scholarly journals Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves.

1986 ◽  
Vol 103 (2) ◽  
pp. 521-534 ◽  
Author(s):  
J D Black ◽  
J O Dolly
Keyword(s):  
Plasma Membrane ◽  
Neuromuscular Junction ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Type A ◽  
Nerve Trunk ◽  
Metabolic Inhibitors ◽  
Motor Nerve Terminal ◽  
Electron Microscopic Autoradiography

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

scholarly journals Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis.

1986 ◽  
Vol 103 (2) ◽  
pp. 535-544 ◽  
Author(s):  
J D Black ◽  
J O Dolly
Keyword(s):  
Nerve Terminal ◽  
Time Course ◽  
Motor Nerve ◽  
Endocytic Pathway ◽  
Metabolic Inhibitors ◽  
Motor Nerve Terminal ◽  
Specific Cell ◽  
Nerve Terminals ◽  
Electron Microscopic Autoradiography ◽  
Silver Grains

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

scholarly journals Increased Transmitter Release and Aberrant Synapse Morphology in a Drosophila Calmodulin Mutant

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 265-274 ◽  
Author(s):  
LaChelle Arredondo ◽  
Heidi B Nelson ◽  
Kathy Beckingham ◽  
Michael Stern
Keyword(s):  
Nerve Terminal ◽  
Calcium Binding ◽  
Motor Nerve ◽  
K Channel ◽  
Motor Nerve Terminal ◽  
Larval Neuromuscular Junction ◽  
Eukaryotic Organisms ◽  
And Function ◽  
Calmodulin Mutant

Abstract The ubiquitous calcium-binding protein calmodulin (CaM) has been implicated in the development and function of the nervous system in a variety of eukaryotic organisms. We have generated mutations in the single Drosophila Calmodulin (Cam) gene and examined the effects of these mutations on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. Larvae exhibit increased neurotransmitter release from the motor nerve terminal at low [Ca2+] in the presence of the K+ channel-blocking drug quinidine. In addition, synaptic bouton structure at motor nerve terminals is altered. These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC). Furthermore, previous in vitro studies of mutant Cam3c1 demonstrated that although its Ca2+ affinity is decreased, Cam3c1 protein can activate CaMKII, CaMAC, and CaM-activated phosphatase calcineurin in a manner similar to wild-type CaM. Thus, the Cam3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target distinct from CaMKII or CaMAC.

scholarly journals Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins fromBothrops marajoensis(Marajó Lancehead) Snake Venom

2016 ◽  
Vol 2016 ◽  
pp. 1-10
Author(s):  
Antonio Lisboa ◽  
Rodolfo Melaré ◽  
Junia R. B. Franco ◽  
Carolina V. Bis ◽  
Marta Gracia ◽  
...  
Keyword(s):  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Acetylcholine Receptors ◽  
Motor Nerve ◽  
Synaptic Proteins ◽  
Muscle Fibres ◽  
Motor Nerve Terminal ◽  
Transmission Electron ◽  
Skeletal Muscle Fibres

Neuromuscular preparations exposed toB. marajoensisvenom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposedin vitroto BmjeTX-I and BmjeTX-II toxins fromB. marajoensisvenom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Steroid anaesthetics: inhibition of depolarization–secretion coupling at the mouse motor nerve terminal

1980 ◽  
Vol 58 (10) ◽  
pp. 1221-1228 ◽  
Author(s):  
P. Pennefather ◽  
E. Puil ◽  
D. M. J. Quastel
Keyword(s):  
Neuromuscular Junction ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Motor Nerve Terminal ◽  
Nerve Terminals ◽  
Potassium Depolarization ◽  
End Plate

The coupling between nerve terminal depolarization and quantal secretion of acetylcholine at the mouse neuromuscular junction was estimated by measuring the multiplication of the frequency of miniature end-plate potentials (m.e.p.p.s) produced by increasing the concentration of calcium in the medium from 0.1 to 1.0 mM in the presence of 15 mM potassium. Depolarization–secretion coupling was inhibited by the anaesthetic steroids progesterone, pregnanedione, and alphaxalone. The nonanaesthetic steroid Δ16-alphaxalone also inhibited depolarization–secretion coupling with the same potency as alphaxalone. This result indicates that inhibition of depolarization–secretion coupling in nerve terminals is unlikely to play a major role in the production of anaesthesia.

scholarly journals Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1898
Author(s):  
Cristina Pinto ◽  
Viviana Pérez ◽  
Jessica Mella ◽  
Miguel Albistur ◽  
Teresa Caprile ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Motor Nerve Terminal ◽  
In Ovo ◽  
Wnt Ligands ◽  
Postsynaptic Differentiation

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

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