Voltage- and time-dependent action of histrionicotoxin on the endplate current of the frog muscle

Histrionicotoxin, a toxin isolated from skin secretions of a Colombian arrow poison frog, Dendrobates histrionicus, decreased the amplitude and time-course of the endplate current, and altered the voltage dependence of the half-decay time. In addition, the toxin produced a characteristic nonlinearity in the current-voltage relationship of the endplate current when 3-s voltage conditioning steps were used. Reduction in time of the conditioning steps to 10 ms made the current-voltage relationship linear. The decrease in peak amplitude of the endplate current (epc) produced by histrionicotoxin measured during long hyperpolarizing conditioning steps was fitted by a single exponential function. The calculated rate constants ranged from 0.03 to 0.14 s-1 and varied with membrane potential at hyperpolarizing levels. The voltage- and time-dependent action of histrionicotoxin does not require an initial activation of receptors by acetylcholine (ACh). The characteristic of the current-voltage relationship can be accounted for by the observed voltage and time dependency of the attenuation of the endplate current amplitude in the presence of histrionicotoxin during long conditioning steps. These effects of histrionicotoxin on the peak amplitude, and on the voltage and time dependence of the epc were concentration-dependent and slowly reversible upon washing out the toxin. Thus, the voltage- and time-dependent action of histrionicotoxin at the endplate is related to an increase in the affinity between the toxin and the ACh receptor-ionic channel complex. This increase in affinity is postulated to be due to a conformational change of the macromolecule in the presence of histrionicotoxin which is demonstrated to be relatively slow, i.e., on the order of tens of seconds.     

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 113–119, January–February, 1988.     

The rising phase of the miniature endplate current at the frog neuromuscular junction

(1) The rising phase of minature endplate currets was recorded at the frog's neuromuscular junction using both the two electrode voltage clamp and a single external electrode, or Strickholm, voltage clamp. (2) The $\text{Q}$(10) of the miniature endplate current rising phase was 2.3 in a variety of solutions selected to alter presynaptic behavior. (3) Increasing the solution's viscosity by an amount sufficient to slow the diffusion coefficient of acetylcholine by a third has no effect on the duration of the rising or the decay phase. This solution does seem to further slow the miniature endplate current decay phase, but not the rising phase, after inhibition of the acetylcholinesterase. (4) As the membrane potential is made more positive, the miniature endplate current rising phase is prolonged, with an $\text{e-}\text{fold}$ slowing per 170 mV change. (5) It is concluded that neither presynaptic nor subsynaptic events determine the rising phase of miniature endplate currents at the frog neuromuscular junction. Rather, the limiting step occurs within the membrane and is most likely a change in the binding constant of the receptor for the acetylcholine molecule.     

The permeability of the endplate channel to organic cations in frog muscle

The relative permeability of endplate channels to many organic cations was determined by reversal-potential criteria. Endplate currents induced by iontophoretic "puffs" of acetylcholine were studied by a Vaseline gap, voltage clamp method in cut muscle fibers. Reversal potential changes were measured as the NaCl of the bathing medium was replaced by salts of organic cations, and permeability ratios relative to Na+ ions were calculated from the Goldman-Hodgkin-Katz equation. 40 small monovalent organic cations had permeability ratios larger than 0.1. The most permeant including NH4+, hydroxylamine, hydrazine, methylamine, guanidine, and several relatives of guanidine had permeability ratios in the range 1.3--2.0. However, even cations such as imidazole, choline, tris(hydroxymethyl)aminomethane, triethylamine, and glycine methylester were appreciably permeant with permeability ratios of 0.13--0.95. Four compounds with two charged nitrogen groups were also permeant. Molecular models of the permeant ions suggest that the smallest cross-section of the open pore must be at least as large as a square, 6.5 A x 6.5 A. Specific chemical factors seem to be less important than access or friction in determining the ionic selectivity of the endplate channel.     

Block of endplate channels by permeant cations in frog skeletal muscle

Motor endplates of frog semitendinosus muscles were studied under voltage clamp. Current fluctuations induced by iontophoretic application of acetylcholine were analyzed to give the elementary conductance, gamma , and mean open time, tau , of endplate channels. Total replacement of the external Na+ ion by several other metal ions and by many permeant organic cations changed both gamma and tau . Except with NH4+ ions, the gamma values with foreign test ions were all smaller than expected from the independence relation and their previously measured permeability ratios. The more hydrophobic ions gave the smallest gamma values. Foreign permeant cations also depress gamma when mixed with Na+ ions. These effects could be interpreted in terms of binding of ions to a saturable site within the endplate channel as they pass through. The site for organic ions would have a hydrophobic component. Similar evidence is given for a metal ion binding site on the cytoplasmic end of the channel accessible to internal ions. Most foreign cations also shortened tau when applied externally. The changes of gating did not seem to be correlated with changes in gamma . Thus there is no evidence for control of tau by ions bound within the pore.     

Effect of a polarizing current on receptor potential of the isolated frog muscle spindle

The effect of a polarizing current on electrical activity of the isolated frog muscle spindle was studied. A depolarizing current increased the frequency and reduced the amplitude of afferent spike activity, both spontaneous and evoked by mechanical stimulation. A hyperpolarizing current produced the opposite effect. The amplitude of the receptor potential in response to a mechanical stimulus varied as a linear function of the intensity of the polarizing currentA. A. Ukhtomskii Physiological Institute, Leningrad State University. Translated from Neirofiziologiya, Vol. 5, No. 1, pp. 95–101, January–February, 1973.     

Effect of antibodies to glial glycolipid antigens on miniature endplate potentials

No more than 4% of the miniature endplate potentials (MEPP) recorded in preparations of frog cutaneous pectoral muscle kept in Ringers solution or treated with nonimmune rabbit serum were atypical, having a normal amplitude but abnormally retarded (almost doubled) time course. The percentage of atypical MEPP more than tripled after processing preparations with galactocerebroside (GalC) antiserum. Armine-induced blockade of synaptic acetylcholinesterase increased the rate of "giant" MEPP occurrence (but not of those with normal amplitude plus protracted time course). Mechanisms possibly underlying the increased percentage of atypical MEPP during GalC action on Schwann cells are discussed.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 217–223, March–April, 1989.     

Structure and ultrastructure of the frog motor endplate

Summary The frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, active zones, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by specific sites in which intramembranous particle aggregations occur at relatively high packing density (7500/m²). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N), The Swiss National Foundation for Scientific Research (Grants Nr. 3 82372 and 3 77472) and the Dr. Eric Slack-Gyr Foundation Zürich.     

Critical quantum content for shortening of endplate currents in the frog skeletal muscle.

The decay time of endplate currents was followed during progressive lowering of quantum content of endplate responses by reduced Ca2+. A certain critical value of about 100 quanta was found, when the decay of endplate currents remained constant even though the quantal content was reduced further.     

Resting membrane potentials and miniature postsynaptic potentials of frog intrafusal muscle fibers]

Electrophysiological charact eristics of frog intrafusal muscle fibers were studied by means of the intracellular potential recording. Analysis of distribution of the amplitude of miniature postsynaptic potentials suggests the nonuniformity of the intrafusal muscle fibers. A conclusion was drawn on the presence of three types of intrafasal muscle fibers in frog muscle spindle: single-innervated, multiple innervated, and fibers with both types of innervation.     

Atypical endplate miniature potentials in the frog neuromuscular junction after modification of the intercellular matrix and osmotic exposures]

In frog cutaneous-pectoris muscles the frequency of slowly rising atypical miniature endplate potentials (MEPPs) was significantly enhanced after collagenase (0.1%) treatment. Treatment with trypsin, hyaluronidase, hyper- and hypoosmotic solutions caused no changes in slowly rising MEPP (frequency in muscle fibers with intact acetylcholinesterase (AChE). Inhibition of AChE caused appearance of giant MEPPs. Acceleration of acetylcholine diffusion from synaptic cleft after treatment with hyaluronidase decreased giant MEPP frequency demonstrating their dependence upon nonhydrolyzed acetylcholine in synaptic cleft. The relation between slowly rising MEPPs and activity of synaptic Schwann cells in discussed.     

Effect of bemegride on root potentials of the frog spinal cord

The effect of the convulsant bemegride (β-ethyl-β-methylglutarimide) on spinal-root potentials was investigated in frogs. After intravenous injection in subconvulsant doses (5–12 mg/kg) bemegride caused rapid depression of the dorsal-root potentials evoked by stimulation of the neighboring dorsal or ventral root. Their amplitude fell by 55–67% 3–6 min after bemegride injection. The action of bemegride was reversible and the amplitude of the dorsal-root potentials returned to its initial level within 1 h. Ventral-root potentials showed greater fluctuations of amplitude after injection of bemegride than in the control. Bemegride is evidently an effective agent blocking depolarization of primary afferents in the frog spinal cord.     

Effect of muscle relaxants on the motor endplate of diabetic and glucocorticoid pretreated rats

The susceptibility to d-tubocurarine, gallamine, pancuronium, succinylcholine, and decamethonium of the motor endplate innervated by the anterior tibial nerve was studied in alloxan diabetic rats and in rats pretreated with cortisone and dexamethasone. The sensitivity to various muscle relaxants of cholinergic receptors in the motor endplate of alloxan diabetic and glucocorticoid-treated rats was changed. Beside alterations in affinity, in some cases the kinetics of action were also altered as compared to controls. The phenomenon is suggested to be brought about by a modulator substance circulating in the blood of alloxan diabetic and glucocorticoid-treated rats.     

The computation of simulated endplate potentials

A method is described for computing simulated subthreshold responses (.e. endplate potentials) for the Falk-Fatt (1964) cable model of a muscle fibre due to a punctate change in ionic conductance. The method has been applied to a comparison of the Falk-Fatt and classical models. Comparison with experimental results suggests that the model is adequate to account for the response of the mouse muscle fibre, in which the endplate is highly localized. For frog muscle there are larger discrepancies, which may be due to the extended endplate in this species.     

Some statistical properties of the miniature endplate potentials

I will demonstrate that series of miniature endplate potentials (m.e.p.ps) showing a high proportion of so-called giant m.e.p.ps (g.m.e.p.ps) have different statistical structures from series where the proportion of g.m.e.p.ps is low. The nature of the different structures will be discussed on the basis of two statistical models, one for the distribution of the m.e.p.p. amplitudes and one for the series of point events occurring in time.     

Modelling of miniature endplate current

From analyzing the mathematical model of miniature endplate current (MEPC) generation described previously an optimal set of parameters was found providing the best match between the results of stimulation and experimentally obtained findings. The time course of MEPC in the controls, after cholinesterase inhibition, and following a bungarotoxin-induced reduction in the density of unoccupied cholinoreceptors were described within the framework of the model. This model also statisfactorily describes voltage-dependent current decay, and the currentvoltage relationship of MEPC, as well as the kinetics of giant MEPC observed during cholinesterase inhibition. The influence of the parameters of the model on model MEPC is also examined. The good match between the results of modelling and experimental findings leads to the conclusion that the model gives a true picture of different processes contributing to the generation of MEPC.S. V. Kurashov Medical Institute, Kazan', Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya Vol. 20, No. 3, pp. 390–397, May–June, 1988.