Electric Lobe Research Articles

Stimulus-evoked changes in RNA translation in cholinergic neurons

The electromotor system of Torpedo marmorata was used to investigate effects of evoked neuronal activity on protein synthesis in neuronal perikarya. At different times after electrical or mechanical stimulation, fish were killed and the RNA of the electric lobes isolated and used to prime a cell-free translation system. The radioactively labeled synthesis products were separated by 2-D electrophoresis and the patterns obtained from different specific RNAs were compared. Several proteins exhibited time-dependent changes in rate of synthesis as indicated by the relative intensity of their autoradiographic spots. These proteins appear/disappear in a specific way, indicating a modulated translational response to neuronal activity.

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

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.

Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: I. Solubility and aggregation properties.

We report an analysis of the solubility and hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. We distinguish globular nonamphiphilic forms (Gna) from globular amphiphilic forms (Ga). The Ga forms bind micelles of detergent, as indicated by the following properties. They are converted by mild proteolysis into nonamphiphilic derivatives. Their Stokes radius in the presence of Triton X-100 is approximately 2 nm greater than that of their lytic derivatives. The G2a forms fall in two classes. Class I contains molecules that aggregate in the absence of detergent, when mixed with an AChE-depleted Triton X-100 extract from electric organ. AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which is also detectable in detergent-soluble (DS) extracts of electric lobes and spinal cord. Class II forms never aggregate but only present a slight shift in sedimentation coefficient, in the presence or absence of detergent. This class contains the AChE G2a forms of plasma and of the low-salt-soluble (LSS) fractions from spinal cord and electric lobes. The heart possesses a BuChE G2a form of class II in LSS extracts, as well as a similar G1a form. G4a forms of AChE, which are solubilized only in the presence of detergent and aggregate in the absence of detergent, represent a large proportion of cholinesterase in DS extracts of nerves and spinal cord, together with a smaller component of G4a BuChE. These forms may be converted to nonamphiphilic derivatives by Pronase. Nonaggregating G4a forms exist at low levels in the plasma (BuChE) and in LSS extracts of nerves (BuChE) and spinal cord (AChE).

Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: II. Electrophoretic variants and phosphatidylinositol phospholipase C-sensitive and -insensitive forms.

We report an electrophoretic analysis of the hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. In charge-shift electrophoresis, the rate of electrophoretic migration of globular amphiphilic forms (Ga) is increased at least twofold when the anionic detergent deoxycholate is added to Triton X-100, whereas that of globular nonamphiphilic forms (Gna) is not modified. The G2a forms of the first class, as defined by their aggregation properties, are converted to nonamphiphilic derivatives by phosphatidylinositol phospholipase C (PI-PLC) and human serum phospholipase D (PLD). AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which also exists in very small quantities in detergent-solubilized extracts of electric lobes and spinal cord. They present different electrophoretic mobilities, so that each of these tissues contains a distinct "electromorph," or two in the case of electric organs. The G2a forms of the second class (AChE in plasma, BuChE in heart), as well as G4a forms of AChE and BuChE, are insensitive to PI-PLC and PLD but may be converted to nonamphiphilic derivatives by Pronase.

Effect of acetylhomocysteine thiolactone on nucleolar cytology and lipofuscinogenesis in electric lobe neurons.

Treatment with acetylhomocysteine thiolactone significantly reduces the cellular level of lipofuscin in neurons of the electric lobe of Torpedo marmorata. At the same time, this drug produces a 45% decrease in nucleolar volume in these neurons, reflecting decreased cellular synthetic activity.

L-Glutamate binding sites in the cerebellum and electric lobes of Torpedo marmorata

1. 1. The Cl −/Ca 2+ independent l-[ 3H]glutamate binding sites have been studied in frozen sections of cerebellum and electric lobes of the fish Torpedo marmorata. 2. 2. In vitro autoradiography showed that labelling was concentrated in the molecular layer of cerebellum and homogeneously distributed in the electric lobes. 3. 3. Kinetics of l-[ 3H]glutamate binding revealed a specific and saturable binding in both regions. 4. 4. The pharmacological characterization showed that quisqualate was the most potent displacer of the l-[ 3H]glutamate binding in both regions of Torpedo marmorata brain.

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.

Monospecific antiserum against 5′-nucleotidase from Torpedo electric organ: Immunocytochemical distribution of the enzyme and its association with Schwann cell membranes

The cellular and subcellular distribution of 5′-nucleotidase in tissues of the electric ray Torpedo marmorata has been investigated by means of an antiserum raised against the native enzyme purified from the electric organ. As revealed by immunohistochemistry the enzyme is associated with the surface of the axons of the electric nerves and of spinal nerves. Using the post-embedding colloidal gold technique at the electron-microscopical level 5′-nucleotidase could be located at the plasma membrane of the Schwann cells including the myelin and the fine processes covering the terminal axon ramifications. Also the perineurial sheath of the axons inside the electric organ is 5′-nucleotidase positive. The plasma membrane of the axon and the terminal axon region or the postsynaptic membrane do not contain 5′-nucleotidase. Immunoprecipitation studies using polyacrylamide beads suggest that the ecto-Ca 2+- or -Mg 2+-adenosine 5′-triphosphatase previously ascribed to synaptosomes of the Torpedo electric organ is not associated with the same membranes as 5′-nucleotidase. Within the electric organ the dorsal plasma membrane of the electroplaque cell, blood capillaries and the connective tissue layer surrounding the columns of electroplaque cells also bind the antibodies. In central nervous tissue solely blood vessels show immunofluorescence. Within the electric lobe both the surface of the electromotor neurons as well as the myelinated axons giving rise to the electric nerve are negative. This also applies to the axons of the optic nerve suggesting that the antiserum is Schwann cell specific, and does not bind to a potential oligodendroglial 5′-nucleotidase. In peripheral tissue the surface of skeletal muscle fibres as well as that of individual myofibrils bind the anti-5′-nucleotidase antibodies. Our results demonstrate that the Schwann cell plasma membrane, including myelin, contains 5′-nucleotidase and that one can distinguish by means of a specific antiserum between Schwann cell and oligodendroglia plasma membranes. The functional significance of the association of 5′-nucleotidase with Schwann cells along the entire surface of axons including the synaptic region as well as with other parts of the electric tissue is discussed regarding its catalytic activity and also the possibility that this surface glycoprotein may be involved in mediating cellular interactions.

Expression of an omega-conotoxin-sensitive calcium channel in Xenopus oocytes injected with mRNA from Torpedo electric lobe.

Xenopus laevis oocytes were injected with poly(A)+ RNA isolated from the electric lobe of Torpedo californica. Six to nine days after mRNA injection of the oocytes a cadmium-sensitive inward current could be detected in oocytes bathed in a calcium- and chloride-free solution containing 40 mM barium. This inward current could be distinguished from the native barium current of control oocytes by its high sensitivity to blockade by cadmium ions and its inhibition by omega-conotoxin, a peptide neurotoxin from Conus geographicus. Neither the current of control cells nor that of injected cells was detectably affected by nisoldipine (1 microM) or nitrendipine (1 microM). However, the barium current of control oocytes showed appreciably more inactivation (in the barium solution used for recording) than the omega-conotoxin-sensitive current that develops in mRNA-injected oocytes. Culturing of mRNA-injected oocytes in medium containing actinomycin D failed to prevent the appearance of the omega-conotoxin-sensitive current. These results support the conclusion that mRNA from Torpedo electric lobe is translated to produce an additional calcium channel in Xenopus oocytes. The features of this channel suggest that it may be the same type of calcium channel that controls transmitter release at nerve endings in Torpedo electroplax.

Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adult Torpedo ocellata

The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G 2 form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G 2 form in back muscle is sensitive to PIPLC, whereas the G 4 tetrameter of Torpedo brain is completely resistant.

Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adult Torpedo ocellata.

The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G2 form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G2 form in back muscle is sensitive to PIPLC, whereas the G4 tetramer of Torpedo brain is completely resistant.

Cloning of cDNA derived from mRNA of the electric lobe of Torpedo marmorata and selection of putative cholinergic-specific sequences.

With the aim of selecting cDNA sequences expressed in neurons utilizing exclusively cholinergic synaptic transmission, cDNA derived from Torpedo electric lobe was cloned and screened by hybridization with probes originating from another brain region known for its low content of cholinergic neurons (cerebellum) and a nonneuronal tissue (muscle). This method led to the isolation of 18 clones among 3,200 showing no hybridization with probes other than those derived from electric lobe. These clones can therefore be considered to represent sequences involved in the expression of cholinergic function in neurons.

Presence of vasoactive intestinal polypeptide-like immunoreactivity in the cholinergic electromotor system of Torpedo marmorata.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was detected in the cholinergic electro-motor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP-like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle-specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP-like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (-50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP-like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse-phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP-like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A +) RNA in the lambda phage expression vector λgtll. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, λAChEl, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that λAChEl contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.

Incorporation of acetate into acetylcholine, acetylcarnitine, and amino acids in the Torpedo electric organ.

The metabolism of acetate was investigated in the nerve-electroplaque system of Torpedo marmorata. In intact fragments of electric organ, radiolabeled acetate was incorporated into acetylcholine (ACh), acetylcarnitine (ACar), and three amino acids: aspartate, glutamate, and glutamine. These compounds were identified by TLC, high-voltage electrophoresis, column chromatography, and enzymic tests. The system responsible for acetate transport and incorporation into ACh displayed a higher affinity but a lower Vmax than that involved in the synthesis of ACar and amino acids. Choline, when added to the medium, increased the rate of acetate incorporation into ACh but decreased (at concentrations greater than 10(-5) M) that into ACar and amino acids. Monofluoroacetate slightly depressed ACh and ACar synthesis from external acetate but inhibited much more the synthesis of amino acids. During repetitive nerve stimulation, the level of the newly synthetized [14C]ACh was found to oscillate together with that of endogenous ACh, but the level of neither [14C]ACar nor the 14C-labeled amino acids exhibited any significant change as a function of time. This means that there is probably no periodic transfer of acetyl groups between ACh and the investigated metabolites in the course of activity. Acetate metabolism was also tested in the electric lobe (which contains the cell bodies of the neurons innervating the electric organ) and in Torpedo synaptosomes (which are nerve terminals isolated from the same neurons). Radioactive pyruvate and glutamine were also assayed in some experiments for comparison with acetate. These observations are discussed in connection with ACh metabolism under resting and active conditions in tissues where acetate is the preferred precursor of the neurotransmitter.

Is propionylcholine present in or synthesized by electric organ?

When small blocks comprising four columns of electrocytes were excised from electric organs of Torpedo marmorata after stimulation in vivo via the electric lobe at 1 Hz for 1 h and allowed to recover at 20-22 degrees C for several hours in medium containing 100 microM d4 choline and 500 microM propionate, small quantities of propionylcholine amounting to no more than 1% of the endogenous acetylcholine of the tissue could be detected in tissue extracts by gas chromatography-mass spectrometry (GCMS). Kinetic studies demonstrated that there was no nonexchangeable propionylcholine in the tissue and in the absence of added propionate, propionylcholine levels were less than 0.2% of tissue acetylcholine. Vesicular propionylcholine amounted to less than 0.5% of vesicular acetylcholine and the distribution of d0 and d4 propionylcholine suggested that an appreciable proportion (up to one-third) of this could be an artifact of preparation for GCMS determinations. Propionylcholine formation during extraction and demethylation of an artificial mixture of acetylcholine, choline, and propionate was indeed detected. It is concluded that propionylcholine has no significance as an endogenous or as a false transmitter at this terminal, in conformity with the work of Sheridan et al. [Z. Zellforsch. 74, 281-307 (1966)] but in contrast to the report of O'Regan [J. Neurochem. 39, 764-772 (1982)].

Creatine phosphokinase: isoenzymes in Torpedo marmorata.

Creatine phosphokinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) is the major constituent of the "low-salt-soluble" proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43 000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti-(electric organ creatine phosphokinase) antibodies are specific for the muscle-type enzyme and do not cross-react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine-receptor-enriched membranes show that this enzyme is as the "low-salt-soluble" electric organ enzyme of the muscle-specific type. In vitro translation of electric organ poly(A)-rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain-type polypeptides are synthesized. The subunits of the brain-specific enzyme differ in molecular mass (Mr approximately equal to 42000) and isoelectric properties (pI approximately equal to 7.0-7.2). The unexpected finding that the brain forms are more basic than the muscle-specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion-exchange chromatography properties.

Torpedo electromotor system development: neuronal cell death and electric organ development in the fourth branchial arch.

The fourth branchial arch of Torpedo marmorata has been examined at the light and electron microscopic level during development. Of interest was the determination of the extent of electric organ tissue reported to be present in this arch and its possible relationship to electromotoneuron cell death in the electric lobes. The main electric organ of the torpedo is derived from the hyoid and first three branchial arches and is innervated by four major electromotor nerves. Extensive electromotoneuron cell death occurs in the electric lobes and most notably in the posterior poles. This feature could be due to a tendency for these neurons to innervate the fourth branchial arch where little or no electric tissue is formed. Our findings support this conclusion but are not entirely consistent with the idea that a population mismatch has occurred. This is because cell death precedes the genesis of the target cells. The presence of innervated differentiated electric tissue in this arch is also reported, leading to the conclusion that Torpedo marmorata possesses an accessory electric organ.

Morphological evaluation of the lipofuscinolytic effect of acetylhomocysteine thiolactone

The acetylhomocysteine thiolactone, a free radical scavenger drug able to activate the superoxide dismutase, shows a lytic effect on neuronal lipofuscin. The study was performed in a nervous tissue particularly rich in lipofuscin, the electric lobe of Torpedo marmorata. It is shown that the drug induces the migration of pigment towards the blood vessels and glial cells which remove them.

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1–8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1–8 monoclonal antibody, it was possible to show that they are different proteins. The C1–8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1–8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1–8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.