Cell Tiss. Res. 178, 155-167 (1977)
Cell and Tissue Research i:' by Springer-Verlag 1977
The Morphology of the Oval Nuclei of Neonatal Torpedo marmorata Geoffrey Q. Fox Department of Neurochemistry, Max-Planck-lnstitut fiir biophysikalische Chemie, G6ttingen, Germany
Summary. The morphology of the oval nucleus of neonatal Torpedo marmorata is described at the light and electron microscopic level of examination. The nucleus is unique relative to other central electromotor centers of electric fish so far described being bilaterally symmetrical, composed of two nerve cell types, and possessing no gap junctions between neurons and their processes. This particular structural plan presents difficulties in accounting for presumed synchronous discharge since it has been strongly argued that electrotonic coupling by means of gap junctions is the primary process by which synchronization is accomplished. Close membrane apposition and dendritic bundling, common features within the nucleus, are discussed as possible alternative structural correlates.
Key words: Electromotor system - Oval nucleus - Electron microscopy - Torpedo marmorata.
Synapses, junctions
Introduction This report concerns a morphological examination of the oval nucleus of neonatal Torpedo marmorata. This fish has a highly synchronized electric system (Fessard, 1961; Szabo, 1961) but little is known concerning the structure or function of its central electromotor components. Central " c o m m a n d " and " p a c e m a k e r " nuclei in electric fish are generally single nuclei located midventrally within the primitive brain stem regions of mesencephalon, medulla, or cervical spinal cord (Bennett, 1970) and represent phylogenetically old structures. In addition to initiating the discharge signal they provide the necessary Send offprint requests to." Dr. Geoffrey Q. Fox, Department of Neurochemistry, Max-Planck-lnstitut ftir biophysikalische Chemie, Am Fassberg, 3400 G6ttingen, Federal Republic of Germany Acknowledgements: I would like to express my appreciation to Drs. V.P. Whittaker, J.-R. Wolff, G.D. Pappas and M.V.L. Bennett for their critical evaluation of the manuscript and to W.D. K6tting and H. Ellermeier for their technical assistance
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s y n c h r o n i z a t i o n so t h a t t h e final d i s c h a r g e is o f m a x i m u m e f f e c t i v e n e s s . T h e oval nuclei are central c o m p o n e n t s of the m o t o r division a n d are located within the a n t e r i o r medulla. T h e y project to the electric lobes, p a i r e d nuclei c o m p o s e d o f g i a n t e l e c t r o m o t o r n e u r o n s w h i c h in t u r n d r i v e t h e p e r i p h e r a l e l e c t r o c y t e s t h a t m a k e u p t h e e l e c t r i c o r g a n . T h e i r c e n t r a l l o c a t i o n as well as t h e i r p o s i t i o n in t h e e l e c t r o m o t o r p a t h w a y s u g g e s t s t h a t t h e s e n u c l e i s e r v e e i t h e r a " c o m m a n d " or " p a c e m a k e r " function.
Material and Methods Newborn Torpedo marmorata, approximately 120 mm in length and 50 g in weight, were used in this study. Neonatal animals were chosen primarily because of our overall interest in the development of the electromotor system. These animals are well developed at birth, swimming well and exhibiting behavior patterns similar to those of the adults. Their electric organs are functional at this time and display mature discharge patterns (Krenz, 1976); thus our assumption is that the electromotor system is synchronized. The animals were anaesthetized with MS222 (Sandoz, 0.5 g/liter of sea water), the pericardial cavity exposed, and a cannula inserted through the heart into the conus arterious. Fifty milliliters of 1% paraformaldehyde and 2.5% glutaraldehyde buffered to pH 7.0 with 0.43 M sodium cacodylate (approximately 900 mOsmols) were perfused into the animals with a hand-held syringe and allowed to fix the tissue for two hours at room temperature. An additional fifty milliliters of 5% glutaraldehyde in the same buffer was then perfused and the animals were set aside for an additional three hours. The brain was removed and the desired tissue blocks cut and washed in several rinses of fresh buffer. The tissue blocks were postfixed in 0.43 M sodium cacodylate-buffered 1% osmium tetroxide for two hours at room temperature. An en block staining procedure consisting of an overnight washing in cold 0.4 M sodium acetate buffer (pH 5.5) followed by 1% uranyl acetate in 0.25 M sodium acetate (pH 5.5) for four hours in the cold and dark was routinely used to enhance the membrane contrast. Dehydration in graded ethanols (50% 100%) and embedding in Epon 812 completed the preparation for the electron microscope. Golgi-stained material was prepared by a chloral formaldehyde procedure (Ramon-Moliner, 1957; Frontera, 1964). The following solution was perfused through anaesthetized animals: potassium dichromate (5 g), chloral hydrate (5 g), formalin (10 ml of 10%), glutaraldehyde (5 ml of 25%), and water (75 ml). After three hours the brains were removed and immersed in the above solution for 2~4 days changing the solution daily. The brains were then placed in 0.75% silver nitrate for four days in the dark at room temperature also changing the solution daily. The brains were dehydrated in ethanol and embedded in a soft mixture of Epon 812. Sections (0.1 0.2 mm) were cut on a sledge microtome and mounted on glass slides for viewing. A Zeiss drawing apparatus mounted on a Zeiss Standard binocular microscope was used for making composite drawings of the Golgi stained material. Routine light microscopy was made from Toluidine Blue-stained sections (1 lam) cut from the material prepared for electron microscopy. Thin sectioned material for the electron microscope was doubly stained with aqueous uranyl acetate and lead citrate (Reynolds, 1963). Viewing and photography was done with a Zeiss Photomicroscope II and a Jeol 100B electron microscope.
Results Gross Structure T h e o v a l n u c l e i a r e t w o in n u m b e r a n d s i t u a t e d b i l a t e r a l l y in t h e m i d - v e n t r a l , r o s t r a l m e d u l l a . E l l i p s o i d a l in s h a p e , e a c h n u c l e u s m e a s u r e s a p p r o x i m a t e l y 1.2 m m ( a n t e r i o r - p o s t e r i o r ) b y 0.5 m m in d i a m e t e r . T h e g e n e r a l s t r u c t u r e o f
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Fig. 1. Transverse section through the anterior medulla of newborn Torpedo showing an oval nucleus. Horizontal (H) and vertical (V) large class neurons make up a central core region. Their axons are directed medially and exit the nucleus as bundles (A). Dendrites from the neuronal population form a dense cortical layer ((7) around the nucleus. Toluidine Blue-stained 1 lam section. (Bar 0.1 mm) Fig. 2. Horizontal section through junctional region between core and dendritic cortex. Large class neurons (L), small class neurons (S) and dendritic bundles (d) are seen. Toluidine Blue-stained 1 ~tm section. ( B a r = 30 lam)
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Figs. 3-5. Reconstructed composite drawing from Golgi stained material showing three aspects of the oval nucleus. Dotted line roughly outlines the core region. It is clear from the sagittal (3) and horizontal (4) planes of section that the large class neurons assume two orientations-vertical and horizontal respectively, a fact obscured when considering only the transverse (5) plane. The small neurons (5') tend to assume only a horizontal orientation within the nucleus. The electromotor neurons (e) within the electric lobe of Figure 5 are approximately 65 gm in diameter
each nucleus consists o f a p e r i k a r y o n - r i c h core, a s u r r o u n d i n g dendritic cortex a n d a m e d i a l a x o n a l zone (Fig. 1). T h e nuclei are c o m p o s e d o f two n e u r o n a l p o p u l a t i o n s o f large a n d small cells a n d also neuroglia. The large n e u r o n s a p p e a r to p r e d o m i n a t e n u m e r i c a l l y a n d f o r m the core region whereas the small n e u r o n s are f o u n d a l o n g the p e r i p h e r y o f the core a n d to a lesser extent within the dendritic c o r t e x (Fig. 2). N e u r o g l i a cells are d i s t r i b u t e d t h r o u g h o u t the entirety o f each nucleus.
Core Region T h e core region is p r i m a r i l y c o m p o s e d o f the cell b o d i e s o f a p o p u l a t i o n o f large, elongate, r o u g h l y cylindrical n e u r o n s ( a p p r o x i m a t e l y 150 l a m x 30 gm). The m a j o r axis o f these n e u r o n s lies within the transverse plane (Figs. 3-5) but m a y be o r i e n t e d in either a h o r i z o n t a l or vertical direction (Figs. 1, 3 5) with the vertically o r i e n t e d n e u r o n s a s s u m i n g a m o r e p e r i p h e r a l p o s i t i o n with respect to the h o r i z o n t a l neurons. Cells o f b o t h o r i e n t a t i o n s a p p e a r m o r p h o l o g -
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Fig. 6. Ultrastructural detail of small class neuron showing nucleus (N), slightly organized endoplasmic reticulum and two axo-somatic synapses. (Bar= 1 lam) Fig. 7. Large class neuron with nucleus (N), Golgi complex (G), lysosome (/), and non-laminar endoplasmic reticulum with intervening channels (c) containing fibrils. (Bar = 1 lain)
ically similar with regard to their size, distribution of processes, and organelle composition. The cell body contains large areas of short, nonoriented formations of endoplasmic reticulum most of which are free of bound ribosomes (Fig. 7). Many free ribosomes and ribosomal rosettes are found within the intervening spaces (Fig. 7). These Nissl areas are surrounded by mitochondria and often separated from one another by clear channels filled with fibrillar and tubular elements or by extensive systems of Golgi membranes (Fig. 7). Densely stained, irregular membrane-boundedlysosomes are also a common feature (Fig. 7). These organelle systems are also present throughout the primary dendrites but not within the initial axonal segment, which is composed of neurofilaments, mitochondria, vesicles and smooth reticular membranes. Single cilia have also been seen on the cell bodies with the basal body contained within a Golgi complex. The nucleus is spherical and approximately 25 btm in diameter containing a single (Figs. 1, 2) or sometimes several darkly staining nucleoli. All of these large cells appear to project their axons medially, with the intitial segment contained within the core region (by definition). Collateral branches of unknown length are often seen on the initial segments (Fig. 8). The dendrites, in contrast, project in other than medial directions, the major thrust being lateral and in a transverse (dorso-ventral) plane (Figs. 3-5). As seen in Golgi preparations, the primary dendrites emerge from all aspects of the cell body (Figs. 3-5), some in very close proximity to the initial axonal
Fig. 8. Cotlateral axonaI branch of the initial segment (/) of a large neuron axon. Two axo-axonic synapses (arrows) containing spherical vesicles are present. ( B a r - 1 gm) Fig. 9. Distal portion of a primary dendritic branch from a large class neuron located in the peripheral region between the core and dendritic cortex. Characteristic dendritic organelles are p r e s e n t - e n d o p l a s m i c reticulum, ribosomes, tubules and fibrils, mitochondria and large lysosomes. Several axo-dendritic synapses of the asymmetric variety with spherical and dense core vesicles are seen (arrows). (Bar = 1 gin) Fig. 10. An en passant axon making numerous synaptic contacts (A) with neural elements in its path: (L) large neuron cell body, (D1 z) dendrites. Asymmetric contact zone, spherical vesicles and axial fibrillar and tubular core are seen. Close membrane apposition is present between (D1) and (D3) with a specialized desmosomal region (arrow). ( B a r = 2 gm) Fig. 11. Details of closely adjacent large neuron cell bodies, showing a desmosomal contact between a somatic spine (S) and the perikaryal membrane. N m n e r o u s axo-somatic synapses are present. (Bar = 1 gin)
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segment. They are typically straight and remain unbranched for long distances. Secondary dendrites are less commonly seen and tertiary branches only occasionally encountered. Dendritic spines are rarely seen. Synapses are found on the cell body (Fig. 11), primary dendrites (Fig. 9) and initial segments of axons and axonal collaterals (Fig. 8).
Small Neurons
A lesser number of small fusiform neurons (cell bodies approximately 20 i.Lm in diameter) with spherical (13 gm in diameter) or occasionally oval nuclei are present within the peripheries of the core region and the dendritic cortex (Fig. 2). They are also oriented in the transverse plane with a major dendritic process directed medially and another laterally and occasional second order branches projecting off these two primary processes (Fig. 3-5). No data are as yet available concerning the character of the axon. Within the cytoplasm the endoplasmic reticulum is evenly distributed, somewhat oriented into stacks and partially studded with ribosomes (Fig. 6). Golgi complexes are few in number and less extensive than those seen in the large neurons. Free ribosomes and ribosomal rosettes, mitochondria, lysosomes, and occasional tubules, filaments and vacuoles make up the remaining cytoplasm (Fig. 6). Axo-somatic (Fig. 6) and axo-dendritic synapses are present in small numbers.
Dendritic Cortex
A cortical layer composed of the dendritic arborizations from both the large and small neurons surrounds much of the core region (Fig. 1). Horizontal sections through this region reveal dendritic bundling and suggest that the dendritic architectonics are oriented along a dorso-ventral axis in the transverse plane (Figs. 2, 12). The cortex is intersected by small caliber myelinated and unmyelinated axons, many of which are oriented along a rostro-caudal axis. Axo-dendritic synapses are established on the core dendrites by axons from these unidentified pathways and others. Axons also pass through the cortex into the core region, presumably to synapse upon the cell bodies of the large neurons.
Medial Axonal Region
The axons of the large neurons exit from the medial aspect of the nucleus (Fig. 1). Their initial segments converge within the core to form small bundles, becoming myelinated at the junction of the core and the medial axonal region (by definition). These axons are from 7 to 9 ~tm in diameter at the first myelin internode. Smaller caliber afferent fibers are also present within these bundles and pass into the core, terminating on neural elements. Individual fibers may
Fig. 12. Horizontal section through dendritic cortex showing density and spacial orientation of the dendritic bundles. Close membrane apposition and desmosomal contacts between adjacent dendrites are c o m m o n features (A). Axo-dendritic synapses are also present in large numbers. (Bar = 4 gm) Fig. 13. Higher power view of a typical dendritic m e m b r a n e apposition as illustrated in the preceding figure. Close non-specialized membrane apposition with intervening 8 10 n m space changes to a desmosomal-like zone with an approximate 20 n m space and heavy subjacent densities. ( B a r = 0.5 p.m) Fig. 14. A region illustrating two c o m m o n synaptic structures of the oval nucleus and also close m e m b r a n e apposition. Axo-dendritic en passant synapses (A) and an individual axo-dendritic synapse (B) have formed upon the same dendrite (D). Close membre appositions ( < 15 nm) (arrows) exist throughout. ( B a r = 1 gm)
Figs. 15-18 illustrate the commonly encountered examples of gap junctions within the core and dendritic cortex region of the oval nucleus Fig. 15. Gap junction (arrow) between pericapillary cell (P) and neural element. Capillary lumen is above. (Bar=0.5 gm) Fig. 16. Gap junction (arrow) between two presumptive glial processes in close vicinity to an axodendritic synapse. (Bar=0.25 gin) Fig. 17. Gap junction (arrow) between densely stained oligodendrocyte process and unidentifiable neural process. (Bar=0.5 lam) Fig. 18. Similar to Figure 17 but illustrating a non-typically extensive junction. (Bar=0.25 gm)
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be seen branching off these bundles as they project medially but it is not known whether these are afferent or efferent axons. At the raphe, few fibers are seen to cross but more typically to bend either dorsally or ventrally and apparently to travel within the raphe region. No communication between the two nuclei has been noted.
Synapses and Junctions Chemical synapses and gap junctions are present within the oval nuclei. Chemical synapses are formed from individual and en passant boutons. The individual boutons are derived from myelinated and unmyelinated axons entering the nucleus (dendritic cortex and core) from lateral or medial directions. Terminal boutons contain spherical (Figs. 6, 8 11, 14, 16) and occasional pleomorphic vesicles and establish axo-somatic (Figs. 6, 11), axo-dendritic (Fig. 9) and axoaxonic (Fig. 8) synapses. Contact with multiple post-synaptic elements is common. En passant boutons also are derived from myelinated and unmyelinated axons seemingly entering the nucleus from all directions. Nodules along the unmyelinated axon cylinder contain spherical synaptic vesicles that make asymmetric synaptic contact with many of the neural elements in their path (Fig. 10). Desmosomal contacts are numerous between all unmyelinated neural processes in the nucleus. They consist of various amounts of dense granular accumulations along the subjacent membrane surfaces, with the intervening intercellular space (approximately 20 nm) often containing a somewhat less dense amorphous material (Fig. 13). Adjacent to these contacts as well as to synapses but common throughout the entirety of the nucleus are regions of close neural membrane apposition characterized by intercellular spaces within a range of 6-12 nm and by the absence of intervening glial processes (Figs. 12-14). Gap junctions containing an approximate 2 to 4 nm gap and of up to 1.5 IJm in length have been identified in both the dendritic cortex and core regions of the nucleus (Figs. 15-18). Though frequently in close proximity to chemical synapses (Fig. 16), the majority of gap junctions are formed between unidentifiable cytoplasmic elements, presumably glia. Oligodendrocytes (Figs. 17, 18) and perivascular cells (Fig. 15) have been identified in the formation of some of these junctions but no neuronal elements have yet been seen in this connection.
Discussion
The question remains unanswered concerning the functional role of the oval nuclei. That is, do these nuclei represent a " c o m m a n d " or " r e l a y " center within the electromotor system of Torpedo ? The physiological evidence remains equivocal indicating only that these nuclei lie within the electromotor pathways (Fessard and Szabo, 1953) and that the large neurons are able to elicite a rhythmic discharge following a single afferent stimulus (Fessard and Szabo,
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1954). Whether the neuron population within these nuclei can mutually reach a threshold and discharge more or less synchronously, as would be expected of a " c o m m a n d " center, or whether they simply pass along an already synchronous signal, as would be expected of a " r e l a y " center, is not known. The oval nuclei are also a somewhat anomalous electromotor component apparently consisting of two long-axoned neuron species in contrast to the single long-axoned neuron composition typical in most electric fish (Szabo, 1961; Bennett et al., 1967a, b, c; Nakajima, 1970; Roberts and Ryan, 1975; Pappas et al., 1975). The large class neurons are clearly long-axoned and are the major if not sole effectors of the electric lobe (Eessard and Szabo, 1953, 1954; Szabo, 1961). The small class neuron is here postulated as having a long axon based on a classification relying on dendroarchitectonics and cellular form (Ram6n-Moliner, 1968, 1969, 1975; Leontovich, 1975), necessary due to our present ignorance concerning the true character of its axon. It is tempting to speculate that this nuclear structure may be somehow related to the different avoidance and prey-catching discharge patterns of this fish (Belbenoit, 1976; Krenz, 1976). It appears a reasonable assumption that some degree of synchronous activity is present within these nuclei as it has been shown that the electric lobe neurons discharge synchronously (Fessard and Szabo, 1953). Gap junctions have been found to be highly correlated with central motor components that exhibit synchrony and are electrotonically coupled (Bennett et al., 1967a, b, c; Meszler et al., 1972, 1974; Pappas et al., 1975). Gap junctions have not been found between neurons within either neonatal or adult (unpublished observations) Torpedo oval nuclei. This finding has also been reported in the skate (Raia clavata and Raia batis) command nucleus (Nakajima, 1970), though interpreted as further support of the gap junction-synchrony hypothesis as the skate discharges asynchronously. The absence of gap junctions in two genera of elasmobranch fish suggests that a phylogenetic difference between these and Teleost fish may exist. It has been proposed by the Scheibels (1975) that dendritic bundling (a common feature in Torpedo oval nuclei) is the repository of central programs for sequential motor activities. Related to this proposal is the concept of close membrane apposition and its possible structural relationship to electrotonicity (Grundfest, 1969; Sotelo, 1975). Electrical interaction has been demonstrated between cat lumbosacral motoneurons (Nelson, 1966) and frog spinal motoneurons (Grinnel, 1966; Magherini et al., 1976), both of which exhibit dendritic bundles (Matthews et al., 1971; Stensaas and Stensaas, 1971). The problem of whether gap junctions and/or close membrane appositions are responsible for the electrotonicity observed needs further study for gap junctions, though rarely seen, have been demonstrated in both of these animals by ZeiglgS.nsberger and Reiter (1974) and Sotelo and Taxi (1971) respectively. Electrotonic (ephaptic) transmission can theoretically occur if three conditions are met: 1) large areas of membrane apposition, 2) not more than 15 nm apart and 3) of low resistance (Bennett and Auerbach, 1969). Extensive close membrane appositions, well below the 15 nm theoretical limit, are common features between all elements (neural and glial) within the oval nuclei. These
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feature seems n o t to be a fixation artefact as it has been observed in adult tissue ( u n p u b l i s h e d observations) as well as in nuclei of n e w b o r n fish subjected to hypo- a n d h y p e r o s m o t i c fixation c o n d i t i o n s ( u n p u b l i s h e d observations). N o data are available c o n c e r n i n g the resistance of these m e m b r a n e s . It w o u l d a p p e a r at this p o i n t that gap j u n c t i o n s are the most likely m o r p h o l o g i c a l correlate of electrotonicity but that they may be present a n d required in so few n u m b e r s that specialized methods, such as intracellular dye injections, are necessary for their d e m o n s t r a t i o n . S y n c h r o n y m a y also be achieved by m e c h a n i s m s i n h e r e n t in the structure a n d f u n c t i o n i n g of axons. M a n y studies are presently available which indicate that the a x o n is far more sophisticated t h a n a simple cable c o n d u c t o r a n d that electric fish i n c o r p o r a t e a variety of design features in their axonal structure by which s y n c h r o n o u s m e c h a n i s m s can be explained (see review by W a x m a n , 1975). I n m a n y of these examples it would seem that structural m o d i f i c a t i o n of the a x o n is utilized to m a i n t a i n s y n c h r o n y rather t h a n to generate it. The afferent axons which give rise to en passant synapses a n d the efferent axons emerging from the oval nuclei in discrete spacially oriented b u n d l e s suggest two possible sources in which to pursue this particular point.
References Belbenoit, P. (Personal communication) (1976) Bennett, M.V.L.: Comparative physiology: electric organs. Ann. Rev. Physiol. 32, 471 528 (1970) Bennett, M.V.L., Auerbach, A.A.: Calculation of electrical coupling of cells separated by a gap. Anat. Rec. 163, 152 (Abstr.) (1969) Bennett, M.V.L., Nakajima, Y., Pappas, G.D. : Physiology and ultrastructure of electrotonic junctions. III. Giant electromotor neurons of Malapterurus electricus. J. Neurophysiol. 30, 209-235 (1967) Bennett, M.V.L., Pappas, G.D., Aljure, E., Nakajima, Y. : Physiology and ultrastructure of electrotonic junctions. II. Spinal and medullary electromotor nuclei in mormyrid fish. J. Neurophysiol. 30, 180-208 (1967) Bennett, M.V.L., Pappas, G.D., Jimbnez, M., Nakajima, Y.: Physiology and ultrastructure of electrotonic junctions. IV. Medullary electromotor nuclei in gymnotid fish. J. Neurophysiol. 30, 236-300 (1967) Fessard, A. : La synchronisation des activit6s ~l~mentairesdans les organes des poissons electriques. In: Bioelectrogenesis (Chagus and de Carvalho, eds.), 202 211. Amsterdam: Elsevier Publ. 1961 Fessard, A., Szabo, Th. : Sur l'organisation anatomofonctionelle des lobes 61ectriques de la Torpille. J. de Physiologie 45, 114-117 (1953) Fessard, A., Szabo, Th.: Etude microphysiologique du neurone intermediaire d'une chaine reflexe disynaptique. C.R. Soc. Biol. (Paris) 148, 281 284 (1954) Frontera, J.C.: Improved Golgi-type impregnation of nerve cells. Anat. Rec. 148, 371-372 (1964) Grinnell, A.D. : A study of the interaction between motoneurons in the frog spinal cord. J. Physiol. (Lond.) 182, 612 648 (1966) Grundfest, H.: Synaptic and ephaptic transmission. In: The structure and function of nervous tissue (Bourne, G.H., ed.), Vol. II, pp. 463 491. London-New York: Academic Press 1969 Krenz, W.D.: (Personal communication) (1976) Leontovich, T.A.: Quantitative analysis and classification of subcortical forebrain neurons. Golgi Cent. Symp. Proc. (Santini, M., ed.), pp. 101 122. New York: Raven Press 1975 Magherini, P.C., Prect, W., Schwindt, P.C. : Evidence for electrotonic coupling between frog motoneurons in the in situ spinal cord. J. Neurophys. 39, 474-483 (1976)
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Matthews, M.A., Willis, W.D., Williams, V.: Dendrite bundles in lamina IX of cat spinal cord: A possible source for electrical interaction between motoneurons. Anat. Rec. 171, 313 328 (1971) Meszler, R.M., Pappas, G.D., Bennett, M.V.E. : Morphological demonstration of electrotonic coupling of neurons by way of presynaptic fibres. Brain Res. 36, 412 415 (1972) Meszler, R.M., Pappas, G.D., Bennett, M.V.L.: Morphology of the electromotor system in the spinal cord of the electric eel, Electrophorus electricus. J. Neurocytol. 3, 251 261 (1974) Nakajima, Y.: Fine structure of the medullary c o m m a n d nucleus of the electric organ of the skate. Tissue and Cell 2, 47 58(1970) Nelson, P,G.: Interaction between spinal motoneurons of the cat. J. NeurophysioI. 29, 275 287 (1966) Pappas, G.D., W a x m a n , St.G., Bennett, M.V.L.: Morphology of spinal electromotor neurons and presynaptic coupling in the gymnotid Sternarchus alb(/i'ons. J. Neurocytol. 4, 469 478 (1975) Ram6n-Moliner, E.: A chlorate-formaldehyde modification of the Golgi method. Stain Technol. 32, 105 116 (1957) Ram6n-Moliner, E.: The morphology of dendrites, ln: The structure and function of nervous tissue (Bourne, G.H., ed.), Vol. I, pp. 205 267. London-New York: Academic Press 1968 Ram6n-Moliner, E. : The leptodendritic neuron: its distribution and significance. Ann. N.Y. Acad. Sci. 167, 65 70 (1969) Ram6n-Moliner, E.: Specialized and generalized dendritic patterns. Golgi Cent. Syrup. Proceed. (Santini, M., ed.), pp. 87 100. New York: Raven Press 1975 Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 212 (1963) Roberts, B.L., Ryan, K.P. : Cytological features of the giant neurons controlling electric discharge in the ray, Torpedo. J. Mar. Biol. Ass. U.K. 55, 123-131 (1975) Scheibel, M.E., Scheibel, A.B.: Dendrites as neuronal couplers: the dendrite bundle. Golgi Cent. Syrup. Proceed. (Santini, M., ed), pp. 347 354. New York: Raven Press 1975 Sotelo, C. : Morphological correlates of electrotonic coupling between neurons in m a m m a l i a n nervous system. Golgi Cent. Syrup. Proceed. (Santini, M., ed.), pp. 355 365. N e w Y o r k : Raven Press 1975 Sotelo, C., Taxi, J.: Ultrastructural aspects of electrotonic junctions in the spinal cord of the frog. Brain Res. 17, 137 141 (1970) Stensaas, L.J., Stensaas, S.S.: Light and electron microscopy of motoneurons and neuropile in the amphibian spinal cord. Brain Res. 31, 67 84 (1971) Szabo, Yh.: Anatomo-physiologie des centres nerveux sp6cifiques de quelques organes 61ectriques. In: Bioelectrogenesis (Chagus, de Carvalho, eds.), pp. 185 201. A m s t e r d a m : Elsevier Publ. 1961 W a x m a n , S.G.: Integrative properties and design principles of axons. Int. Rev. Neurobiol. 18, I 40 (1975) Zeiglg~insberger, W., Reiter, Ch. : ]nterneuronal movement of procion yellow in cat spinal neurones. Exp. Brain Res. 20, 527 530 (1974)
Accepted December 19, 1976
Cell and Tissue Research i:' by Springer-Verlag 1977
The Morphology of the Oval Nuclei of Neonatal Torpedo marmorata Geoffrey Q. Fox Department of Neurochemistry, Max-Planck-lnstitut fiir biophysikalische Chemie, G6ttingen, Germany
Summary. The morphology of the oval nucleus of neonatal Torpedo marmorata is described at the light and electron microscopic level of examination. The nucleus is unique relative to other central electromotor centers of electric fish so far described being bilaterally symmetrical, composed of two nerve cell types, and possessing no gap junctions between neurons and their processes. This particular structural plan presents difficulties in accounting for presumed synchronous discharge since it has been strongly argued that electrotonic coupling by means of gap junctions is the primary process by which synchronization is accomplished. Close membrane apposition and dendritic bundling, common features within the nucleus, are discussed as possible alternative structural correlates.
Key words: Electromotor system - Oval nucleus - Electron microscopy - Torpedo marmorata.
Synapses, junctions
Introduction This report concerns a morphological examination of the oval nucleus of neonatal Torpedo marmorata. This fish has a highly synchronized electric system (Fessard, 1961; Szabo, 1961) but little is known concerning the structure or function of its central electromotor components. Central " c o m m a n d " and " p a c e m a k e r " nuclei in electric fish are generally single nuclei located midventrally within the primitive brain stem regions of mesencephalon, medulla, or cervical spinal cord (Bennett, 1970) and represent phylogenetically old structures. In addition to initiating the discharge signal they provide the necessary Send offprint requests to." Dr. Geoffrey Q. Fox, Department of Neurochemistry, Max-Planck-lnstitut ftir biophysikalische Chemie, Am Fassberg, 3400 G6ttingen, Federal Republic of Germany Acknowledgements: I would like to express my appreciation to Drs. V.P. Whittaker, J.-R. Wolff, G.D. Pappas and M.V.L. Bennett for their critical evaluation of the manuscript and to W.D. K6tting and H. Ellermeier for their technical assistance
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s y n c h r o n i z a t i o n so t h a t t h e final d i s c h a r g e is o f m a x i m u m e f f e c t i v e n e s s . T h e oval nuclei are central c o m p o n e n t s of the m o t o r division a n d are located within the a n t e r i o r medulla. T h e y project to the electric lobes, p a i r e d nuclei c o m p o s e d o f g i a n t e l e c t r o m o t o r n e u r o n s w h i c h in t u r n d r i v e t h e p e r i p h e r a l e l e c t r o c y t e s t h a t m a k e u p t h e e l e c t r i c o r g a n . T h e i r c e n t r a l l o c a t i o n as well as t h e i r p o s i t i o n in t h e e l e c t r o m o t o r p a t h w a y s u g g e s t s t h a t t h e s e n u c l e i s e r v e e i t h e r a " c o m m a n d " or " p a c e m a k e r " function.
Material and Methods Newborn Torpedo marmorata, approximately 120 mm in length and 50 g in weight, were used in this study. Neonatal animals were chosen primarily because of our overall interest in the development of the electromotor system. These animals are well developed at birth, swimming well and exhibiting behavior patterns similar to those of the adults. Their electric organs are functional at this time and display mature discharge patterns (Krenz, 1976); thus our assumption is that the electromotor system is synchronized. The animals were anaesthetized with MS222 (Sandoz, 0.5 g/liter of sea water), the pericardial cavity exposed, and a cannula inserted through the heart into the conus arterious. Fifty milliliters of 1% paraformaldehyde and 2.5% glutaraldehyde buffered to pH 7.0 with 0.43 M sodium cacodylate (approximately 900 mOsmols) were perfused into the animals with a hand-held syringe and allowed to fix the tissue for two hours at room temperature. An additional fifty milliliters of 5% glutaraldehyde in the same buffer was then perfused and the animals were set aside for an additional three hours. The brain was removed and the desired tissue blocks cut and washed in several rinses of fresh buffer. The tissue blocks were postfixed in 0.43 M sodium cacodylate-buffered 1% osmium tetroxide for two hours at room temperature. An en block staining procedure consisting of an overnight washing in cold 0.4 M sodium acetate buffer (pH 5.5) followed by 1% uranyl acetate in 0.25 M sodium acetate (pH 5.5) for four hours in the cold and dark was routinely used to enhance the membrane contrast. Dehydration in graded ethanols (50% 100%) and embedding in Epon 812 completed the preparation for the electron microscope. Golgi-stained material was prepared by a chloral formaldehyde procedure (Ramon-Moliner, 1957; Frontera, 1964). The following solution was perfused through anaesthetized animals: potassium dichromate (5 g), chloral hydrate (5 g), formalin (10 ml of 10%), glutaraldehyde (5 ml of 25%), and water (75 ml). After three hours the brains were removed and immersed in the above solution for 2~4 days changing the solution daily. The brains were then placed in 0.75% silver nitrate for four days in the dark at room temperature also changing the solution daily. The brains were dehydrated in ethanol and embedded in a soft mixture of Epon 812. Sections (0.1 0.2 mm) were cut on a sledge microtome and mounted on glass slides for viewing. A Zeiss drawing apparatus mounted on a Zeiss Standard binocular microscope was used for making composite drawings of the Golgi stained material. Routine light microscopy was made from Toluidine Blue-stained sections (1 lam) cut from the material prepared for electron microscopy. Thin sectioned material for the electron microscope was doubly stained with aqueous uranyl acetate and lead citrate (Reynolds, 1963). Viewing and photography was done with a Zeiss Photomicroscope II and a Jeol 100B electron microscope.
Results Gross Structure T h e o v a l n u c l e i a r e t w o in n u m b e r a n d s i t u a t e d b i l a t e r a l l y in t h e m i d - v e n t r a l , r o s t r a l m e d u l l a . E l l i p s o i d a l in s h a p e , e a c h n u c l e u s m e a s u r e s a p p r o x i m a t e l y 1.2 m m ( a n t e r i o r - p o s t e r i o r ) b y 0.5 m m in d i a m e t e r . T h e g e n e r a l s t r u c t u r e o f
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Fig. 1. Transverse section through the anterior medulla of newborn Torpedo showing an oval nucleus. Horizontal (H) and vertical (V) large class neurons make up a central core region. Their axons are directed medially and exit the nucleus as bundles (A). Dendrites from the neuronal population form a dense cortical layer ((7) around the nucleus. Toluidine Blue-stained 1 lam section. (Bar 0.1 mm) Fig. 2. Horizontal section through junctional region between core and dendritic cortex. Large class neurons (L), small class neurons (S) and dendritic bundles (d) are seen. Toluidine Blue-stained 1 ~tm section. ( B a r = 30 lam)
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3 \,
/
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i
Figs. 3-5. Reconstructed composite drawing from Golgi stained material showing three aspects of the oval nucleus. Dotted line roughly outlines the core region. It is clear from the sagittal (3) and horizontal (4) planes of section that the large class neurons assume two orientations-vertical and horizontal respectively, a fact obscured when considering only the transverse (5) plane. The small neurons (5') tend to assume only a horizontal orientation within the nucleus. The electromotor neurons (e) within the electric lobe of Figure 5 are approximately 65 gm in diameter
each nucleus consists o f a p e r i k a r y o n - r i c h core, a s u r r o u n d i n g dendritic cortex a n d a m e d i a l a x o n a l zone (Fig. 1). T h e nuclei are c o m p o s e d o f two n e u r o n a l p o p u l a t i o n s o f large a n d small cells a n d also neuroglia. The large n e u r o n s a p p e a r to p r e d o m i n a t e n u m e r i c a l l y a n d f o r m the core region whereas the small n e u r o n s are f o u n d a l o n g the p e r i p h e r y o f the core a n d to a lesser extent within the dendritic c o r t e x (Fig. 2). N e u r o g l i a cells are d i s t r i b u t e d t h r o u g h o u t the entirety o f each nucleus.
Core Region T h e core region is p r i m a r i l y c o m p o s e d o f the cell b o d i e s o f a p o p u l a t i o n o f large, elongate, r o u g h l y cylindrical n e u r o n s ( a p p r o x i m a t e l y 150 l a m x 30 gm). The m a j o r axis o f these n e u r o n s lies within the transverse plane (Figs. 3-5) but m a y be o r i e n t e d in either a h o r i z o n t a l or vertical direction (Figs. 1, 3 5) with the vertically o r i e n t e d n e u r o n s a s s u m i n g a m o r e p e r i p h e r a l p o s i t i o n with respect to the h o r i z o n t a l neurons. Cells o f b o t h o r i e n t a t i o n s a p p e a r m o r p h o l o g -
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Fig. 6. Ultrastructural detail of small class neuron showing nucleus (N), slightly organized endoplasmic reticulum and two axo-somatic synapses. (Bar= 1 lam) Fig. 7. Large class neuron with nucleus (N), Golgi complex (G), lysosome (/), and non-laminar endoplasmic reticulum with intervening channels (c) containing fibrils. (Bar = 1 lain)
ically similar with regard to their size, distribution of processes, and organelle composition. The cell body contains large areas of short, nonoriented formations of endoplasmic reticulum most of which are free of bound ribosomes (Fig. 7). Many free ribosomes and ribosomal rosettes are found within the intervening spaces (Fig. 7). These Nissl areas are surrounded by mitochondria and often separated from one another by clear channels filled with fibrillar and tubular elements or by extensive systems of Golgi membranes (Fig. 7). Densely stained, irregular membrane-boundedlysosomes are also a common feature (Fig. 7). These organelle systems are also present throughout the primary dendrites but not within the initial axonal segment, which is composed of neurofilaments, mitochondria, vesicles and smooth reticular membranes. Single cilia have also been seen on the cell bodies with the basal body contained within a Golgi complex. The nucleus is spherical and approximately 25 btm in diameter containing a single (Figs. 1, 2) or sometimes several darkly staining nucleoli. All of these large cells appear to project their axons medially, with the intitial segment contained within the core region (by definition). Collateral branches of unknown length are often seen on the initial segments (Fig. 8). The dendrites, in contrast, project in other than medial directions, the major thrust being lateral and in a transverse (dorso-ventral) plane (Figs. 3-5). As seen in Golgi preparations, the primary dendrites emerge from all aspects of the cell body (Figs. 3-5), some in very close proximity to the initial axonal
Fig. 8. Cotlateral axonaI branch of the initial segment (/) of a large neuron axon. Two axo-axonic synapses (arrows) containing spherical vesicles are present. ( B a r - 1 gm) Fig. 9. Distal portion of a primary dendritic branch from a large class neuron located in the peripheral region between the core and dendritic cortex. Characteristic dendritic organelles are p r e s e n t - e n d o p l a s m i c reticulum, ribosomes, tubules and fibrils, mitochondria and large lysosomes. Several axo-dendritic synapses of the asymmetric variety with spherical and dense core vesicles are seen (arrows). (Bar = 1 gin) Fig. 10. An en passant axon making numerous synaptic contacts (A) with neural elements in its path: (L) large neuron cell body, (D1 z) dendrites. Asymmetric contact zone, spherical vesicles and axial fibrillar and tubular core are seen. Close membrane apposition is present between (D1) and (D3) with a specialized desmosomal region (arrow). ( B a r = 2 gm) Fig. 11. Details of closely adjacent large neuron cell bodies, showing a desmosomal contact between a somatic spine (S) and the perikaryal membrane. N m n e r o u s axo-somatic synapses are present. (Bar = 1 gin)
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segment. They are typically straight and remain unbranched for long distances. Secondary dendrites are less commonly seen and tertiary branches only occasionally encountered. Dendritic spines are rarely seen. Synapses are found on the cell body (Fig. 11), primary dendrites (Fig. 9) and initial segments of axons and axonal collaterals (Fig. 8).
Small Neurons
A lesser number of small fusiform neurons (cell bodies approximately 20 i.Lm in diameter) with spherical (13 gm in diameter) or occasionally oval nuclei are present within the peripheries of the core region and the dendritic cortex (Fig. 2). They are also oriented in the transverse plane with a major dendritic process directed medially and another laterally and occasional second order branches projecting off these two primary processes (Fig. 3-5). No data are as yet available concerning the character of the axon. Within the cytoplasm the endoplasmic reticulum is evenly distributed, somewhat oriented into stacks and partially studded with ribosomes (Fig. 6). Golgi complexes are few in number and less extensive than those seen in the large neurons. Free ribosomes and ribosomal rosettes, mitochondria, lysosomes, and occasional tubules, filaments and vacuoles make up the remaining cytoplasm (Fig. 6). Axo-somatic (Fig. 6) and axo-dendritic synapses are present in small numbers.
Dendritic Cortex
A cortical layer composed of the dendritic arborizations from both the large and small neurons surrounds much of the core region (Fig. 1). Horizontal sections through this region reveal dendritic bundling and suggest that the dendritic architectonics are oriented along a dorso-ventral axis in the transverse plane (Figs. 2, 12). The cortex is intersected by small caliber myelinated and unmyelinated axons, many of which are oriented along a rostro-caudal axis. Axo-dendritic synapses are established on the core dendrites by axons from these unidentified pathways and others. Axons also pass through the cortex into the core region, presumably to synapse upon the cell bodies of the large neurons.
Medial Axonal Region
The axons of the large neurons exit from the medial aspect of the nucleus (Fig. 1). Their initial segments converge within the core to form small bundles, becoming myelinated at the junction of the core and the medial axonal region (by definition). These axons are from 7 to 9 ~tm in diameter at the first myelin internode. Smaller caliber afferent fibers are also present within these bundles and pass into the core, terminating on neural elements. Individual fibers may
Fig. 12. Horizontal section through dendritic cortex showing density and spacial orientation of the dendritic bundles. Close membrane apposition and desmosomal contacts between adjacent dendrites are c o m m o n features (A). Axo-dendritic synapses are also present in large numbers. (Bar = 4 gm) Fig. 13. Higher power view of a typical dendritic m e m b r a n e apposition as illustrated in the preceding figure. Close non-specialized membrane apposition with intervening 8 10 n m space changes to a desmosomal-like zone with an approximate 20 n m space and heavy subjacent densities. ( B a r = 0.5 p.m) Fig. 14. A region illustrating two c o m m o n synaptic structures of the oval nucleus and also close m e m b r a n e apposition. Axo-dendritic en passant synapses (A) and an individual axo-dendritic synapse (B) have formed upon the same dendrite (D). Close membre appositions ( < 15 nm) (arrows) exist throughout. ( B a r = 1 gm)
Figs. 15-18 illustrate the commonly encountered examples of gap junctions within the core and dendritic cortex region of the oval nucleus Fig. 15. Gap junction (arrow) between pericapillary cell (P) and neural element. Capillary lumen is above. (Bar=0.5 gm) Fig. 16. Gap junction (arrow) between two presumptive glial processes in close vicinity to an axodendritic synapse. (Bar=0.25 gin) Fig. 17. Gap junction (arrow) between densely stained oligodendrocyte process and unidentifiable neural process. (Bar=0.5 lam) Fig. 18. Similar to Figure 17 but illustrating a non-typically extensive junction. (Bar=0.25 gm)
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be seen branching off these bundles as they project medially but it is not known whether these are afferent or efferent axons. At the raphe, few fibers are seen to cross but more typically to bend either dorsally or ventrally and apparently to travel within the raphe region. No communication between the two nuclei has been noted.
Synapses and Junctions Chemical synapses and gap junctions are present within the oval nuclei. Chemical synapses are formed from individual and en passant boutons. The individual boutons are derived from myelinated and unmyelinated axons entering the nucleus (dendritic cortex and core) from lateral or medial directions. Terminal boutons contain spherical (Figs. 6, 8 11, 14, 16) and occasional pleomorphic vesicles and establish axo-somatic (Figs. 6, 11), axo-dendritic (Fig. 9) and axoaxonic (Fig. 8) synapses. Contact with multiple post-synaptic elements is common. En passant boutons also are derived from myelinated and unmyelinated axons seemingly entering the nucleus from all directions. Nodules along the unmyelinated axon cylinder contain spherical synaptic vesicles that make asymmetric synaptic contact with many of the neural elements in their path (Fig. 10). Desmosomal contacts are numerous between all unmyelinated neural processes in the nucleus. They consist of various amounts of dense granular accumulations along the subjacent membrane surfaces, with the intervening intercellular space (approximately 20 nm) often containing a somewhat less dense amorphous material (Fig. 13). Adjacent to these contacts as well as to synapses but common throughout the entirety of the nucleus are regions of close neural membrane apposition characterized by intercellular spaces within a range of 6-12 nm and by the absence of intervening glial processes (Figs. 12-14). Gap junctions containing an approximate 2 to 4 nm gap and of up to 1.5 IJm in length have been identified in both the dendritic cortex and core regions of the nucleus (Figs. 15-18). Though frequently in close proximity to chemical synapses (Fig. 16), the majority of gap junctions are formed between unidentifiable cytoplasmic elements, presumably glia. Oligodendrocytes (Figs. 17, 18) and perivascular cells (Fig. 15) have been identified in the formation of some of these junctions but no neuronal elements have yet been seen in this connection.
Discussion
The question remains unanswered concerning the functional role of the oval nuclei. That is, do these nuclei represent a " c o m m a n d " or " r e l a y " center within the electromotor system of Torpedo ? The physiological evidence remains equivocal indicating only that these nuclei lie within the electromotor pathways (Fessard and Szabo, 1953) and that the large neurons are able to elicite a rhythmic discharge following a single afferent stimulus (Fessard and Szabo,
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1954). Whether the neuron population within these nuclei can mutually reach a threshold and discharge more or less synchronously, as would be expected of a " c o m m a n d " center, or whether they simply pass along an already synchronous signal, as would be expected of a " r e l a y " center, is not known. The oval nuclei are also a somewhat anomalous electromotor component apparently consisting of two long-axoned neuron species in contrast to the single long-axoned neuron composition typical in most electric fish (Szabo, 1961; Bennett et al., 1967a, b, c; Nakajima, 1970; Roberts and Ryan, 1975; Pappas et al., 1975). The large class neurons are clearly long-axoned and are the major if not sole effectors of the electric lobe (Eessard and Szabo, 1953, 1954; Szabo, 1961). The small class neuron is here postulated as having a long axon based on a classification relying on dendroarchitectonics and cellular form (Ram6n-Moliner, 1968, 1969, 1975; Leontovich, 1975), necessary due to our present ignorance concerning the true character of its axon. It is tempting to speculate that this nuclear structure may be somehow related to the different avoidance and prey-catching discharge patterns of this fish (Belbenoit, 1976; Krenz, 1976). It appears a reasonable assumption that some degree of synchronous activity is present within these nuclei as it has been shown that the electric lobe neurons discharge synchronously (Fessard and Szabo, 1953). Gap junctions have been found to be highly correlated with central motor components that exhibit synchrony and are electrotonically coupled (Bennett et al., 1967a, b, c; Meszler et al., 1972, 1974; Pappas et al., 1975). Gap junctions have not been found between neurons within either neonatal or adult (unpublished observations) Torpedo oval nuclei. This finding has also been reported in the skate (Raia clavata and Raia batis) command nucleus (Nakajima, 1970), though interpreted as further support of the gap junction-synchrony hypothesis as the skate discharges asynchronously. The absence of gap junctions in two genera of elasmobranch fish suggests that a phylogenetic difference between these and Teleost fish may exist. It has been proposed by the Scheibels (1975) that dendritic bundling (a common feature in Torpedo oval nuclei) is the repository of central programs for sequential motor activities. Related to this proposal is the concept of close membrane apposition and its possible structural relationship to electrotonicity (Grundfest, 1969; Sotelo, 1975). Electrical interaction has been demonstrated between cat lumbosacral motoneurons (Nelson, 1966) and frog spinal motoneurons (Grinnel, 1966; Magherini et al., 1976), both of which exhibit dendritic bundles (Matthews et al., 1971; Stensaas and Stensaas, 1971). The problem of whether gap junctions and/or close membrane appositions are responsible for the electrotonicity observed needs further study for gap junctions, though rarely seen, have been demonstrated in both of these animals by ZeiglgS.nsberger and Reiter (1974) and Sotelo and Taxi (1971) respectively. Electrotonic (ephaptic) transmission can theoretically occur if three conditions are met: 1) large areas of membrane apposition, 2) not more than 15 nm apart and 3) of low resistance (Bennett and Auerbach, 1969). Extensive close membrane appositions, well below the 15 nm theoretical limit, are common features between all elements (neural and glial) within the oval nuclei. These
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feature seems n o t to be a fixation artefact as it has been observed in adult tissue ( u n p u b l i s h e d observations) as well as in nuclei of n e w b o r n fish subjected to hypo- a n d h y p e r o s m o t i c fixation c o n d i t i o n s ( u n p u b l i s h e d observations). N o data are available c o n c e r n i n g the resistance of these m e m b r a n e s . It w o u l d a p p e a r at this p o i n t that gap j u n c t i o n s are the most likely m o r p h o l o g i c a l correlate of electrotonicity but that they may be present a n d required in so few n u m b e r s that specialized methods, such as intracellular dye injections, are necessary for their d e m o n s t r a t i o n . S y n c h r o n y m a y also be achieved by m e c h a n i s m s i n h e r e n t in the structure a n d f u n c t i o n i n g of axons. M a n y studies are presently available which indicate that the a x o n is far more sophisticated t h a n a simple cable c o n d u c t o r a n d that electric fish i n c o r p o r a t e a variety of design features in their axonal structure by which s y n c h r o n o u s m e c h a n i s m s can be explained (see review by W a x m a n , 1975). I n m a n y of these examples it would seem that structural m o d i f i c a t i o n of the a x o n is utilized to m a i n t a i n s y n c h r o n y rather t h a n to generate it. The afferent axons which give rise to en passant synapses a n d the efferent axons emerging from the oval nuclei in discrete spacially oriented b u n d l e s suggest two possible sources in which to pursue this particular point.
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Matthews, M.A., Willis, W.D., Williams, V.: Dendrite bundles in lamina IX of cat spinal cord: A possible source for electrical interaction between motoneurons. Anat. Rec. 171, 313 328 (1971) Meszler, R.M., Pappas, G.D., Bennett, M.V.E. : Morphological demonstration of electrotonic coupling of neurons by way of presynaptic fibres. Brain Res. 36, 412 415 (1972) Meszler, R.M., Pappas, G.D., Bennett, M.V.L.: Morphology of the electromotor system in the spinal cord of the electric eel, Electrophorus electricus. J. Neurocytol. 3, 251 261 (1974) Nakajima, Y.: Fine structure of the medullary c o m m a n d nucleus of the electric organ of the skate. Tissue and Cell 2, 47 58(1970) Nelson, P,G.: Interaction between spinal motoneurons of the cat. J. NeurophysioI. 29, 275 287 (1966) Pappas, G.D., W a x m a n , St.G., Bennett, M.V.L.: Morphology of spinal electromotor neurons and presynaptic coupling in the gymnotid Sternarchus alb(/i'ons. J. Neurocytol. 4, 469 478 (1975) Ram6n-Moliner, E.: A chlorate-formaldehyde modification of the Golgi method. Stain Technol. 32, 105 116 (1957) Ram6n-Moliner, E.: The morphology of dendrites, ln: The structure and function of nervous tissue (Bourne, G.H., ed.), Vol. I, pp. 205 267. London-New York: Academic Press 1968 Ram6n-Moliner, E. : The leptodendritic neuron: its distribution and significance. Ann. N.Y. Acad. Sci. 167, 65 70 (1969) Ram6n-Moliner, E.: Specialized and generalized dendritic patterns. Golgi Cent. Syrup. Proceed. (Santini, M., ed.), pp. 87 100. New York: Raven Press 1975 Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208 212 (1963) Roberts, B.L., Ryan, K.P. : Cytological features of the giant neurons controlling electric discharge in the ray, Torpedo. J. Mar. Biol. Ass. U.K. 55, 123-131 (1975) Scheibel, M.E., Scheibel, A.B.: Dendrites as neuronal couplers: the dendrite bundle. Golgi Cent. Syrup. Proceed. (Santini, M., ed), pp. 347 354. New York: Raven Press 1975 Sotelo, C. : Morphological correlates of electrotonic coupling between neurons in m a m m a l i a n nervous system. Golgi Cent. Syrup. Proceed. (Santini, M., ed.), pp. 355 365. N e w Y o r k : Raven Press 1975 Sotelo, C., Taxi, J.: Ultrastructural aspects of electrotonic junctions in the spinal cord of the frog. Brain Res. 17, 137 141 (1970) Stensaas, L.J., Stensaas, S.S.: Light and electron microscopy of motoneurons and neuropile in the amphibian spinal cord. Brain Res. 31, 67 84 (1971) Szabo, Yh.: Anatomo-physiologie des centres nerveux sp6cifiques de quelques organes 61ectriques. In: Bioelectrogenesis (Chagus, de Carvalho, eds.), pp. 185 201. A m s t e r d a m : Elsevier Publ. 1961 W a x m a n , S.G.: Integrative properties and design principles of axons. Int. Rev. Neurobiol. 18, I 40 (1975) Zeiglg~insberger, W., Reiter, Ch. : ]nterneuronal movement of procion yellow in cat spinal neurones. Exp. Brain Res. 20, 527 530 (1974)
Accepted December 19, 1976
