Some aspects of the structural organization of the spinal cord of Gymnotus carapo (Teleostei, gymnotiformes) II. The motoneurons

JOURNALOF

ULTRASTRUCTURE

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MOLECULAKSTRUCTURE

RESEARCH

101,224-235 (1988)

Some Aspects of the Structural Organization of the Spinal Cord of Gymnotus carapo (Teleostei, Gymnotiformes) II. The Motoneurons ~.TRUJILLO-CEN~ZANDC.

BERTOLOTTO

Division of Compurutive Neurounatomy, Institute de Investigaciones Bioldgicas Avenida Italiu 3318, Montevideo, Uruguuv

“Clemente Estable. ”

Received December 2. I988 The use of horseradish peroxidase as neuronal marker has allowed us to distinguish in the spinal cord of the weakly electric fish Gymnom curupo two main populations of motoneurons: the periependymal motoneurons which innervate the axial musculature and the ventral motoneurons which innervate the appendicular muscles. The ventral horns were explored by means of conventional staining procedures and divided into four dorsoventral zones (I-IV). The largest motoneurons lying in the periependymal gray have been tentatively identified as the “primary motoneurons” (PM) of this species. The PMs are located forming two columns at each side of the central canal. They are characterized by their large size (N-7.5 pm), the occurrence of four or five thick dendritic trunks, and the peculiar intraspinal pathways of their axons. The dendritic trunks originate exclusively from the lateral surface of each neuron and project toward the ipsilateral, most dorsal neuropiles of the ventral horns (zones I and II). The axons follow ventromedial courses, close to the ipsilateral Mauthner fiber. The ventral motoneurons innervating the muscles of the anal fin are located within zones III and IV of the anterior horns. They are arranged in groups distributed along the cord; each group consists of 15-20 small- and medium-size neurons. The muscles of the pectoral fins receive their innervation from motoneurons lying in the ventral portion of the transitional zone between the medulla and the spinal cord. Three kinds of nerve terminals have been found impinging on the motoneuron somata and dendrites: (a) terminals with the fine structural characteristics of chemical synapses. (b) terminals identified as “mixed junctions” (chemical and electrical), and (c) terminals containing flat vesicles (proposed as serving inhibitory functions). Adjacent nerve terminals show zones in which the plasma membranes lie closely apposed. At these levels there are membrane pentalaminar patches (gap junctions) similar to those found at the level of the mixed junctions. These patches may facilitate, by means of electrotonic coupling, the synchronized activity of several synaptic terminals. The functional properties of the motoneurons of Gymnotus remain unexplored. These morphological studies have been initiated to support forthcoming electrophysiological investigations. C 1988 Academic Press. Inc.

Gymnotoid fish perform, together with well-studied electrogenic activity, peculiar swimming movements which permit one to distinguish them from other groups of teleosts. It was recognized early (Garden, 1774; cited by Ellis, 1913) that these peculiar movements are mediated by a particularly long anal fin. By means of its undulating motion, the animal progresses, retreats, or remains stationary counteracting water currents. While these movements are performed, the electrogenic organ (EO) remains straight, without experiencing major flexures. According to Lissmann (1958),

this feature is an important requirement for detecting changes in autogenerated electric fields. Like other teleosts, gymnotoid fish are also able to make forward thrusts and turns which demand both contractions of the trunk-tail muscles and vigorous strokes of the pectoral fins. The present paper, which complements a previous one dealing with electromotor neurons (EMNs) (Trujillo-Cenoz et al., 1986), is concerned with the location, morphology, and synaptology of the main classes of motoneurons occurring in the spinal cord of Gymnotus carapo. 224

0889- 1605/88 $3 .oo Copyright0 1988by Academic Press. Inc All rights ofreproduction in any form reserved.

STRUCTURAL

ORGANIZATION

Three main muscular groups can be recognized in this kind of fish: (a) axial muscles which participate in the forceful flip of the trunk-tail region (they are dorsoventrally arranged in no less than seven longitudinally oriented fascicles (Ellis, 1913)), (b) muscles moving the pectoral fins, and (c) numerous slender muscles whose harmonic contractions produce the wave motion of the anal fin (there is a pair of small oblique muscles inserted on each side of a single anal ray). Retrograde labeling of the nerve cell bodies with horseradish peroxidase (HRP) was used to locate the different motoneuron pools and to obtain information concerning the morphology of the different types of spinal motoneurons. The fine structure of the synaptic terminals was explored by means of electron microscope techniques.

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chloride as chromogen. Adams’s intensification procedure (Adams, 1977) was routinely employed for light microscope studies. For electron microscopy the nerve tissues were posttixed with 1% 0~0, in phosphate buffer. The pieces were dehydrated in the graded alcohol or acetone series followed by 100% acetone. Durcupan ACM (Fluka) or Vestopal W were used as embedding materials. When HRP-labeled neurons were utilized, transillumination of the blocks proved helpful in achieving a precise trimming of the cutting faces. Several series of sections from different spinal cord regions were obtained with an LKB Ultratome and subsequently doubly stained with uranyl acetate and lead citrate. The ultrathin sections were mounted on one-hole Sjostrand-type grids (1 x 2 mm), covered with Formvar films. Semithin sections (OS-1 urn) cut at different planes (transverse, horizontal, and parasagittal) were stained with boraxic methylene blue and examined under a light microscope. Additional technical details can be found in our previous paper dealing with the electromotor neurons of G. carupo (TrujilloCen6z et al., 1986). RESULTS

MATERIALS

AND METHODS

Fifty G. carapo were used in this investigation. The specimens were collected from a lake located in the southeast region of Uruguay (“Laguna de1 Sauce,” Departamento de Maldonado). All surgical procedures were performed on deeply ether-anesthetized fish, kept oxygenated by water passing through the gills. Horseradish peroxidase (Sigma, Type VI) was used as neuronal tracer. To achieve HRP labeling of the spinal motoneurons two technical approaches were employed: (a) insertion of HRP-soaked micropellets in the different muscular groups (the pellets were made with “Microsponge Surgery,” Alcon Laboratories, Inc., and were soaked in a 50% aqueous solution of HRP); (b) transections of the ventral spinal roots, followed by the application of HRP crystals on the proximal stumps of the severed fibers. The first procedure secured specific labeling of the different motoneuron pools while the second yielded Golgi-like images of all classes of neurons projecting their axons outside the spinal cord. These two technical approaches complement each other, allowing both the unambiguous identification of the different neuronal groups and the morphological analysis of their constituent units. After S-10 days of survival, the anesthetized animals were fixed by perfusion with a phosphate-buffered dialdehyde mixture, consisting of l’% paraformaldehyde and 1% glutaraldehyde (0.1 M, pH 7.4). Selected segments of the spinal cord were Vibratome-sectioned at 60-100 urn in the transverse, horizontal, or parasagittal planes. Peroxidase activity was revealed using 3’-3’diaminobenzidine tetrahydro-

A. Light Microscopy (1) The neuroarchitecture

of the cord.

General information concerning the neuroarchitecture of the cord was obtained from the study of semithin, methyleneblue-stained sections cut following different planes. The same technique also proved helpful in our attempts to perform a detailed, systematic analysis of the ventral horns. As previously reported for other teleosts, the gray matter appears in cross sections, as an inverted Y (Beccari, 1943). At most levels of the cord the two dorsal horns are represented by a single, vertical plate, continuous with the periependymal gray (Fig. 1). Surrounding the central canal, there are three main columns of neurons: a dorsal, consisting of EMNs supplying the electrogenic organ, and two laterals containing the motoneurons innervating the axial musculature. The ventral or anterior horns are seen as two obliquely oriented gray matter plates, which converge toward the ventral midline. The white matter consists of three main fields: a ventral funiculus and two lateral funiculi.

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FIG. I. This microphotographic collage shows the neuroarchitecture of the spinal cord of G. curapo as it appears in transverse, semithin sections stained with methylene blue. The gray matter is seen as an inverted Y with the two dorsal horns forming a single medial plate continuous with the periependymal region. The somata of two PMs (PM) lie at both sides of the central canal (arrow). The distribution of the neuronal bodies and the characteristics of the neuropile fields taken into account, each ventral horn was divided into four dorsoventral zones (I-IV) (for details see text). EM, electromotor neuron: MF. Mauthner fibers; VM, ventral motoneurons innvervating the anal fin; VR. ventral root. x 208.

Taking into account the number of myelinated fibers as well as the distribution of the neuron somata it is possible to recognize in each ventral horn four dorsoventral zones (I-IV in Fig. 1). Zone I consists of a narrow neuropile layer emitting dorsal and lateral projections toward the adjacent white matter. Minute and small myelinated axons have been consistently found at this level. Zone II appears as a wide homogeneously stained area, practically devoid of both myelinated axons and neuron somata.

Zone III contains groups of small- and medium-size neuronal bodies; it shows a neuropile band with bundles of myelinated axons. Zone IV, which occupies the most ventral portion of the horn, also contains neuron somata and small-diameter myelinated axons. The histological organization described has not been found at the most caudal levels of the cord. In these terminal segments (composing the last 30 or 40% of the fish length) a less differentiated neural organization which reflects the plastic po-

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tentialities of the organexists. Therefore, a descriptionof thesemost caudalportions of the cord will be included in a forthcoming paper addressingthe regeneration of the electromotor system of Gymnotus carupo (Echagtieet al., in preparation). (2) Neurons innervating

the axial muscu-

future. When the HRP-embeddedpellets were introduced within the axial muscular masses, the retrograde transported enzymes appearedlocated within large neurons (their sizesrangingbetween50 and 75 pm) lying in the periependymalgray. These neuronsform bilateral, continuouscolumns clearly identified except at most caudalsegments of the cord. When stainedwith boraxic methylene blue, the cell bodies show large, conspicuous, Nissl bodies. The geometry of these cells was revealed by means of massive, Golgi-like, HRP staining. As shown in Fig. 2 (top) each neuron bears four or five thick dendritic trunks which, after crossing the wedge of white matter separatingthe periependymalgray from the ventral horn, project toward the ipsilateral zones I and II. The horizontal sections show that each one of these main dendritic trunks gives rise to two secondary brancheswhich run in opposite, longitudinal directions (Fig. 2 (bottom). Some of them reachmore than 300km in length and overlap with those stemming from neighboring dendritic trunks. These featuresdetermine the occurrence,within zonesI and II of the ventral horns, of several dorsoventral dendritic strata. In turn, the secondary branchesare the origin of slender,less conspicuousdendtitic processeswhich after traveling short, transverse pathways, ramify in the neuropile. It is worth noting that neither the main dendritic trunks nor their branches bear spines. The correspondingaxon arisesfrom the ventral pole of the cell and follows a dorsoventralpathway close to the medial surfaceof the ipsilateral Mauthner axon. Similar features have beenfound in nonelectric teleostsand the first amyelinic portion of this process has been interpreted as a peculiar kind of

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ventral dendrite“connected in series” with the efferent axon (Tiegs, 1931; Diamond, 1971).At the level of each ventral root, the axons stemming from these large neurons constitute a small, well-defined population of six to eight axons, with diameters ranging between 8 and 10 pm. On the basis of the aforementioned characteristics the large motoneurons of the periependymal gray have beententatively identified as the primary motoneurons(PM) of G. carupo. (3) Neurons innervating

the analfin

mus-

cles. The nervessupplyingthe tiny muscles that move the anal fin originate from metamericallydistributed neuronalgroups, locatedwithin zonesIII and IV of the ventral horns (Fig. 3 (top and bottom)). Each group consists of a variable number (usually 15-17)of small- and medium-size neurons (the somatarangingbetween10and 25 pm) which project their slender dendritic shafts toward zones I and II of the ipsilatera1ventral horn. The small-diameteraxons (below 4 pm) leave the cord through the metamerically correspondentroots. (4) Neurons innervating the pectoral fin muscles. These neurons form a well-

characterized population occupying the most caudal region of the medulla and also invading the first spinal segments(Fig. 4 (left and right)). As expected for neurons innervating the appendicularmusculature, the cell bodies are located in the most ventral regions of the gray matter. Like the neuronscommandingthe anal tin, eachcell bearsa singledendritic shaft which projects toward the most dorsal neuropile fields (zonesI and II at spinal cord level) where they ramify profusely. Since all their axons leave the central nervous system at spinal cord levels, those originating from motoneuronslying in the medulla must travel relatively long distances within the white matter before reachingtheir corresponding exiting sites. B. Electron Microscopy

Consideringthat regardingthose aspects concernedwith the fine structure of syn-

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FIG. 2. (Top) Transverse section of the spinal cord showing a HRP-stained PM. Note the presence of various dendritic trunks (arrows) projecting toward zones 1 and II of the ipsilateral horn. The axon (Ax) arises from the ventral pole of the cell and follows a ventromedial pathway passing close to the Mauthner fiber (outlimits of its sheath indicated by a dotted line). Complementary information concerning branching patterns of the PMs can be obtained from the horizontal section shown in the bottom figure. Each main dendritic trunk (DT) divides into two secondary branches which run in longitudinal, opposite directions. The asterisk indicates the central canal. (Top) x 672; (bottom) x 504.

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FIG. 3. The anal fin muscles are innervated by groups of ventral motoneurons metamerically distributed along the cord. One of these groups, consisting of approximately 20 nerve cells, is shown in the microphotograph (top) obtained from a horizontal section. The main geometrical characteristics of these neurons can be seen in the camera lucida drawing shown in the bottom figure. Note that each neuron bears a single dendritic shaft which ramifies in zones I and II of the ipsilateral horn. HRP-labeled material; MF, Mauthner fibers. (Top) x 450; (bottom) x 68.

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FIG. 4. These two camera lucida drawings show the location of the motoneuron pool innervating the pectoral fin muscles. (Left) Section passing through the most rostra1 spinal cord level. (Right) Section passing through the caudal region of the medulla. HRP-labeled material. MF, Mauthner fibers. x 60.

apses we found no differences between the different classes of motoneurons, the subject has been covered under a unified description. Exploration of the somata, dendritic trunks, and secondary branches showed the neuronal plasma membrane practically covered by a palisade of synaptic terminals. Many of them exhibited the fine structural characteristics usually associated with chemical transmission. In other synaptic junctions, however, the zones identified as the “active sites” of chemical synapses were found intermixed with areas in which the close apposition of the synaptic membranes determined the formation of pentalaminar patches (Fig. 5). Such patches (identified with gap junctions) seem to represent low-resistant areas mediating the

electrotonic coupling of cells (for a review on the subject see Hertzberg et al., 1981). Therefore, these synapses, like those occurring on the EMNs (Trujillo-Cenoz et ul., 1986), should be classified on morphological grounds as “mixed junctions” mediating both chemical and electrical transmission (Bodian, 1972). Pentalaminar plaques identical to those occurring at the mixed junctions have also been found at the zones where adjacent synaptic knobs contact each other without the interposition of glial elements (Fig. 6). In addition, some of the terminals with the morphological characteristics of chemical synapses contain, instead of the usual cumuli of round vesicles, a small population of more flat, oval vesicles associated with less apparent synaptic densities (Fig. 5).

STRUCTURAL

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FIG. 5. Synaptic terminals with the fine structural characteristics of “mixed junctions” have been consistently found impinging on the motoneuron somata. One such terminal (MJ) can be seen contacting the soma of a PM. Note the presence of membrane densities and clusters of vesicles accompanying areas in which the synaptic cleft is inappreciable. At this zone pre- and postsynaptic membranes are seen as a single pentalaminar profile (arrowhead and inset). This “mixed junction” lies between two terminals (1, and I,) containing flat or oval vesicles. x 55 400; inset: Vestopal-embedded material, x 130 000.

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FIG. 6. This electron micrograph shows four synaptic terminals (l-4) contacting the soma of one of the ventraJ motoneurons (VM) innervating the anal fin. The neuron was retrograded, marked with HRP introduced within the bundles of anal fin muscles by means of a HRP-soaked micropellet. The arrows indicate lysosomes containing the HRP-chromogen reaction product. Note that terminals 3 and 4 show a zone (arrowhead) in which the adjacent plasma membranes lie in close apposition. When these zones are studied in Vestopal-embedded material a pentalaminar membrane pattern 150 A in width can be distinguished (inset). x 27 300; inset: x 93 300.

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Axon terminals with this kind of vesicle by Myers (1985)in the periependymalgray have been consideredas servinginhibitory of zebrafish embryos (Bruchydunio rerio). functions (Uchizono, 1965, 1967;Bodian, The putative PMs of Gymnotus, like those 1972). of Bruchydunio, project their axons(or, according to Diamond (1971), ventral denDISCUSSION drites) toward the medial zone of the cord, The neurohistologicalorganizationof the closeto the ipsilateral Mauthner fiber. The spinal cord of Gymnotus shows features light and electron microscope investigacommon to most teleostsand also peculiar- tions carried out by Diamond (1971)in the ities typical of gymnotoid fish. Like non- goldfish and the tenth tend to indicate that electric specimens, their spinal motoneu- the first portion of the ventral processcould rons can be divided into two major groups: have a single large dendritic spine in very (a) neuronsinnervatingthe axial musclesof close relation to the Mauthner axon collatthe trunk-tail region and (b) neurons sup- eral. In Gymnotus, however, we have not plying the appendicularmusculature. The observed the postulated connections beaxial muscles receive their innervation tween these ventral processesof the mofrom large motoneurons which constitute toneuronsand the collaterals of the Mauthtwo columns at each side of the central ca- ner axons. nal. The presenceof motoneuronsin this The morphologyand spatial organization regionof the teleost cord was notedby Van of the PMs of Gymnotus follow patterns Gehuchten(1895)working on young speci- also found in other teleosts(Fetcho, 1986). mens of T. furio. He describedthe geome- Conversely, the ventral motoneurons intry of the cells without mentioning the des- nervatingthe anal fin are arrangedin a typtiny of their axons. Severalyears later, in- ical gymnotoid fashion. As observed by terest concerningthe functional properties Baillet-Derbin (1984)in the related genus of the Mauthner cells also promoted inves- Eigenmunniu, the anal fin neuronsare distigations on motoneuronscircuitry. It was tributed along the ventral portions of the then noted that the collaterals of the cord, forming well-defined, metameric Mauthner axonsseemto establishcontacts clusters. The slenderdendritic shafts ariswith the ventral processesof “certain large ing from theseneuronsproject to the dorsal motoneurons,which we shall call the pri- regions of the ipsilateral horn. Contrasting mary ones” (Diamond, 1971). The term with the PMs they do not form dendritic “primary motoneuron” derivesfrom devel- strata but terminate by means of less oropmentalstudiescarriedout on amphibians dered, less extended apical bouquets. (Herrick and Coghill, 1915;Coghill, 1926); These neurohistological features permit it was coined to identify the population of one to postulate that the PMs are able to early differentiated spinal motoneurons integrate information from large afferent subservingthe first trunk-tail movementsof fields, while the smaller ventral motoneuthe larvae. As developmentproceeds,an- rons may deal with more localized fiber inother set of motoneurons, involved in the puts. withdrawal reflex of the limbs, undergoes As suggestedby the electrical models differentiation at more ventral regions of (Lissmann, 1958),the operation of “active the cord. From the order of their develop- electroreceptive systems” requires a parment they have been called “secondary ticular type of propulsion, compatible with motoneurons” (Youngstrom, 1940). Our a certain degreeof rigidity of the EO. This observationson Gymnotusstrongly suggest is accomplished in the unrelated species that the largest motoneurons innervating Gymnurchus niloticus and G. curupo, by a the axial musclesrepresentthe PMs of this similar mechanism:the undulationof a very species.They are similar to the PMs found long unpairedfin (the dorsal fin in Gymnur-

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thus and the anal fin in Gymnotus). As already mentioned, in gymnotoids the hypertrophy of the anal fin is associated with the presence of numerous discrete, multineuronal units distributed along most of the cord. The synaptology of the various kinds of neurons is very complex. The somata and the main dendritic trunks are covered by synaptic terminals exhibiting different fine structural characteristics, usually correlated with different synaptic mechanisms. As already mentioned, it is possible to recognize three types of synaptic junctions: (a) terminals with round vesicles and welldeveloped membrane specializations (presynaptic grids and postsynaptic densities); (b) terminals with flat vesicles and less evident membrane specializations; and (c) mixed junctions which show clusters of round vesicles, pre- and postmembrane densities, and also pentalaminar membrane patches. It is now generally accepted that these pentalaminar patches correspond to “gap junctions” subserving electrical coupling between cells. In fish, mixed junctions have been identified in the medullary electromotor nuclei (Bennett rf ul., 1967). on the spinal electromotor neurons (BailletDerbin and Denizot, 1980; Denizot et al., 1983; Trujillo-Cenoz et ui., 1986), on the spinal motoneurons (Christensen, 1976; Bennett et al., 1978; Baillet-Derbin and Denizot, 1980), and also on the Mauthner cells (Nakajima and Kohno, 1978). Electrical coupling seems to be the predominant mechanism of transmission in the electromotor system of weakly electric fish (Bennett et al., 1967) and also at the level of the synapsis between vestibular fibers (club endings) and the Mauthner neurons (Faber et al., 1980). A dissimilar situation seems to occur in the spinal cord of the lamprey where the postsynaptic responses. mediated by morphologically identified mixed junctions, show both electrical and chemical components (Rovainen, 1974; Christensen, 1976). It is worthwhile to mention that close apposition of membranes conforming to pen-

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BERTOLOTTO

talaminar profiles has also been found between adjacent nerve terminals impinging on the same motoneuron. If these pentalaminar plaques behave as low-resistance bridges, electrical coupling of several nerve terminals may occur. This may synchronize the activation of the nerve terminals thus increasing the effectiveness of the presynaptic volleys. Nevertheless, the presence of gap junctions cannot be accepted as unequivocal evidence of electrical coupling (Hertzberg et al., 1981). If terminals containing flat or oval vesicles are considered to represent inhibitory synapses (Uchizono, 1965, 1967). it is no surprise to find them on motoneuron surfaces. Particularly for PMs, it is a wellknown fact that activation of motoneurons of one side (via the ipsilateral Mauthner axon) elicits crossed inhibition (Yasargil and Diamond, 1968). It is reasonable to speculate that inhibitory phenomena could also play an important role during the generation of the wave motion of the anal tin. Understanding the functional properties of the motoneurons circuits is a demanding task which requires the complementary use of physiological and neurohistological procedures. The present. purely morphological report represents a first step directed toward supporting future electrophysiological investigations. This research project was supported by the Commission of the European Communities under Contract CII tL165.U (H). The Electron Microscope was donated by the Ministry of Culture of Japan. The authors acknowledge the help of Drs. 0. Macadar and D. L>orenzo. for critical reading and discussion of the manuscript. REFERENCES ADAMS, S. C. (1977) Neuroscience 2, 141-146. BAILLET-DERBIN, C. (1984) Brain Res. 295, 384-390. BAILLET-DEREIIN. C., ANT) DENIZOT, J. P. (1980) in Cercle Francais de Biol. Cellufaire, 5 tme Colloque Annuel, Paris. BECCARI, N. (1943) Neurologia Comparata, Sansoni, Firenze. BENNETT, M. V. L., PAPPAS. G. D., GIMBNEZ, M.. AND NAKAJIMA. Y. (1967) J. Neurophysiol. 30,236300.

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