Development of the electromotor system in Torpedo marmorata: Cationic staining of the electric organ

Summary The electric organs of embryonic Torpedo marmorala have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neuntes during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.     

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.     

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     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.     

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.     

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.     

Structural changes at pure cholinergic synaptosomes during the transmitter release induced by A-23187 inTorpedo marmorata

Summary Pure cholinergic synaptosomes isolated from the electric organ ofTorpedo marmorata were stimulated by calcium ionophore A-23187. The effect of time course of stimulation on the changes in intramembrane particles (IMPs) on presynaptic membranes was studied by quickfreezing and aldehyde-fixation freeze-fracture. We showed that the decrease of small-particle density at the P-face and the increase of large-particle density at the E-face was maximum after 30 sec of A-23187 stimulation. Later, the density of synaptic vesicles decreased. We suggest that the redistribution of IMPs on the presynaptic membrane and acetylcholine (ACh) release from pure cholinergic synaptosomes have a similar time course when triggered by A-23187     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na⁺ + K⁺)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C₁₂E₈). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K⁺-phosphatase or (Na⁺ + K⁺)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the α- and β-subunits. The (Na⁺ + K⁺)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis α-toxin-Sepharose columns. The data suggest that the α-subunit of (Na⁺ + K⁺)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [¹²⁵I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC₅₀), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC₅₀. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.     

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     

High-affinity, sodium-gradient-dependent transport of choline into vesiculated presynaptic plasma membrane fragments from the electric organ of Torpedo marmorata and reconstitution of the solubilized transporter into liposomes

Vesiculated fragments of presynaptic plasma membranes have been isolated from the purely cholinergic electromotor nerve terminals of Torpedo marmorata. Synaptosomes, generated from the terminals by homogenization, were separated on a discontinuous Ficoll gradient and then lysed by osmotic shock at 2 degrees C, pH 8.5 in the presence of 0.1 mM MgCl2. These conditions for lysis were optimal for choline transport. Electron micrographs of lysed synaptosomes showed vesiculated membranes with diameters smaller than those of synaptosomes; occasionally, synaptic vesicles were observed attached to them. Intact mitochondria or synaptosomes and basal laminae were not present. High-affinity (KT = 1.7 microM) uptake of choline into these vesiculated membrane fragments showed: an absolute dependence on the Na+ gradient (outside greater than inside), a transient Na+-gradient-dependent accumulation of choline over the equilibrium concentration (over-shoot), electrogenicity and rheogenicity, since the uptake was further stimulated in the presence of a Na+ gradient by valinomycin, dependence on the presence of external Cl-, and partial dependence on a Cl- gradient (outside greater than inside), high-affinity (Ki = 25 nM) inhibition by hemicholinium-3 and temperature sensitivity. The plasma membranes were further purified by centrifugal density gradient fractionation on a 4-12% Ficoll gradient. Several enzymes and polypeptides copurified with the specific binding sites for choline present in the membranes. The fraction with the most binding sites was one denser than 12% Ficoll. This was also the fraction richest in acetylcholinesterase, 5'-nucleotidase and polypeptides of relative molecular mass, Mr (X 10(-3)) of greater than 200, 140, 68 (doublet), 57, 54 and 28. Acetylcholinesterase was positively identified as a Mr 68 000 component by immune blot. By contrast the ouabain-sensitive ATPase showed a negative correlation with choline binding sites. When the solubilized proteins of the vesiculated membranes were transferred to liposomes, they conferred on the latter the capacity to take up choline in a manner closely resembling its transport in natural membranes but with an initial (one minute) rate of uptake approximately 10-times greater per mg of protein. Several proteins were selectively transferred to the liposomes including ones of Mr (X 10(-3)) 34, 42, 47, 54, 60, 68, 92, 160 and greater than 200. The polypeptides of Mr (X 10(-3)) 140, 57 and 28 were lost in the transfer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical localization of a synaptic-vesicle antigen in a cholinergic neuron under conditions of stimulation and rest

Summary An antiserum against a specific component (a glycosamino glycan) of the cholinergic synaptic-vesicle of Torpedo marmorata has been used to investigate the localization of the component in the cell body, its movement within the electromotor axon and its fate within the nerve terminal upon electrical stimulation. After immunofluorescent staining, spots are observed throughout the cytoplasm of the lobe perikarya, although they are concentrated in the region of the axon hillock. Ligation of the electromotor nerves leading from the lobe to electric organ produces a proximal build-up of material which stains readily with the antivesicle antiserum, indicating that the vesicle antigen is transported from the cell body to the nerve terminal. A marked increase in indirect immunofluorescent staining of the electric organ is observed in the nerve ending upon electrical stimulation. We interpret this result as fusion of the vesicles with the presynaptic plasma membrane and exteriorization of the vesicle antigen to the extracellular space, thereby facilitating its staining. After recovery of the system the fluorescence declines, a result that is consistent with the reinternalization of the vesicle antigen into the core of reformed vesicles. The results support a mechanism whereby vesicles recycle within the nerve terminal and transmitter is released by exocytosis.     

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.     

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: A comparative study

Summary By means of light-microscopic immunocyto-chemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcom-missural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptidic compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, to E.M.R. and A.O.; Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile, to E.M.R.; Grant 187 from FONDECYT, Chile, to C.R.Y.; and Grant Ko 758/3-1 from the Deutsche Forschungsgemeinschaft, Federal Republic of Germany, to H.W.K.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Organization of the nervous system in Opisthorchis viverrini investigated by histochemical and immunohistochemical study

The structure and organization of the nervous system has been documented for various helminth parasites. However, the neuroanatomy of the carcinogenic liver fluke, Opisthorchis viverrini has not been described. This study therefore investigated the organization of the nervous system of this fluke using cholinesterase activity, aminergic and peptidergic (FMRFamide-like peptides) immunostaining to tag major neural elements. The nervous system, as detected by acetylcholinesterase (AchE) reaction, was similar in newly excysted metacercariae, migrating juveniles and adult parasites. In these stages, there were three pairs (dorsal, ventral and lateral) of bilaterally symmetrical longitudinal nerve cords and two cerebral ganglia. The ventral nerve cords and the cerebral ganglia were well-developed and exhibited strong AchE reactivity, as well as aminergic and FMRFamide-like immunoreactivity. Numerous immunoreactive nerve cell bodies were observed around the inner surface of the ventral sucker. Fine FMRFamide-like peptides immunopositive nerve fiber was rarely observed. Overall, the organization of the nervous system of O. viverrini is similar to other trematodes.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

Application of capillary gas chromatography to the study of hydrolysis of the nerve agent VX in rat plasma

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate] a as well as its P---S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53±4% of 374 μM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P---S bond (K_m=2.5 mM and V_max=13.3 nmol min⁻¹ of rat plasma). The activity was totally inhibited by 1 mM Hg²⁺ and was also inhibited by metal chelators.