Reversibility and mode of action of Black Widow spider venom on the vertebrate neuromuscular junction

Black widow spider venom (BWSV) stimulates transmitter release and depletes synaptic vesicles from muscles bathed in a sodium free medium containing 1 mM EGTA. However, frog neuromuscular junctions treated with BWSV in glucosamine Ringer's and post-treated with antivenin recover normal function. This suggests that probably the permanent block of neuromuscular transmission is due to changes in permeability of the nerve ending plasma membrane to cations such as Na+. When BWSV is applied in a medium lacking divalent cations and containing 1 mM EGTA, in most of the cases no effect is observed. We found that this inhibition can be overcome in three ways: (a) by adding divalent cations to the medium; (b) by increasing the tonicity of the medium with sucrose; (c) by raising the temperature of the medium. These results suggest that the lack of divalent cations influences the membrane fluidity. Moreover, in view of the report by Yahara and Kakimoto-Sameshima (1977. Proc. Natl. Acad. Sci. U.S.A. 74:4511--4515) that hypertonic media induce capping of surface receptors in lymphocytes and thymocytes, we think that these data further support the hypothesis that BWSV stimulates release by a dual mode of action; namely, it increases the nerve ending permeability to cations and also stimulates release directly via a process of redistribution of membrane components, a process which may also inhibit vesicle recycling.     

Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.     

The action of ionophores at the frog neuromuscular junction

The ionophores A23187 and X537A have markedly different actions on the MEPP frequency recorded at the frog neuromuscular junction. A23187 has no significant effect at 9–17°C, but causes a small increase in MEPP frequency at 6°C. At 25°C, on the other hand, A23187 causes a marked and progressive rise in MEPP rate. It is suggested that, in spite of increased Ca²⁺ influx associated with application of the ionophore, the presynaptic terminals can maintain [Ca²⁺]_i constant at 9–17°C, although [Ca²⁺]_i rises at higher and lower temperatures, causing an increase in frequency of MEPPs. As previously reported by Kita and Van der Kloot (5) X537A causes a dramatic increase in MEPP frequency, but it is suggested that its action is more complex and probably involves an increase in Na⁺ permeability.     

The ionic dependence of black widow spider venom action at the stretch receptor neuron and neuromuscular junction of crustaceans

The effects of black widow spider venom (BWSV) on the crayfish stretch receptor and the lobster neuromuscular junction were examined. In crayfish stretch receptor neurons, BWSV caused a slight hyperpolarization followed by a large depolarization. The venom-induced depolarization of the strech receptor was caused by an increase in membrane conductance to Na⁺ and Ca²⁺. Black widow spider venom also caused an increase in the frequency of miniature inhibitory postsynaptic potentials recorded in the strech receptor. The ability of BWSV to increase the frequency of miniature excitatory postsynaptic potentials (MEPSPs) at the lobster neuromuscular junction was dependent on the divalent cation composition of the bathing medium. Ringer solutions containing Ca²⁺ supported the greatest venom-induced increase in MEPSP frequency, Mg²⁺ and Mn²⁺ supported a moderate increase in MEPSP frequency, while Co²⁺ and Zn²⁺ blocked this venom effect entirely. Black widow spider venom did not block axonal conduction in lobster walking leg axons or in the axon of the crayfish stretch receptor. The results suggest that in crustaceans, BWSV interacts specifically with membrane of the soma-dendritic region of the stretch receptor and with nerve terminal membrane, causing an increase in Na⁺ and Ca²⁺ conductance.     

beta-Bungarotoxin antagonizes the effect of alpha-latrotoxin from black widow spider venom on the neuromuscular junction

Sustained contraction of the chick biventer cervicis nerve-muscle preparations evoked by alpha-latrotoxin was antagonized quickly by beta-bungarotoxin. This effect of beta-bungarotoxin was dependent on its phospholipase A2 activity. In contrast, pancreatic phospholipase A2 was ineffective even at a much higher dose. It is concluded that alpha-latrotoxin needs intact presynaptic membrane to exert its effect.     

Effect of thiamine on the processes responsible for acetylcholine secretion in the frog neuromuscular synapses

The effect of vitamin B₁ (thiamine, 10^–10–10^–3 M) on direct (transmitter secretion) and recurrent (resynthesis of the transmitter and its storage in synaptic vesicles) processes of acetylcholine (ACh) secretion was studied in the frog neuromuscular junction. In Ca²⁺-containing extracellular medium, the facilitatory effects of thiamine and -latrotoxin (an increase in the frequency of miniature end-plate potentials, MEPP) were additive, regardless of the sequence of their application. After partial exhaustion of the synaptic vesicle stores caused by -latrotoxin (2 nM) in Ca²⁺-free extracellular medium, thiamine accelerated the Ca²⁺-induced recovery of the ACh secretion. In the presence of thiamine, there were two phases in the dependence of quantum content of an end-plate potential (EPP) on stimulation frequency, which are typical of the effects of Sr²⁺ and Ba²⁺ on the ACh secretion. Under conditions of depression and postdepression recovery, the effect of thiamine on the time course of the changes in EPP amplitude was similar to that produced by Ba²⁺. Possible mechanisms of the effects of vitamin B₁ on the processes responsible for the ACh secretion and the dependence of the MEPP frequency on the concentrations of thiamine and thiamine diphosphate are discussed in light of the above results.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 291–298, July–August, 1994.     

Anti-curare action of stannous ion in the frog neuromuscular junction

For the purpose of elucidating the mechanism of action of stannous ion (Sn2+), we investigated effects of stannous chloride (SnCl2) on the twitch and on the electrical phenomena in the muscle fiber. Sciatic nerve-sartorius muscle preparations from the bullfrog were used as the material. Effect of SnCl2 was examined on the twitch partially inhibited by pretreatment with d-tubocurarine. SnCl2 (1-100 microM) antagonized d-tubocurarine and enhanced the twitch dose-dependently. Tartaric acid, which is the solvent used for SnCl2 solution, had no augmentative effect on the twitch, even at a concentration as high as 250 microM. SnCl2 (1-50 microM) increased the amplitude of the endplate potential; that is, it exerted an anti-curare action. The resting potential and the membrane resistance of the muscle fiber were not altered by 30 microM SnCl2. These findings lead to the conclusion that Sn2+ enhances the twitch by increasing the endplate potential of the muscle fibers.     

Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses

The localization of Ca2+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged omega-conotoxin GVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent alpha-bung-arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane.     

Funnel web spider venom produces spontaneous action potentials in nerve

Venom from the lethal Australian spider, $\text{Atrax robustus}$, causes fasciculation of muscles $\text{in vivo}$ and in isolated diaphragms in mice. Spontaneous end-plate potentials were recorded in muscle fibres exposed to the venom and associated spontaneous electrical activity could also be recorded from the phrenic nerve. It was proposed that the venom produces muscle fasciculation by causing abnormal, spontaneous, repetitive firing of motor nerves. The mechanism of this action was investigated in aplysia neurones. The venom produced abnormal, spontaneous, repetitive inward currents in voltage clamped neurones and changed the current-voltage characteristics of the surface membrane. It is suggested that the basic mode of action of Funnel-web venom is to change the electrical field in nerve membrane.     

The action of black widow spider venom on cholinergic mechanisms in synaptosomal preparations of rat brain cortices

The neurochemical activity of black widow spider venom (BWSV) was investigated using a synaptosomal preparation (P₂) of rat cerebral cortices. In a P₂ preparation pre-incubated with [3H]-choline in order to label acetylcholine (ACh), BWSV caused a marked release of radioactive ACh which was calcium independent. The onset of the action of BWSV was very rapid, i.e. in a 20 sec incubation it caused a release of the transmitter. BWSV was also shown to be a potent inhibitor of the high affinity uptake system for choline, producing about 50% inhibition at a concentration of 3.75 μg protein/m1. These findings explain in the observed electrophysiological effects of the venom.     

The channel-forming component of the Theridiidae spider venom neurotoxins

It is known that Steatoda (Lityphantes) paykulliana and Latrodectus mactans tredecimguttatus spider venoms are toxic to mammals and insects. These venoms act presynaptically eliciting massive release of transmitters. They also form channels in bilayer lipid membranes (BLM) that are selective for cations. Venoms of both spider species were fractionated by gel filtration on a Sephadex G-100 column. The fraction obtained were tested on neuromuscular preparations of frog and locust and on BLM. A fraction of low molecular weight components (about 5000 daltons and less) was disclosed. This fraction showed presynaptic and channel-forming effects similar to those of crude venoms and of high molecular weight toxin fractions, obtained simultaneously from these venoms. It was shown that channels formed in BLM by crude venoms and its different fractions are identical. Also, it was found that the low molecular weight channel-forming component is a construction element of high molecular weight toxins. On the basis of data obtained a toxin structure model of the Theridiidae family spider venoms was proposed.     

The electrophysiology and pharmacology of lobster neuromuscular synapses

Effects of drugs on resting potential, membrane resistance, and excitatory and inhibitory postsynaptic potentials (e.p.s.p.'s and i.p.s.p.'s) of lobster muscle fibers were studied using intracellular microelectrodes Acetylcholine, d-tubocurarine, strychnine, and other drugs of respectively related actions on vertebrate synapses were without effects even in 1 per cent solutions (10^- w/v). Gamma-aminobutyric acid (GABA) acted powerfully and nearly maximally at 10^-7 to 10^-6 w/v. Membrane resistance fell two- to tenfold, the resting potential usually increasing slightly. This combination of effects, which indicates activation of inhibitory synaptic membrane, was also produced by other short chain ω-amino acids and related compounds that inactivate depolarizing axodendritic synapses of cat. The conductance change, involving increased permeability to Cl^-, by its clamping action on membrane potential shortened as well as decreased individual e.p.s.p.'s. Picrotoxin in low concentration (ca. 10^-7 w/v) and guanidine in higher (ca. 10^-3 w/v) specifically inactivate inhibitory synapses. GABA and picrotoxin are competitive antagonists. The longer chain ω-amino acids which inactivate hyperpolarizing axodendritic synapses of cat are without effect on lobster neuromuscular synapse. However, one member of this group, carnitine (β-OH-GABA betaine), activated the excitatory synapses, a decreased membrane resistance being associated with depolarzation. The pharmacological properties of lobster neuromuscular synapses and probably also of other crustacean inhibitory synapses appear to stand in a doubly inverted relation to axodendritic synapses of cat.     

Activity-dependent elimination of neuromuscular synapses

At developing neuromuscular synapses in vertebrates, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. At the same time, a single input is strengthened and maintained throughout adult life, consistently recruiting muscle fibers to contract even at rapid firing rates. Work over the last decade has led to an understanding of some of the cell biological mechanisms that underlie competition and how these culminate in synapse elimination. We discuss current ideas about how activity modulates neuromuscular synaptic competition, how competition leads to synapse loss, and how these processes are modulated by cell-cell signaling. A common feature of competition at neuromuscular as well as CNS synapses is that temporally correlated activity seems to slow or prevent competition, while uncorrelated activity seems to trigger or enhance competition. Important questions that remain to be addressed include how patterns of motor neuron activity affect synaptic strength, what is the temporal relationship between changes in synaptic strength and structure, and what cellular signals mediate synapse loss. Answers to these questions will expand our understanding of the mechanisms by which activity edits synaptic structure and function, writing permanent changes in neural circuitry.