. R. hebd. Sc. Acad. Sei. (Paris), 248 (1959) 3488-3489. 11 Szabo, T., Anatomo-physiologie des centres nerveux specifiques de quelques organes 61ectriques. In Chagas C. and Paes de Carvalho, A. (Eds.) Bioelectrogenesis, Amsterdam, Elsevier, 1961, pp. 185-201. 12 Szabo, T. and Enger, P. S., Pacemaker activity of the medullary command nucleus controlling electric organs in high-frequency gymnotid fish, Z. vergl. Physiol., 49 (1964) 285-300. 13 Szabo, T. and Ravaille, M., Synaptic structure of the lateral line lobe nucleus in mormyrid fish, Neurosci. Lett., 2 (1976) 127-131. 14 Szabo, T., Sakata, H. and Ravaille, M., An electrotonic coupled pathway in the central nervous system of some teleost fish, Gymnotidae and Mormyridae, Brain Research, 95 (1975) 459474. 15 Tokunaga, A., Akert, K., Sandri, C. and Bennett, M. V. L., Cell types and synaptic organisation of the medullary electromotor nucleus in a constant frequency weakly electric fish, Sternarchus albifrons, J. comp. NeuroL, 172 (1980) 4074-426. 16 Watanabe, A. and Takeda, K., The change of discharge frequency by a.c. stimulus in a weakly electric fish, J. exp. BioL, 40 (1963) 57-66.
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Elsevier Biomedical Press
Research Reports
S Y N A P T I C O R G A N I Z A T I O N I N T H E P A C E M A K E R N U C L E U S OF A M E D I U M - F R E Q U E N C Y W E A K L Y E L E C T R I C FISH, E I G E N M A N N I A SP.
K. ELEKES* and T. SZABO Laboratoire de Physiologie Nerveuse, Departement de Neurophysiologie Sensorielle, CNRS, 91190 Gif sur Yvette (France) and Biological Research Institute of the Hungarian Academy o f Sciences, Tihany (Hungary)
(Accepted September 17th, 1981) Key words: synaptic contacts - - pacemaker nucleus - - weakly electric fish - - electro n microscopy
SUMMARY The medullary c o m m a n d nucleus (MCN) of the medium-frequency weakly electric fish, Eigenmannia sp., contains two types of neurones, namely large and small cells, which are embedded in a neuropile of large and small myelinated fibers. Using serial semi-thin and ultra-thin sectioning, combined with H R P labelling it has been established that both cell types possess rich dendritic arborization and large myelinated axons. Only the axons of the large cells leave the nucleus and these constitute the unique output of the MCN. Axon branching has been observed only in the axons of small cells and their collaterals show an exclusively intranuclear course. Two types of synaptic terminals have been found on large as well as on small cells: (1) large club endings forming both gap (electrotonic)junctions and polarized chemical synapses, which often appear at the same junction constituting morphologically mixed synapses; and (2) small bouton-like terminals forming exclusively chemical synaptic contacts. N o differences between the two neuron types could be detected with respect to the arrangement of the synaptic contacts: club endings and small bouton-like terminals synapse on dendritic processes as well as on perikarya, while the unmyelinated initial segments were always found to be free of synaptic contacts. Large and small cells were found to be simultaneously connected by the same club ending or small bouton-like terminal: in the case of club endings by means of gap junctions and chemical synapses, whereas in the case of boutons by chemical synapses only. Club endings sometimes form gap junctions with each other. The possible role of these * Present address: The Biological Research Institute of the Hungarian Academy of Sciences, H 8237 Tihany, Hungary. 00~6-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
268 unusual synaptic connections in local synchronization is suggested. Club endings originate from the large axons of small cells, while small bouton-like terminals originate from the fine myelinated fibers of extranuclear origin. In Eigenmannia, small cells, being connected to large cells as well as to each other by axo-somatic and axodendritic synapses, can be considered as the pacemaker cells of the MCN whereas large cells are relay cells. Small bouton-like terminals may convey exogeneous impulses towards the MCN exerting modulatory effects at both pacemaker and relay cell levels. The greater variety of ultrastructural correlates established in the MCN of Eigenmannia, in comparison with Sternarchus 5 (see also ref. 16), suggests increased modulation possibilities in the former fish's EOD behaviour.
INTRODUCTION The electric organ in all species of weakly electric fish discharges electric impulses of which the frequency is characteristic for different species, in many species, and in particular in gymnotids, the rhythmic discharge depends on a specific nucleus, the medullary command nucleus (MCN), situated in the rhombencephalic reticular formation. Several investigationsl0,1~, 1~ showed that the spinal electromotor neurons innervating the electric organ are directly excited with the MCN. In agreement with earlier data concerning the morphological organization of the MCN in two gymnotid species, Sternarchus and Eigenmannia r2, it has recently been shown by H R P injection into the spinal cord that only large (relay) cells of the MCN send their axon to the spinal electromotor neurons6,1L Furthermore, it has also been found following semithin serial sectioning in Sternarchus albifrons 5 that large (relay) cells failed to send off collaterals within the nucleus, while the axons of small (pacemaker) cells, which exhibit rich branching patterns, never leave the MCN. The ultrastructure of the MCN was first investigated in gymnotids by Bennett et al. 1 in a medium-frequency pulse species Steatogenys sp. and in a medium-frequency wave species Sternopygus sp. Club endings and small boutons of unidentified origin were localized on large (relay) as well as small (pacemaker) cells. In Sternopygus, electrotonic (gap)junctions between pacemaker cell dendrites were also seen. Recently detailed microscopic investigations were performed on the MCN of the high frequency Sternarchus albifronsS, 1~. Both small (pacemaker) and large (relay) cells are richly provided with different kinds of synaptic terminals: club endings with electrotonic junctions and small boutons with polarised chemical synapses. Elekes and Szabo 5 have furnished additional data as to the origin of these two types of synaptic terminals: large club endings originate from thick myelinated axon collaterals of the pacemaker cells, while small bouton-like terminals take their origin from thin myelinated fibers entering the nucleus from neighboring medullary areas. On the basis of these findings, the synaptic connections between the neuronal components of Sternarchus MCN have been established. The aim of the present work was to perform a detailed ultrastructural analysis on the MCN ofEigenmannia sp., a medium-frequency wave species gymnotid. Such an analysis would make it possible to compare the synaptology of the MCN in two
269 weakly electric fish with medium (Eigenmannia) and high frequency (Sternarchus) EOD discharges. This comparison is of particular interest since Eigenmanniaexhibits a more complex electrical behaviour than Sternarchus during social encountersa,S, la. MATERIALSAND METHODS Adult specimens, 8-12 cm long, of the weakly electric fish Eigenmanniasp. were used. After anesthesia with tricaine methanesulfonate (MS 222, Sandoz, 50 rag/l), the animals were perfused through the heart with a solution containing 2 ~ glutaraldehyde and 2 ~ formaldehyde buffered with 0.1 M phosphate at pH 7.2, for 45-60 min. The brains were thereafter immersed in the same solution overnight at 4 °C. A small part containing the MCN was dissected out of the medulla, washed in phosphate blaffer for 1 h and post-fixed in 2 ~ OsO4 buffered with 0.1 M phosphate for 2 h at 4 °C. After dehydration, tissue samples were embedded in araldite and cut in serial semi-thin and ultra-thin sections on an LKB III ultrotome. Semi-thin sections were stained with 1 ~o toluidine blue while ultra-thin sections were stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Siemens Elmiskop 102. For HRP labelling, a 50~o solution of the enzyme was injected through a micropipette by pressure into the caudal part of the spinal cord. After a survival time of 10-14 days the animals were perfused and fixed as described above. For light microscopical investigations, the brains were cut on a Vibratome. The sections were treated with 3,3'-DAB for 20 min and the HRP reaction product was developed with H202. The mounted sections were lightly counterstained by cresyl violet. For electron microscopy, 60-/~m sections, cut on Vibratome and treated with DAB and H202, were postfixed with 2 ~ OsO4 for 60 min, then dehydrated and embedded as described above. Ultra-thin sections either without or following double staining with uranyl acetate and lead citrate were observed in a TESLA BS 500 electron microscope. RESULTS The MCN of the weakly electric fish, Eigenmanniasp., is situated in the reticular formation of the medulla just below the Mauthner cell axons. In semi-thin transverse sections it shows a heart-like form; it is separated from the neighbouring medullary area by large bundles of myelinated axons showing intense toluidine blue staining (Fig. 1). Ventrally, this bordering layer is constituted of extremely thick myelin sheathed axons, while dorsally the limit is formed by thin myelinated fibers (Fig. 1). The nucleus itself consists of a network of large and thin myelinated fibers in which two types of nerve cells are embedded: large cells with a diameter of 65-70/~na and small cells with a diameter of 30-35/,m (Fig. 1). The two cell types are more or less intermingled, but rather more large cells were found in the lateral part of the nucleus. Capillaries are present throughout in the MCN forming a dense vascularization. Large cells, with centrally located spherical nuclei, possess an initial segment about 20/zm long. The myelinated axon never gives off collaterals either within the
270
Fig. 1. Semi-thin section from a MCN of Eigenmannia. Large (arrows) and small cells (double arrows) are embedded in a neuropile composed by myelinated axons. Region of the bordering layer of myelinated fibers through which large cell axons leave the nucleus is marked by arrowheads; see also Fig. 3. Scale: 50/~m.
271 nucleus or outside the nucleus in the neighboring medullary area. Concordantly, after H R P injection we failed to observe labelled axons sending off collaterals either in semithin or ultra-thin sections. However, HRP-labelled myelinated axon profiles were found in the ventral bordering layer of the M C N mentioned above (Fig. 3). These correspond to the large cell axons which course towards the spinal electromotor neurons. Ten to 14 days after spinal injection, only large cells were found to be labelled in the M C N (see ref. 6). While small cells were always free of reaction product (Fig. 4). H R P labelling also revealed a large number of dendritic arborization of the large cells, appearing in the light microscope as a fine network throughout the nucleus. Electron microscopical analysis reveals that the initial axon segments of the large cells is practically free of synaptic terminals but covered by glia processes (Fig. 2). In contrast, the cell body of the large cells is partly covered with club endings and small bouton-like terminals, partly with a multi-layered sheath of glial processes. On large cells, club endings and bouton-like terminals were also seen on primary dendrites as well as at some distance from the cell body (Figs. 5 and 6). HRP-labelled small dendritic processes made synaptic contact mainly with bouton-like terminals; club endings were less frequently seen at this level (Figs. 5 and 6). A considerable part of the dendritic surface is covered by glial processes (Fig. 6). Besides H R P labelling, identification of large cell dendrites was possible on the basis of glycogen content: large cells appear practically free of glycogen granules whereas small cells have always a large amount of glycogen (Figs. 10a, 12 and 17). Apart from their smaller size, the morphology of the small cells differs from the large ones in that that the initial segments of their axons are about twice as long (40 #m) and that their axons send off several collaterals, not far from the beginning of the myelin sheath (Fig. 7). The small cells have several short primary dendrites branching almost immediately at their origin (Fig. 9). The ultrastructural aspects of the small cells differ from those of the large ceils. A large amount of complex glycogen granules is found in their perikarya, mainly concentrated in the peripheral cytoplasm (Figs. 9 and 12). Consequently, glycogen granules are also seen in the dendritic processes, as well as in the axoplasm of the initial segment where they were uniformly distributed. In contrast, glycogen granules Fig. 2. Electron micrograph of the initial segment (IS) of a large cell axon. No synaptic terminals, but densely packed glial processes can be seen on its surface. C, capillary. Scale: 2/zm. Fig. 3. HRP-labelled (arrows) large cell axon (A) surrounded by thickly myelinated axons separating the MCN from the neighboring medullary areas. Unstained section. Scale: 2/~m. Fig. 4. Light microscopic view of large cells labelled retrogradely by HRP injection into the spinal cord. One cell only weakly labelled (arrow). Note the rich dendritic arborization of the two intensely labelled cells. Dark-field micrograph. Scale: 30/tm. Fig. 5. A club ending (CE) forms chemical synapses (arrowheads) on a HRP-labelled dendritic process of a large cell (RD). Simple glycogengranules and rER elements (asterisk) are present in the dendrite. Scale: 0.5/~m. Fig. 6. HRP-labelled dendritic process (RD) with 3 small bouton-like terminals (asterisk) forming chemical synapses. Note the presence of simple glycogen granules in the dendrite. Arrowhead, rER elements. G, glia processes. Scale: 0.5 ktm.
272
Fig. 7. Light micrographs of a small cell seen in serial semi-thin sections, a: note the long initial axon segment (IS). Arrow points to the axon profile which appears as a typical collateral of the myelinated axon in Fig. 7b. b: arrow points to the branching point of the myelinated axon. P, perikaryon; C, capillary. Scale: 10/~rn. Fig. 8. Low power electron micrograph of the initial segment (IS) of a small cell axon. No terminals are present on the IS membrane which is completely covered by glial processes. Arrows indicate glycogen in the myelinated part of the axon (A). C, capillary. Scale: 2 ibm. Inset: high-power electron microscopic detail of the surface of small cell initial segment (IS). Note medium electron-dense material in the intercellular cleft and high electron-dense material attached to the inner surface of the 1S membrane (arrowheads). Scale: 0.1 /tm.
273 in the myelinated axoplasm were often clustered just below the myelin sheath (Fig. 8). The distribution o f synaptic terminals on the different parts o f the small cells, i.e. perikaryon, initial axon segment and dendrites was f o u n d to be similar to that observed on the large cells. The long initial axon segment is completely free o f synaptic terminals and connections and it is covered by a multilayered glial sheath. At lowpower magnification a strikingly increased electron density o f the initial axon m e m b r a n e is seen. At higher magnification a finely granulated electron-dense material was f o u n d in the intracellular cleft between the axolemma and the glial m e m b r a n e ; a layer o f dense material o f irregular thickness and o f granulated structure is attached to the inner surface o f the initial axon segment m e m b r a n e (Fig. 8 and inset). The perikaryon o f the small cells bears several bouton-like terminals and a few club endings f r o m thick myelinated axons. However, a large part o f the cell b o d y is covered with multilayered glial sheath. In contrast, the dendritic processes receive
Fig. 9. Three club endings (arrows) synapse on a bifurcating primary dendrite (D) of a small cell (PC). One of the club endings (filled asterisk) simultaneously connects the small cell dendrite (D) and a large cell dendritic process (open asterisk). C, capillary. Scale: 2/~m. Fig. 10. Synaptic terminals on small cell dendrites, a: large club ending (CE) and bouton-like terminals (arrows) on a small cell dendrite (D). Several large granular vesicles are present in neighboring bouton (asterisk). M, mitochondria. Scale: 0.5 #m. b: enlarged detail of Fig. 10a showing two gap junctions (arrows) of the club ending and a polarized chemical synapse of a bouton (arrowheads) on the dendrite (D). Several large granular vesicles are present in the neighbouring bouton (asterisk). M, mitochondria. Scale: 0.2/~m. Inset: high-power magnification of the gap junction shown in Fig. 10b. Scale: 0.05/~m.
274
Fig. 11. Unusual nodal synaptic ending of a large bifurcating myelinated axon (A): a thin axoplasmic preterminal protrusion (arrow) contacts with a typical terminal (filled asterisk) filled with mitochondria. The terminal forms a gap junction (arrowhead) with a small cell dendrite (D). Open asterisk: glial processes. Scale: 1 itm. Fig. 12. A small (PC) and a large (RC) cell are connected by two en passant terminals (asterisks) of the same large myelinated axon (A). N, nucleus; C, capillary. Scale: 2/J,m. Upper inset: in the course of serial sectioning, one of the terminals (open asterisk) appears as a typical club ending forming a gap junction (arrow) with the large cell (RC). Scale: 1/~m. Bottom inset: high-power magnification of the gap junction indicated by arrow in the upper inset. Scale: 0.05/zm.
275
Fig. 13. A club ending (CE) simultaneously connecting a large cell dendrite (open asterisk) with a chemical synapse (arrow) and a small cell perikaryon (filled asterisk) with a gap junction (double arrow). Note the spine-like postsynaptic profile (arrowhead) of the large cell dendrite at the chemical synapse. G, glial processes. Scale: 0.5 #m. Inset: high-power magnification of the gap junction indicated by double arrow in Fig. 13. Scale: 0.05/~m. Fig. 14. Electron micrograph of a club ending (CE) forming a mixed synapse on a large cell dendrite. The polarized chemical synapse is indicated by arrow, the gap junction by double arrow and a desmosome-like attachment by an arrowhead. Scale: 0.2 #m. Fig. 15. Gap junction (between arrows) between two club endings (CE). M, mitochondria. Scale: 0.25 /tm. Figs. 16 and 17. Ultrastructure and origin of small bouton-like terminals. Fig. 16. Fine myelinated fiber (A) synapses with a bouton-like terminal perikaryon (asterisk). C, capillary. Scale: 2 #m. Fig. 17. Small bouton-like terminal (asterisk) connect simultaneously by synapses the dendrite of a large cell (RD) and that of a small (PD) cell. Note granules (arrowheads) in the small cell dendrite and its absence in the mitochondria; G, glial processes. Scale: 0.25/~m.
(arrow) on a small cell means of two chemical the presence of glycogen large cell dendrite. M,
276 many synaptic endings: primary dendrites are covered by club endings and small bouton-like terminals (Fig. 9); smaller dendritic processes form synaptic contacts with several bouton-like terminals and few club endings (Figs. 10, 11 and 17).
Ultrastructure of synaptic terminals and synaptic connections Club endings occurring on both large and small cells take their origin from large myelinated axons (Figs. 9, 10, 11, 12 and 14). They form partly en passant terminals (Figs. 11 and 12). A new form ofen passant terminals was found (Fig. 11) consisting ol a thin (about 0.5/tm in diameter) axoplasmic 'neck' protruding from a node of Ranvier which, after 1-2/ma course, forms a typical club ending filled with mitochondria and surrounded by glial processes. Club endings were mainly characterized by two cellular compartments, namely mitochondria and small (40-50 /~m) clear synaptic vesicles (Figs. 5, 13 and 14). These latter were mostly found clustered in the lateral parts of the terminals. Most of the club endings also contained neurotubules and a few glycogen granules. Club endings with HRP labelling were never found. Club endings formed 3 typical junctional complexes both with large and small cells: gap (electrotonic) junctions, polarized chemical synapses and desmosome-like attachments (Figs. 5, 10, 13, 14 and 15). Polarized chemical synapses in a club ending could be distinguished easily from desmosome-like attachments. The former are characterized by an asymmetric clustering of clear synaptic vesicles, typical postsynaptic paramembranous dense apposition and presynaptic dense projections, whereas the latter show exclusively symmetrical apposition of dense material (Fig. 14). Gap junctions and polarized chemical synapses occur close to each other, having the same postsynaptic membrane and thus form typical mixed synapses (Fig. 14). Both types of contacts were also found in the same plane of section of a club ending, but making synaptic connections with different postsynaptic elements (Fig. 13). Such club endings were seen to synapse on a small cell dendrite with a gap junction and simultaneously with a polarized chemical junction on a large cell dendrite (Figs. 9 and 13). Similar connections were also observed between small and large cell perikarya. Furthermore, small and large cell dendrites or perikarya can also be connected by different club endings or en passant terminals of the same large myelinated axon (Fig. 12). This is the first time that an electrotonic (gap)junction between club endings has been described (Fig. 15), in which either one or both endings simultaneously contact typical postsynaptic element(s), i.e. dendritic profile(s). Small bouton-like terminals forming synaptic contacts both on large and small cells originate from thin myelinated axons entering the nucleus from neighboring medullary areas (Fig. 16). Thin myelinated axons show an electron transparent axoplasm containing only few mitochondria (even in the preterminal segment) and glycogen granules. Several boutons ending on large or small cells have been seen to originate from the same thin myelinated fiber forming terminals or en passant contacts. The boutons are small, with a diameter of about 0.5-1 #m ; besides many small clear spheroid-shaped vesicles of 40-60 nm in diameter, they also contain a few small
277 mitochondria and glycogen granules (Fig. 17). The clear vesicles represent a homogeneous population; a few large (85 nm) granular vesicles were sometimes observed in these boutons (Fig. 17) or in the preterminal myelinated segment of the thin fiber, although in a few cases, the boutons also contained many large, granular vesicles (Fig. 10b). Flattened, clear vesicles were never seen in boutons. Small bouton-like terminals of thin myelinated fibers form only chemical synapses (Figs. 7, 10 and 17). They never synapse on each other but may connect postsynaptic profiles of different origin (Fig. 17). DISCUSSION Our present findings concerning the MCN of Eigenmannia, a wave species, weakly electric fish with medium-frequency EOD, revealed that its general organization is essentially similar to that of Sternarchus albifrons, another wave species electric fish with high-frequency EOD 5. In both, two different neuron types constitute the MCN; these are embedded in a neuropile constituted by large and thin myelinated fibers. Large and small neurons as well as large myelinated fibers have earlier been described in the MCN of several weakly electric fish species1,12; however, the presence of thin myelinated fibers of extranuclear origin was described only recently by Elekes and Szabo 5 in Sternarchus. From the present data, the latter also appear to be a basic neuronal component of the MCN in Eigenmannia, representing the only afferent connections of the nucleus. Concerning the neuronal morphology within the MCN of Eigenmannia, large cells differ from the small ones in that that their axon leaves the nucleus without giving off collaterals, whereas small cell axons exhibit branching patterns; the branches have an exclusively intranuclear course. That only large cell axons project to the spinal cord was also proved by retrograde HRP labelling: following spinal injection of HRP only large cells were labelled. A similar conclusion has been drawn in several other gymnotid species, using Bodian impregnation methods 12, and recently HRP labelling6, as. Dendritic arborization of the large cells were also revealed by HRP labelling as well as by electron microscopic analysis. Thus, in Eigenmannia both neuronal cell types possess a rather large dendritic tree, in contrast to Sternarchus in which only small cells were found to have long arborized dendrites whereas large cells have only short spinelike dendritic processesL The more extensive dendritic development of large cells in Eigenmannia may partly explain the differences between Eigenmannia and Sternarchus concerning the arrangement ofsynaptic terminals on large cells. In Eigenmannia, club endings are also present on dendritic processes, while in Sternarchus, they are concentrated on the perikaryon5. In contrast, we failed to find club endings on the initial segment of large cell axons in Eigenmannia, whereas in Sternarchus the initial segments were completely and exclusively covered by them. This suggests that the coupling between pacemaker and relay cells may be much tighter in Sternarchus, which has a discharge frequency of 1000 c/s than in Eigenmannia with only 300 c/s. Elekes and Szabo 5 proposed that this particular synaptic arrangement perhaps represents the morphological correlate of
278 the reinforcing function of pacemaker impulses in view of the exact timing of large cell activity in the case of high frequency discharge. This explanation also holds for low frequency fish such as Hypopomus in which the initial segment of the large cells is also free from club endings 4. The arrangement of synaptic contacts on the small cells is different in Sternarchus and Eigenmannia in spite of a relatively rich dendritic arborization in both species: club endings and small bouton-like terminals occur on dendrites in Eigenmannia, whereas only small boutons are present on these in
Sternarchus 5. In Eigenmannia, large club endings form gap (electrotonic) junctions and chemical synapses on both large and small cells; they often occur together constituting so-called mixed synapses. Such synaptic contacts were also observed in the M C N of the medium-frequency wave species Sternopygus and in the variable-frequency pulse species Steaogenys as well as in different parts of the electrosensory and electromotor pathway of gymnotid and mormyrid CNS 1,2,9,1a,14. In contrast, we, as well as Tokunaga et al. 15, have failed to find mixed synapses in Sternarchus MCN, where only gap junctions occur on both large and small cellsL Whether these mixed synapses in Eigenmannia represent the morphological correlates of a functionally dual transmission cannot be stated without precise electrophysiological experiments. Recently, the existence of dual transmission at mixed synapses in goldfish Mauthner cell has been proven by cobalt injections at the postsynaptic levelL Another arrangement of
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ventral surface
Fig. 18. Schematic representation of synaptic connections and arrangements in the M C N of the weakly electric fish Eigenmannia sp. P, small pacemaker cell; R, large relay cell.
279 synaptic contacts found in certain club endings in the MCN of Eigenmannia may reflect a form of dual transmission at mixed synapses; the gap junction and polarized chemical synapses of the same club ending can connect different postsynaptic profiles suggesting a different function for the two forms of the neuronal connection of the club ending. The club endings must originate from the small pacemaker cells (Fig. 18), since no large myelinated axons enter the MCN and only the axons of small cells were found to send off collaterals; axons of large cells leave the nucleus without branching. Therefore, one has to conclude that all club endings synapsing both on large and small cells belong to the small cells. Additional indirect evidence may be mentioned in this connection: following retrograde HRP labelling of large cells, labelled club endings were never found, and HRP reaction product was present only in large myelinated fibers leaving the nucleus. Thus, it appears that similarly to the MCN of the highfrequency fish Sternarchus5, small cells in Eigenmannia are connected by their own terminals to each other as well as to the large cells. Supporting the physiological data of Szabo and Enger TM small cells of Eigenmannia MCN can be considered as pacemaker cells responsible for the activation of the large cells, in the same way as has also been described for other gymnotid species1,5. Large cells are relay cells connecting Eigenmannia MCN to the spinal electromotor neurons. Ellis and Szabo 6 recently gave light microscopical evidence of the dendrodendritic connections of large cells. At ultrastructural levels we failed to find any dendro-dendritic connections in Eigenmannia MCN, even in HRP-labelled material. Nor have dendro-dendritic contacts been established in Sternarchus5, though they exist between small cells in Sternopygus1, another medium-frequency wave species. In spite of the absence of electrotonic coupling between MCN neurons, we found club endings of small cells in Eigenmannia which were coupled to each other by gap junctions. Such coupled club endings interconnect postsynaptic elements. Gap junctions between club endings in the MCN or in other parts of the electrosensory--electromotor pathway of different weakly electric fish have not been described until now 1,z,5,9,13-15. Coming from similar origin, these connections may serve for the synchronization of presynaptic activities, or, if from different origins, they may represent morphological correlates for presynaptic modulation. Small bouton-like terminals have their origin in thin myelinated fibers. These fibers are the only afferents to the MCN of Eigenmannia; they mediate afferent information only chemically, but both at pacemaker and relay cell level. In Sternarchus, Tokunaga et al. 15 and Elekes and Szabo 5 ascribed a modulatory role upon the MCN activity to terminals of these thin fibers. Small bouton-like terminals were never seen to synapse with each other, but the same bouton was found to connect pacemaker and relay cell dendrites suggesting that simultaneous modulation at both cellular levels may take place in Eigenmannia. In conclusion (Fig. 18) in the medium-frequency weakly electric fish Eigenmannia small pacemaker cell axons connect with the relay cells by club endings; in addition, the pacemaker cells are interconnected with each other by axo-somatic, axodendritic and axo-axonic connections. The MCN receives extranuclear afferences by
280 means o f thin m y e l i n a t e d fibers o f u n k n o w n origin*. Thus, the synaptic organisation o f the c o n s t a n t m e d i u m - f r e q u e n c y weakly electric fish Eigenmannia sp. is essentially similar to t h a t o f the high-frequency weakly electric fish SternarchusZ, 1.~. However, differences between the two species were f o u n d in certain details, first o f all in the neuronal m o r p h o l o g y and a r r a n g e m e n t a n d also in the ultrastructural aspects o f the synaptic terminals. It can be stated that Eigenmannia M C N possesses a m o r e complex structure. These differences m a y c o r r e s p o n d to the observations that Eigenmannia displays a richer repertoire o f E O D activity m o d u l a t i o n 4. Indeed, Sternarchus exhibits only two types o f m o d u l a t i o n , n a m e l y a j a m m i n g avoidance response with exclusively increasing frequency a n d transient changes (increasing) in E O D rate; b o t h can only be e v o k e d by electrical stimuli3, s, (Behrend, personal c o m m u n i c a t i o n ; Scheich, personal c o m m u n i c a t i o n ) . In contrast, Eigenmannia can p r o d u c e 4 types o f E O D m o d u l a t i o n , n a m e l y j a m m i n g avoidance response with increasing a n d decreasing frequency, transient changes o f E O D rateS, 16 ; Eigenmannia can also stop its E O D ( K i r s c h b a u m , personal c o m m u n i c a t i o n ; Westby, personal c o m m u n i c a t i o n ) . All these m o d u l a t i o n s can be e v o k e d by electrical stimuli s. ACK NOWLEDGEM ENTS This w o r k was p a r t l y s u p p o r t e d by the F o n d a t i o n de la Recherche M e d i c a l e Franqaise and the E u r o p e a n T r a i n i n g P r o g r a m in Brain a n d Behaviour Research. * Note added in proof: afferent fibers to the MCN have been recently traced m Eigenmannia; according to Heiligenberg et al. (Brah7 Research, 211 (1981) 418-423) they originate from the mesencephalic torus semicircularis,
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