Central projections and motor nuclei of the facial, glossopharyngeal, and vagus nerves in the mormyrid fish Gnathonemus petersii.

Most of the information about the anatomy of the fish's cranial nerves was collected in the first two decades of this century. Experimental analysis of the VIIth, IXth, and Xth cranial nerves by modern tract tracing techniques started about 20 years ago. Several species have been investigated to date, including one species of Agnatha (Myxinoidea), two species of elasmobranchs, and species of some orders of Teleostei like Cyprinidae, Siluriformes, Perciformes, and Gadidae. The sensory and motor nuclei of the VIIth, IXth, and Xth cranial nerves of Gnathonemus petersii were studied by anterograde and retrograde axoplasmatic transport of horseradish peroxidase and cobaltous lysine complex. The sensory nuclei form a continuous column of cells in the brain stem extending caudal to the obex. The rostral one-fourth of this column is occupied by the overlapping terminals of the VIIth and IXth nerves. The vagus nerve has 5 roots. The first 4 of these innervate the gills and the fifth supplies viscera. Afferents from the gills terminate ipsilaterally rostral to the obex in topographic order and their terminal fields overlap. Viscerosensory fibers terminate ipsilaterally in the obex region and bilaterally in the commissural nucleus of Cajal. The facial motor nucleus is located rostral to the sensory nucleus. Facial motoneurons have pear-shaped and multipolar perikarya. Their axons form a rostrally directed knee before leaving the brain. The motoneurons of the IXth and Xth nerves have a common cell column. The vagal motoneurons form a periventricular, a medial, and an intermediate cell group rostral to the obex. In the obex region and also caudal to it, a lateral and a caudal group can be distinguished. Vagal motoneurons show a topographic arrangement that is similar to that of the sensory vagal projections. The majority of motoneurons have pear-shaped perikary and ventrolaterally oriented dendrites. In the caudal nucleus the dendrites extend dorsally and overlap the terminals of sensory fibers. The axons form a dorsolaterally directed arch before joining the sensory roots. Since G. petersii uses its electrosensory system primarily for detection of food, its gustatory system is less developed than in other fishes, which possess a large number of taste buds.

[Long ascending fibers in the dorsal column of a teleost fish: a disynaptic pathway connecting sense organs to cerebellum]. / Fibres longues ascendantes dans les colonnes dorsales d'un poisson téléostéen: une voie disynaptique reliant des organes sensoriels au cervelet.

In spite of the generally accepted opinion that long ascending proprioceptive and tactile fibers do not occur in the spinal dorsal columns of teleost fish, it was demonstrated with degeneration and axonal transport tracing methods that such dorsal column fibers exist in the teleost fish Gnathonemus petersii. These fibers are in fact common spinal afferent fibers originating in spinal ganglion cells. They connect the peripheral sense organs with the lateral funicular nuclei (Fl2) in which the dorsal column fibers terminate, directly through the dorsal columns. In contrast to the dorsal column nuclei of higher vertebrates, the Fl2 nuclei do not project to the diencephalic thalamus but to the caudal lobe and the second lobe (C2) of the corpus cerebelli. Thus, sense organs and cerebellum are connected by a disynaptic pathway. Since the caudal lobe projects directly to the electrosensory lobe, that is, to the target of electrosensory afferents, the presence of a disynaptic pathway in G. petersii suggests the existence of a proprioceptive control of the electrosensory input.

A well defined spinocerebellar system in the weakly electric teleost fish Gnathonemus petersii. A tracing and immuno-histochemical study.

Long ascending fiber systems were investigated in the spinal cord of a teleost fish, Gnathonemus petersii. Concomitant results of Fink-Heimer degeneration tracing as well as CaBP28K immunohistochemical labelling demonstrate the existence of a well defined direct pathway from the very lowest spinal level to the caudal lobe of the cerebellum. HRP retrograde labelling shows that this pathway originates in a cellular column located in the most ventral part of the lateral column next to the lateral extremity of the ventral horn. From each spinal segment, the large axons of these cells gather and form a strip shaped tract at the periphery of the lateral column immediately dorsal to the cell column from which they originate. The spinal course of these fibers is ipsilateral; they give off a large number of collaterals to the lateral reticular nucleus. Bypassing the trigeminal motor nucleus, the lateral column tract courses dorsally to the paratrigeminal command associated nucleus between the lateral lemniscus and the nucleus preeminentialis and with a ventro-dorsally oriented large loop, turns in the caudal direction and penetrates into the cerebellar caudal lobe. Running caudally in the dorsal granular layer of the caudal lobe, it shifts more and more medially and crosses the midline whilst decussating with the contralateral tract on the dorsal margin of the molecular layer of the caudal lobe. Finally, the tract splits off and terminates throughout the granular layer of the caudal lobe. The main characteristics of this pathway are similar to those of the ventral spinocerebellar tract of higher vertebrates; it conveys information from all spinal levels directly to the contralateral cerebellum. However, it does not seem to receive direct synaptic input from the periphery, since projection of the dorsal root fibers appears to be limited to the dorsal ipsilateral half of the spinal cord. The appearance of such a pathway in a teleost fish is probably related to the existence of a well developed proprioceptive system in this species.

Serotoninergic neurons in the mormyrid brain and their projection to the preelectromotor and primary electrosensory centers: immunohistochemical study.

Serotonin-containing neurons in the brain of the weak-electric fish Gnathonemus petersii (mormyridae, teleostei) were studied with the aid of immunohistochemical labeling. Study of the central serotoninergic innervation was focused on the structures subserving the command of the electric organ and the first central relay of the electrosensory system. In the midline raphe nuclei, serotoninergic neurons formed a column that stretched from the ventral caudal medulla to the dorsal midbrain, ending caudal to the cerebellar peduncle. In the dorsal tegmentum, serotoninergic neurons were found bilaterally at the anterior margin of the decussation of the lateral lemniscus. Labeled neurons were also present bilaterally immediately anterior to the cerebellar peduncle and also in the pretectal region. In the hypothalamus, many serotoninergic neurons were in contact with the ventricular wall, and a few were present in the preoptic area. This distribution of serotoninergic cell bodies showed many similarities to that in other fish and higher vertebrates but lacked the lateral spread of the serotoninergic raphe system found in the midbrain tegmentum in mammals. Labeled fibers were found in both the preelectromotor medullary relay nucleus and the electromotor command nucleus. These serotoninergic projections were traced to the posterior raphe. Serotoninergic fibers also formed a dense network in the cortex and in the nucleus of the electrosensory lobe, both of which receive primary input from electroreceptors. These results suggest that serotonin may have a role in the modulation of the intrinsic, rhythmic electromotor command and in the gating of electrosensory input.

The use of Quetol 651 for the post-embedding immunohistochemical demonstration of gamma-aminobutyric acid on semithin sections.

Quetol 651 was used as an embedding medium for the demonstration of gamma aminobutyric acid (GABA) in semithin sections by the peroxidase-anti-peroxidase method. In order to demonstrate the immunoreactivity, the embedding medium was partially dissolved using absolute ethanol containing 0.8-1 M NaOH or KOH for 5-7 min. The experimental procedure was elaborated by testing the GABAergic sites in the endings surrounding the small neurones of the anterior exterolateral nucleus of a mormyrid fish and in the pyramidal cells of the electrosensory lateral line lobe of gymnotoid fish by applying anti-GAD (glutamic acid decarboxylase) antiserum. To test the general validity of the use of Quetol 651, GABAergic sites were also identified in the central nervous system of an insect, the honey bee, with anti-GABA and anti-GAD antisera. The intensity of labelling revealed by immunoperoxidase applied to Quetol 651-embedded semithin sections, demonstrated high precision and gave good resolution for light microscopical observations.

Oculomotor system of the weakly electric fish Gnathonemus petersii.

The peripheral and central aspects of the extraocular system were studied in the weakly electric fish Gnathonemus petersii. All six extraocular muscles show a similar composition of large and small fibers grouped characteristically in the proximal and distal regions respectively. The exit of the three extraocular nerves from the brain is similar to that in other vertebrates. However, the intracephalic and intracranial course of the trochlear nerve is unusual, partly because of the extraordinary hypertrophy of the cerebellum. The three nerves course rostrally on the ventral brain surface; the trochlear nerve penetrates the orbital cavity separately from the two other nerves. The fiber-diameter spectrum of each extraocular nerve is bimodal; unmyelinated fibers were not observed in any of the nerves. The location of the extraocular motor nuclei was established by retrograde axonal transport of HRP or cobaltic-lysine complex. The oculomotor nucleus is situated ventral to the posterior pole of the magnocellular mesencephalic nucleus and the trochlear nucleus is found caudal and dorsal to this. The abducens nucleus is situated at the level of the octavolateral efferent nucleus and consists of a single group of cells on each side of the ventral tegmentum. The oculomotor nucleus of G. petersii shows a somatotopic organization. The superior rectus muscle receives a contralateral innervation whereas the inferior rectus and oblique muscles and the internal rectus muscles receive an ipsilateral innervation. The superior oblique muscle is innervated by contralateral trochlear motoneurons and the external rectus by ipsilateral abducens motoneurons. The majority of extraocular motoneurons have piriform perikarya and long beaded dendrites that extend laterally in the oculomotor and abducens nuclei and rostrally in the trochlear nucleus. The terminal dendritic portions of trochlear motoneurons widely overlap with oculomotor dendrites and perikarya. In all three nuclei the axon originates opposite to the main dendrite. Collaterals of the hairpin-bend abducens axons could be identified in a few cases. The oculomotor system of G. petersii appears basically similar to that of other teleosts; the differences observed concern mainly the structure of the abducens nucleus, the intracranial and intracephalic course of the trochlear nerve, and the relatively small number of axons in each nerve.

Convergence of electrotonic club endings, GABA- and serotoninergic terminals on second order neurons of the electrosensory pathway in mormyrid fish, Gnathonemus petersii and Brienomyrus niger (Teleostei).

Previous electrophysiological data indicate that the afferent electrosensory impulses conveyed towards the mesencephalon are blocked in the rhombencephalic electrosensory lateral line nucleus (nELL) by the concomitantly occurring EOD (electric organ discharge) command-associated (corollary) discharge. Electron-microscopic observations and anterograde labeling with horseradish peroxidase show that the primary electrosensory fibers terminate with club endings on the adendritic soma of the nELL cells and form gap junctions with the postsynaptic membrane. The remaining part of the soma and the initial segment membrane of nELL cells are covered with a large number of boutons showing chemical synaptic profiles. The GABA-ergic (gamma-aminobutyric-acid) nature of the majority of the boutons is revealed immunocytochemically by anti-GABA and anti-glutamic acid decarboxylase (anti-GAD) antisera, as seen in the light microscope. Electron-microscopic examination confirms the GABAergic nature of most of the bouton-like terminals, whereas club endings show negative immunoreactivity. In addition, serotonin-immunoreactive fibers and boutons are found in the same nucleus, between and next to the nELL cells. It is suggested that the GABAergic endings are the morphological basis for the inhibition that occurs in the nELL and that is mediated by the corollary discharge.

The mormyrid brainstem--II. The medullary electromotor relay nucleus: an ultrastructural horseradish peroxidase study.

The medullary relay nucleus of the mormyrid weakly electric fish Gnathonemus petersii is a stage in the command pathway for the electric organ discharge. It receives input from the presumed command or pacemaker nucleus and projects to the electromotoneurons in the spinal cord. Its fine structure and synaptology were investigated by electron microscopy. The origin of the terminals contacting the cell membrane of the neurons of this nucleus was determined by horseradish peroxidase (HRP) injections into different brain structures, namely into the bulbar command- and mesencephalic command-associated nuclei. Twenty-five to thirty large cells of about 45 micron in diameter constitute the medullary electromotor relay. Each cell has a kidney-shaped, lobulated nucleus, a large myelinated axon with a short initial segment and several long, richly arborizing primary dendrites. Many, if not all, cells are interconnected with large somatosomatic or dendrosomatic, dendrodendritic and dendroaxonic gap junctions. These junctions often occur in serial or triadic arrangements. The relay cells receive large club endings as well as small boutons. The club endings are found mainly on the soma and primary dendrites and are morphologically mixed synapses. The boutons are characterized by synapses which are only chemical and are distributed all over the cell membrane, but with a definitely higher frequency on secondary dendrites and more distal parts of dendritic processes. Horseradish peroxidase injections into the mesencephalic command-associated nucleus reveal a large number of labelled boutons on the secondary dendrites of the relay cells. Injections into the bulbar command-associated nucleus label the same type of boutons as mesencephalic injections, but also label club endings on relay cell soma and primary dendrites. The results support the conclusion made on the basis of previous light microscopical observations that boutons originate from the bulbar command-associated nucleus, whereas the club endings issue from the presumed pacemaker nucleus (nucleus c). The club endings of the bifurcating axons of this nucleus are labelled by retro- and anterograde transport of horseradish peroxidase; the bifurcating axons project simultaneously to the bulbar command-associated nucleus and the medullary relay nucleus.

New formation of sensory cells in the tuberous organ (Electroreceptor) of Brienomyrus niger (Mormyridae) induced by transection of afferent nerve.

The tuberous organs-cutaneous electroreceptors of the mormyrid fish Brienomyrus niger-were examined, with the light and electron microscope, after sectioning of the afferent nerve of the lateral line. Transection of the afferent nerve leads to the concomitant complete degeneration of all sensory cells, and to a differentiation of new sensory cells from accessory cells, which constitute the platform. Ultrastructural examination of the newly formed sensory cells shows that within a few days these gain the characteristics of normal sensory cells. The rapidly growing cytoplasm is enclosed in a folded membrane; the foldings develop typical dense microvilli. At the same time, in spite of lack of innervation, synaptic bars surrounded by vesicles differentiate in the cytoplasm; these are opposed to the basal cell membrane which is attached to the accessory cell platform. The newly formed sensory cells never reach the size of normally developed sensory cells. Their existence is transitory, for they degenerate, together with the sensory cells, one month after de-afferentiation.

The mormyrid mesencephalon. III. Retinal projections in a weakly electric fish, Gnathonemus petersii.

The optic nerve and the retinal projections were studied in a mormyrid fish, Gnathonemus petersii, by using Fink-Heimer, HRP, cobalt labeling, and autoradiographic tracing techniques. The retinal fibers terminate bilaterally in the following places: suprachiasmatic nucleus, dorsolateral optic nucleus, optic nucleus of the posterior commissure, cortical nucleus, ventral pretectal area, optic tectum, and the accessory optic terminal field. The number of uncrossed fibers is relatively high in the suprachiasmatic nucleus, but negligibly small in the other retinal terminal fields. In the lateral geniculate nucleus and pretectal nucleus only crossed retinal fibers could be detected. The visual system of Gnathonemus is compared to that of other fishes, amphibians, and reptiles and the possible homologies are proposed. The comparison points to the conclusion that the visual system is less developed in Gnathonemus. This nocturnal species lives in turbid waters and has a special electric sense which may permit compensation for the reduced visual capacity.

The mormyrid brainstem. I. Distribution of brainstem neurones projecting to the spinal cord in Gnathonemus petersii. An HRP study.

The sources of the descending spinal tracts were identified in the teleost fish Gnathonemus petersii by retrograde HRP transport. HRP injections were made at two spinal levels, either at level of the caudal end of the dorsal fin, anterior to the electric organ, or at the pectoral fin. In both cases all labeled cells were found in the rhombencephalon and the mesencephalic tegmentum. No labeled cells were observed either in the cerebellum and lateral line lobes or in the dorsal mesencephalon i.e. torus semicircularis and mesencephalic tectum or in the telencephalon. Following caudal spinal injections, the majority of the labeled cells were grouped in a median and a ventrolateral column of the rhombencephalic reticular formation. The latter is composed of three parts corresponding to the nucleus reticularis inferior, medius and superior. Both Mauthner cells, all the cells in the medullary relay nucleus controlling the electric organ discharge and a few cells in the posterior part of the magnocellular octaval nucleus were labeled. In the mesencephalon, four nuclei were identified by HRP labeling: the nucleus of the medial longitudinal fasciculus, the nucleus reticularis mesencephali and the anterior and posterior tegmental mesodiencephalic nuclei. The rostral injections revealed several additional spinal projections from the descending vestibular and tangential nuclei, from the medial part of the magnocellular nucleus and, finally, from the rostral periventricular gray of the mesencephalon. Also, after such injections, a greater number of cells were labeled in the reticular formation, especially in the median column and in the inferior reticular nucleus. The results suggest that the rostral spinal cord has a larger connection with the acoustico-vestibular area and the medullary reticular formation than the caudal spinal cord. In contrast, the mesencephalic nuclei, probably linked to the mesencephalic tectum and the pretectal area, appears to be a coordinating apparatus between the visual system and the trunk/tail musculature. Thus, it appears that teleost fish possess the same basic equipment of descending spinal pathways as higher vertebrates.

Pathways of the electric organ discharge command and its corollary discharges in mormyrid fish.

The motoneurons which innervate the mormyrid electric organ are driven by a descending volley from the medullary relay nucleus. This nucleus does not initiate the electric organ discharge (EOD) but is driven in an obligatory manner by another center, a command nucleus. One goal of the present study was to identify this command nucleus anatomically. A second goal was to determine the pathways by which corollary discharges of the EOD motor command exert their effects on sensory input to the electroreceptive lateral line lobe. Horseradish peroxidase (HRP) was injected into the medullary relay nucleus and other EOD command-related centers. Placement was guided by recording the electrical activity preceding the EOD. A nucleus of smaller cells is found immediately beneath the large cells of the medullary relay nucleus. This nucleus, nucleus C, projects densely to the medullary relay nucleus and is hypothesized here to be the command nucleus. Nucleus C appears to receive input from the mesencephalon and from unspecified sources of input to the nearby reticular formation. Nucleus C projects to the medullary relay nucleus and to a lateral nucleus, the bulbar command-associated nucleus. This nucleus is probably the source of the corollary discharge signals. It projects to the medullary relay nucleus and to the paratrigeminal and mesencephalic command-associated nuclei. The latter two nuclei project to separate regions which in turn project to the electroreceptive lateral line lobe. There are thus at least two different paths by which the presumed EOD command nucleus, nucleus C, can affect the electroreceptive lateral line lobe.

The mormyrid rhombencephalon: I. Light and EM investigations on the structure and connections of the lateral line lobe nucleus with HRP labelling.

The rhombencephalic posterior lateral line lobe nucleus (nLLL) and its connections were investigated in the mormyrid fish Gnathonemus petersii, at light and electron microscopical levels using HRP tracing. The nLLL, constituted on each side of about 1500 large, round shaped, adendritic cells, is located in the intermediate cell and fibre layer exclusively, in the ventrolateral zone of the posterior lateral line lobe. The cells show a complex synaptology: boutons with chemical synapses cover the largest part of the soma and the long initial segment of the axons. In addition each nLLL cell bears generally two club endings which form gap junctions with the postsynaptic membrane. On the unmyelinated portion of the club ending, a particular synaptic complex (= serial synaptic connections) was observed; large endings, bearing boutons with chemical synapses, contact the club ending with gap junctions. HRP injection into either lateral line nerve showed that the club endings represent the peripheral input of the nLLL. This input is exclusively ipsilateral; the anterior and posterior lateral line nerve projection does not seem to overlay at this level. Retrograde labelling of nLLL cells after HRP injection into the anterior mesencephalic exterolateral nucleus (nELa) confirms the electrophysiological results according to which the nLLL projects directly and bilaterally to the nELa. This rhombo-mesencephalic connection is established through club endings of the nLLL axons which form gap junctions with the large cells of the nELa. About two thirds of the nLLL axons form a crossed and one third an uncrossed bundle within the lateral lemniscal pathway. Anterograde transport in the axon collaterals shows that some of the nLLL cells project simultaneously to the ipsi- and to the contralateral nELa permitting this system a high degree of electrosensory information processing.

Anatomical study and HRP identification of electromotoneurons and motoneurons in the spinal cord of Gymnarchus niloticus.

Two types of neurons were identified by light and electron microscopy in the spinal cord of Gymnarchus niloticus following HRP injections at different peripheral sites: one, situated in the mediodorsal region, is labelled after injection into the electric organ; the other, situated in the lateroventral region, is labelled in addition after injections extending beyond to the lateral muscle. The cells of the first type, thus shown to be electromotoneurons (EMNs), are spherical and do not have dendritic processes; some of them are connected by somato-somatic gap junctions. The EMNs are surrounded by dense glial processes and embedded in a network of myelinated axons of large diameter. Synaptic contacts with the EMNs are of axosomatic type, exhibiting mostly gap junctions and, less frequently, mixed or chemical junctions. Some endings establish gap junctions simultaneously, with two electromotoneurons, the somatic membrane of which may be joined. The initial segment does not bear any endings. The second type of cells, identified as motoneurons, are pyriform and have large dendritic processes. The motoneurons are surrounded by myelinated axons of small diameter. They receive axosomatic endings with chemical synapses.

On the course and origin of cranial nerves in the teleost fish Gnathonemus determined by ortho- and retrograde horseradish peroxidase axonal transport.

By horseradish peroxidase (HRP) labelling the course and origin of the II-X cranial nerves are identified in the teleost fish Gnathonemus petersii. Roots as well as motor and sensory nuclei in the viscero- and somatomotor and somatosensory areas are localised. Earlier data of comparative and experimental anatomical observations are completed, partly confirmed or corrected.

Electrosensory systems in the mormyrid fish, Gnathonemus petersii : special emphasis on the fast conducting pathway.

1. Rhombencephalic and mesencephalic structures involved in electroreception were investigated by electrophysiological methods in the weakly electric fish Gnathonemus petersii. 2. The existence of a synchronous response to electric field stimulation of the fish in the mesencephalic exterolateral nucleus (n.ext.-lat.mes) with 2.5-3 ms latency was confirmed. The lateral line lobe nucleus (nLLL) is identified as the rhombencephalic relay for the mesencephalic responses because of the short latency synchronous response in the nLLL obtained by threshold stimulation of the posterior lateral line nerve. Responses in both the nLLL and the n.ext.-lat.mes. appear and their amplitudes increase simultaneously with increasing stimulus intensity. 3. Comparison of latencies supports a three-neuron pathway hypothesis which also agrees well with the various functional properties described. 4. The nLLL-n.ext.-lat.mes. pathway is blocked sharply for a period of 1 ms occurring 3 ms after the electric organ discharge (EOD). This inhibitory period is phase-related to the Mesencephalic Command Associated Signal (MCAS) of Aljure (1946) ; The phase relation is such that no response is observed to the fish's own EOD. 5. Long-lasting responses of 10-12 ms duration to higher stimulation intensities were obtained in the ganglionic layer of the lateral line lobe (LLL). Intensities evoking maximal responses in the nLLL and n.ext.lat.mes. are still threshold stimulation for lateral line lobe responses. 6. Long-lasting responses (of the same order as in the LLL) to the fish' own EOD were observed in the mesencephalic lateral nucleus. Responses to artificial electric pulses were obtained only if delivered in a certain phase realtion to the MCAS. The MCAS displays a facilitating effect on the slow conducting electrosensory system. 7. Results indicate the existence in mormyrids of a double, fast and slow conducting, electrosensory system similar to that of gymnotid fish. The mormyrids can control both of these electrosensory systems by means of the MCAS, the effect of which is opposite for the same time period on the two systems.

Fast conducting electrosensory pathway in the mormyrid fish, Gnathonemus petersii.

A compound action potential of short latency was recorded from the ganglion mesencephali extrolaterale in response to both the fish's own electric organ discharge and electric stimulation of the whole fish or a branch of the posterior lateral line nerve. The time difference between the response in the nerve, 16 mm from its entry into the brain, and the mesencephalic ganglion, which is positioned 10 mm anterior in the CNS, was found to be 1.6 msec. This rapid conduction pathway involves peripheral nerve fibres of high conduction velocity (40 m/sec) and presumably two electric synapses, one at the rhombencephalic and the other at the mesencephalic level.