An ultrastructural analysis of electromotor cell death in Torpedo marmorata and its counterpart in vitro

Summary Naturally occurring neuronal cell death has been investigated in the electromotor system of Torpedo marmorata and compared with neuronal death seen in expiant cultures of tissues from T. marmorata electric lobe. One objective of the study was to determine whether cell death in vitro was morphologically the same as cell death in vivo and to substantiate the validity of using in vitro models for studying naturally occurring cell death. Sequences of degeneration in vitro and in vivo have been established and compared: a single morphological sequence best represents this form of cell death in both conditions. In vivo, dying neurons are seen at all depths of the electric lobe indicating the involvement of different generations. Retrograde degeneration appears to be the first sign of cell death.     

Development of the electromotor system of Torpedo marmorata: Distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization

Summary A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptorrich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electronmicroscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.Abbreviations ACHR nicotinic acetylcholine receptor - ACHE acetylcholinesterase - BSA bovine serum albumin - EMPG extracellular matrix proteoglycan fraction - FITC fluorescein isothiocyanate - FN fibronectin - LN laminin - TBS Tris-HCl-buffered saline - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Further evidence that glycosaminoglycan specific to cholinergic synaptic vesicles recycles during electrical stimulation of the electric organ of Torpedo marmorata

Summary Semiquantitative immunohistochemical methods were used to demonstrate that at least some of the glycosaminoglycan contained within cholinergic synaptic vesicles is recycled during successive electrical stimulations of the electric organ of Torpedo marmorata.     

Effects of ouabain and electrical stimulation on the fine structure of nerve endings in the electric organ of Torpedo marmorata

Summary The cycle of synaptic vesicles was studied in isolated nerve terminals and in the electric tissue of Torpedo marmorata. The synaptosomes, as used in this investigation, were a pure cholinergic subcellular fraction that captured dextran particles as an extracellular marker. This endocytotic phenomenon was enhanced by potassium depolarization. Field electrical stimulation (1 Hz and 10 Hz) of the electric organ induced the appearance of membrane foldings into presynaptic terminals. Morphometric studies showed that the number of synaptic vesicles did not decline until after at least 30 min. On the other hand, at 10 Hz these changes were accompanied by an increase in length of the membrane of the terminal. At 15 min of recovery after prolonged stimulation, there was a great increase in density of synaptic vesicles with a large number of vesicles of small diameter. This increase was accompanied by a decrease of membrane length, suggesting that reformation of vesicles is related to retrieval of membrane. Pharmacological stimulation with ouabain produced changes similar to those of long-term electrical stimulation. These changes in membrane were accompanied by a decrease of the population of synaptic vesicles and a wide variation in their diameters. It is concluded that structural changes reported here could not be correlated with kinetics of the transmitter release.We are grateful to Dr. E. Cañadas, Prof. Dr. D. Ribas and Dr. J. Tomás for valuable help and encouragement. We are indebted to Dr. P. Arté and to the staff of the Acuario de Barcelona del Instituto de Investigaciones Pesqueras for providing specimens of Torpedo marmorata. This investigation was supported by a grant Formación Personal Investigador del Ministerio de Universidades e Investigación     

Changes in Acetylcholinesterase Molecular Forms During the Embryonic Development of Torpedo marmorata

Abstract: Multiple molecular forms of acetylcholinesterase from electric organ and electric lobe of Torpedo marmorata were examined at various developmental stages by sucrose density sedimentation. Four major forms were characterized by their apparent sedimentation coefficients of 6 S, 11 S, 13 S, and 17 S. Embryonic lobe possessed at early stages predominantly the 11 S form. With maturation the 17 S form became the most abundant. The early embryonic stages of the electric organ were characterized by predominating amounts of 6 S and 11 S forms. With differentiation of the postsynaptic membrane of the developing electrocytes, 13 S and 17 S forms replaced the slower-sedimenting forms. Concomitant with the formation of synaptic contacts, a transient increase in the 13 S form was followed by a dramatic accumulation of rapid-sedimenting 17 S form. The establishment of fully functional synapses was accompanied by an increase in the amount of the hydrophobic 6 S form. At birth, equal amounts of 6 S and 17 S form were found, with the other forms present in only trace amounts. The observed characteristic changes correlated with morphological and physiological events, indicating a close functional relationship between the accumulation of the 17 S form and synapse formation and the accumulation of the 6 S form and onset of function.     

Conotoxin MI inhibits the alpha-delta acetylcholine binding site of the Torpedo marmorata receptor

The muscle-type nicotinic receptor has two pharmacologically distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. As reported, alpha-conotoxin MI has greater affinity for the site near the alpha-delta interface of the BC(3)H1 cell receptor but, in the case of the Torpedo californica receptor, displays greater affinity for that near the alpha-gamma interface. To further investigate ligand selectivity, we study the conotoxin MI-Torpedo marmorata receptor interaction. In this work, we show the binding of alpha-conotoxin MI to the T. marmorata receptor and the influence of the antagonist alpha-Bungarotoxin and the agonist carbamylcholine on such binding; in addition, and contrasting with the results for the Torpedo californica receptor, we identify the alpha-delta subunit interface as the high affinity binding site. This is the first work describing different characteristics of the interaction between alpha-conotoxin MI and receptors from different species of the same genus.     

The electromotor system of the electric catfish (Malapterurus electricus): A fine-structural analysis

Summary The electromotor system of the electric catfish (Malapterurus electricus) consists of two large ganglion cells situated in the spinal cord, two single axons containing electric nerves and two large electric organs with several million electroplaque cells. The small, irregularly stacked electroplaque cells possess at their center a crater-like indentation from which a stalk like protrusion arises. Many synaptic contacts derived from a single axon collateral are carried on lobe-like protrusions at the terminal knob of this stalk. The electric nerve consists of a large myelinated axon (diameter: 25 m) surrounded by many layers of connective tissue cells. The two ganglion cells (200 m in diameter) are rich in elements of the rough endoplasmic reticulum, Golgi apparatus and lysosomal structures. The cytoplasm of the soma changes its appearance towards the voluminous axon hillock (50 m in diameter) which these organelles do not enter. The cell soma is perforated in a tunnel-like manner by blood capillaries, axons and processes of glial cells. The cell soma and dendrites are covered with two types of synapse. One type forms mixed chemical and electrical (gap junctions) contacts with intermediate attachment plaques. The other type is only chemical in nature. This system may be useful in the study of an identified vertebrate giant neuron.     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997     

Ganglioside composition of subcellular fractions,including pre- and postsynaptic membranes,fromTorpedo electric organ

Gangliosides were isolated from four subcellular fractions of the electric organ ofTorpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postyynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 g of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.Abbreviations SVs synaptic vesicles - TLC thin-layer chromatography - cholera B-HRP B subunit of cholera toxin linked to horseradish peroxidase     

Gangliosides in acetylcholine receptor-rich membranes fromTorpedo marmorata andDiscopyge tschudii

The ganglioside composition of membranes enriched in nicotinic acetylcholine receptor (AChR) from the electric raysDiscopyge tschudii andTorpedo marmorata has been determined, and compared to that of total electric organ. A ganglioside having the chromatographic mobility of GM2 constitutes the major ganglioside (60%) in totalD. tschudii electric organ, followed by a component with the mobility of GD3 (10%), and a component running just below GD1a (about 12%). Minor constituents running as GM3 (2%) and as polysialogangliosides (comprising 8–15%) were also observed. Purified native membranes ofD. tschudii andT. marmorata displayed a similar profile, except that they were richer in a GM1-like component, and the proportion of GM2-like gangliosides was lower than that in total electric organ. Using a¹²⁵I-cholera toxin overlay assay on neuraminidase-treated high-performance thin layer chromatograms, the presence of GM1, GD1a and trace amounts of GD1b and GT1 (or GQ) were detected inD. Tschudii total membranes. Immunocytochemical trechniques showed the co-localization of gangliosides GQ1c/GT1c/GP1c, recognized by the monoclonal antibody Q211, and the AChR at the ventral, innervated face of the electrocyte.     

Role of Creatine Phosphate in the Discharge of the Electric Organ of Torpedo marmorata

Abstract: The role of creatine phosphate and adenosine triphosphate, as high energy phosphate sources, has been investigated during the discharge and recovery of the electric organ of Torpedo. ATP serves as the immediate source of energy for the biochemical process supporting the electrical activity of the electric organ. Under repetitive stimulation, when the energy demands exceed production, ATP levels are maintained constant at the expense of creatine phosphate. Only when the reservoir of creatine phosphate is depleted do the levels of ATP decrease, and this point corresponds to the state of maximal fatigue of the electric organ. Recovery studies show that the electric organ rapidly recovers the capacity to respond to single pulse stimuli. This recovery is statistically related to the recovery of the levels of ATP and acetylcholine. However, in this phase, the fatiguability of the electric organ is very high since its energy reservoir is still depleted. The complete recovery of the electric organ requires several hours and is closely related to the restoration of the levels of creatine phosphate.     

Cytoarchitectural organization of the electromotor system in the electric catfish (Malapterurus electricus)

Summary The cytoarchitectural organization of the electromotor system of the electric catfish (Malapterurus electricus) was investigated in order to obtain insight into the neuronal reorganization accompanying the functional transition of a presumptive previous motor system to an electromotor system eliciting electric organ discharge. The electric catfish possesses two giant electromotoneurons situated within the rostral spinal cord. Intracellular dye injections have revealed the enormous extension of the dendritic tree of electromotoneurons. About 50 primary dendrites span the entire lateral funicle and intermediate grey matter, and reveal an extensive contralateral projection. The giant dendritic tree (1.2 mm in rostrocaudal direction) presumably receives inputs from all ascending and descending pathways of the spinal cord. Electromotoneurons and motoneurons receive the same type of fibre inputs, and electromotoneurons and interneurons are connected through common presynaptic elements. The innervation pattern of the electromotoneurons and spinal motoneurons is similar. Synaptic terminals with round synaptic vesicles often reveal chemical contacts and gap junctions. Furthermore, dendrites of the two electromotoneurons form juxtapositions (ephapses) with each other and also with spinal interneurons. Our results suggest that the two electromotoneurons are homologous to median (primary) spinal motoneurons and are the central structures of the electromotor system within the central nervous system of the electric catfish. A high capability of information processing can be attributed to the giant dendritic trees from functional considerations. This presumably enables the electromotoneurons to elicit an electric organ discharge in different behavioural contexts with a minimum of functional reorganization.     

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.     

Phase and amplitude maps of the electric organ discharge of the weakly electric fish,Apteronotus leptorhynchus

Summary The electric organ discharge (EOD) potential was mapped on the skin and midplane of several Apteronotus leptorhynchus. The frequency components of the EOD on the surface of the fish have extremely stable amplitude and phase. However, the waveform varies considerably with different positions on the body surface. Peaks and zero crossings of the potential propagate along the fish's body, and there is no point where the potential is always zero. The EOD differs significantly from a sinusoid over at least one third of the body and tail. A qualitative comparison between fish showed that each individual had a unique spatiotemporal pattern of the EOD potential on its body.The potential waveforms have been assembled into high temporal and spatial resolution maps which show the dynamics of the EOD. Animation sequences and Macintosh software are available by anonymous ftp (mordor.cns.caltech.edu; cd/pub/ElectricFish).We interpret the EOD maps in terms of ramifications on electric organ control and electroreception. The electrocytes comprising the electric organ do not all fire in unison, indicating that the command pathway is not synchronized overall. The maps suggest that electroreceptors in different regions fulfill different computational roles in electroreception. Receptor mechanisms may exist to make use of the phase information or harmonic content of the EOD, so that both spatial and temporal patterns could contribute information useful for electrolocation and communication.Abbreviations EOD electric organ discharge - EO electric organ - CV coefficient of variance     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)