Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Summary The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synapto-somes ofTorpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5′-adenylyl imidodiphosphate as substrate and 5′-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of leed pyrophosphate precipitate.     

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     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

Electron microscopic localization of choline acetyl transferase activity in the electric organ of Torpedo marmorata

The light microscopic method for demonstration of choline acetyltransferase (CAT) activity based on the formation of a lead mercaptide of free SH-acetyl Coenzyme A was adapted for electron microscopy. In samples of electric organ of Torpedo marmorata CAT activity was found to be restricted to synaptic vesicles and cysternae. The precipitate formed was mostly fine grained and distributed more or less evenly throughout the vesicles. Generally, the reaction product seemed not to adhere to the inner side of the vesicle membrane. CAT activity was found only in the presynaptic region of the synapse, neither the synaptic cleft nor the postsynaptic region reacted positively. CAT activity was found also within synaptic vesicles in nerve endings prepared from electric organ. Samples of Torpedo brain reacted positively too. Complete suppression of CAT activity with inhibitors, judged on the basis of lead mercaptide deposited, was rather difficult to achieve. From a group of 10 presumed enzyme inhibitors, only 2 compounds reacted satisfactorily, namely trans-1,2-dihydro-2-imino-4-(1-naphthylvinyl)-1-pyridine-ethanol hydrobromide and 5,5-dithio-bis-(2-nitrobenzoic acid) (3,3'-6). On the whole, the results obtained show the viability of the method used and furthermore it offers also some new insight into the turnover of acetylcholine, since it may be deduced from the results that under certain circumstances acetylcholine may be synthesized in synaptic vesicles.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.     

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase. Gel electrophoresis permitted the identification of a major protein component of the presynaptic membrane fraction which had a molecular weight of 67,000. This protein was not found in postsynaptic membrane or synaptic vesicle fractions. Thus it appeared to be specific to the nerve terminal plasma membrane.     

Electron microscopic-cytochemical study of the effect of vinblastine on synaptosome adenylate cyclase in the rat cerebral cortex

Adenylate cyclase activity was demonstrated by means of electron microscopic cytochemistry in rat cortical synaptosomes incubated under various conditions. It was found that vinblastine caused remarkable changes in the reaction product localization: the limiting membrane reaction diminished, but the number of synaptosomes were seen to contain a course granular precipitate filling up almost the whole presynaptic cytoplasm. The regulatory role the microtubules play in the distribution of adenylate cyclase on cellular membrane is discussed.     

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl₂ but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.     

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     

A further contribution to the study of the contact region of Octopus synaptosomes

Summary Octopus synaptosomes have been examined after glutaraldehyde fixation and phosphotungstic acid (PTA) staining of non-osmicated tissue. The results concentrate on the appearance of the contact region between the presynaptic component of synaptosomes and their postsynaptic processes. Membranes have a triple-layered appearance, consisting of an electronopaque internal coat, an electrontranslucent band and an electronopaque external coat. Good examples of this are found in synaptosomal, dendritic and axonal membranes. At specialized synaptic contact regions the external coats of the pre- and postsynaptic membranes coalesce to form a prominent synaptic plate, which has a width of 18 nm and is subdivided into zones of varying electronopacity. It is suggested that this plate is formed from the specialized external coat of the postsynaptic membrane and the unspecialized external coat of the presynaptic membrane. Presynaptic spicules extend from the internal coat of the presynaptic membrane. They are closely associated with elements of the presynaptic network.It is suggested that the synaptic plate is probably composed of mucopolysaccharides, while the relation of the plate to acetylcholinesterase is discussed. It is proposed that functional localization at the synapse is less precise in octopus than vertebrates.I would like to express my thanks to Professors J. Z. Young, F. R. S. and E. G. Gray for their helpful advice, and also Mr. S. Waterman for photographic assistance.     

Electron microscopic demonstration of adenylate cyclase activity in rat cortical synaptosomes

Summary The electron cytochemical demonstration of adenylate cyclase activity was carried out in rat cortical synaptosomes. Reaction product was found in 60–70% of the synaptosomes in three predominant localizations: (i) on the postsynaptic density; (ii) on the outer aspect of the synaptosomal membrane; (iii) inside the synaptosome. Results suggest that in addition to postsynaptic localization adenylate cyclase activity is cytochemically demonstrable also at presynaptic sites.     

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.     

Isolation from Cholinergic Synapses of a Protein That Binds to Membranes in a Calcium-Dependent Manner

A protein that binds to membranes in a calcium-dependent manner between calcium concentrations of 10(-5) and 10(-6) M has been isolated in large amounts (20 mg/kg tissue) from the entirely cholinergic electric organ of Torpedo marmorata. The protein bound reversibly to membrane fractions in a calcium-specific and saturable manner. The protein also bound to lipids isolated from Torpedo electric organ and to clathrin-coated vesicles prepared from pig brain. The protein bound to a Triton X-100-sensitive site. It had an apparent subunit molecular weight of 32,000 by polyacrylamide gel electrophoresis and of 35,900 by amino acid analysis; a broad isoelectric range of 4.8 to 5.5; and 27% of its amino acids after hydrolysis were observed to be aspartic and glutamic acids. Synaptosomes derived from electric organ were enriched in the protein which is probably localised within the nerve ending. It was localised in the synaptic region of the electric organ by means of immunofluorescence. In the electric lobe, discrete patches of fluorescence were seen within the cell bodies that innervate the electric organ. The protein may be involved in the recognition of membranes within the cholinergic neurone. Proteins with similar purification properties were found in all tissues investigated so far, and polypeptides of subunit molecular weight 32,000 were identified in bovine adrenal medulla and guinea pig brain synaptosomes.     

Binding of [3H]Hemicholinium-3 to the High-Affinity Choline Transporter in Electric Organ Synaptosomal Membranes

Sodium-dependent binding of [3H]hemicholinium-3 was observed to be 10-fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium-3 binding was found to be 31 +/- 4 nM and the Bmax, 5.0 +/- 0.2 pmol/mg protein; a Ki of 16 nM was estimated for hemicholinium-3 as a competitive inhibitor of high-affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium-3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high-affinity choline transporter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium-3 binding sites indicated that the high-affinity choline transporter has an apparent turnover number of about 3s-1 at 20 degrees C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high-affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium-3 binding to be of the order of 100/micron2 of synaptosomal membrane.     

Zinc-iodine-osmium impregnation of giant synaptosomes obtained from the hippocampal formation of the rabbit]

The fraction of giant synaptosomes from the r. inferior of the rabbit hippocampus was studied using impregnation with zinc iodide-osmium tetroxide (XIO) reagent and electron microscopy. In this fraction, light and dark synaptosomes were observed. The reaction product was found in the clear-centered synaptic vesicles (200-400 A) as electron-dense structures of different forms and small osmiophilic particles on the vesicular membranes. Dense-cored vesicles and postsynaptic structures were not revealed with ZIO-reagent. The structures revealed with ZIO-reagent in the giant synaptosomes of the hippocampus are supposedly related to stroage of the neurotransmitter-glutamate.     

Two pools of electron cytochemically demonstrable adenylate cyclase activity in cortical synaptosomes

Adenylate cyclase activity was demonstrated by means of electron microscopic cytochemistry in rat cortical synaptosomes incubated under various conditions. It was found that only the reaction at the postsynaptic density could be enhanced by noradrenaline and postmortem storage, while that on the presynaptic limiting membrane was insensitive to these effects but was affected selectively by vinblastine. On these grounds two different pools of cytochemically demonstrable adenylate cyclase activity were distinguished within the synaptic region.     

Isolation of Membrane Fractions with Sodium Channels Sensitive to Veratridine and tetrodotoxin from the Electric Organ of the Eel Electrophorus electricus

The veratridine/tetrodotoxin-sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre- and postsynaptic membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside-out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.     

Adenylate Cyclase of Torpedo Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Abstract: Cholinergic synaptosomes isolated from the electric organ of Torpedo contain membrane-bound adenylate cyclase activity (∼6 pmol/mg proteidmin), which is dependent on the presence of guanine nucleotides. The activity is strongly dependent on temperature and only slightly affected by NaCl. The Torpedo adenylate cyclase is completely inhibited by low levels of free Ca²⁺ (K₀∼ 0.5 μ M ). This effect is not altered by either trifluoperazine or addition of exogenous calmodulin. Ca³⁺ has no effect on the activation step of the adenylate cyclase by guanyl-5'-yl imidodiphosphate (GppNHp), and Mn²⁺ abolishes the Ca²⁺-dependent inhibition of cyclic AMP synthesis. These findings suggest that Ca²⁺ exerts its effect by direct interaction with a site located on the catalytic subunit. Torpedo synaptosomes contain presynaptic inhibitory muscarinic receptors. The binding of muscarinic agonists to the receptors is modulated (to lower affinity) by GTP. However, muscarinic ligands, examined under a variety of assay conditions, have no effect on adenylate cyclase activity. These results suggest that although both the muscarinic receptor and the adenylate cyclase are coupled to G proteins, they either interact with different G proteins or are situated in different regions of the presynaptic membrane.     

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.     

Large-Scale Purification of Torpedo Electric Organ Synaptosomes

Abstract: A procedure for the large-scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium. Two hundred grams of electric organ can be fractionated in a single run, allowing biochemical studies on presynaptic membrane constituents.