Miniature Endplate Currents Research Articles

Sites of action of procaine at the motor end-plate.

The effects of procaine at pH 7.4 and 9.9 were studied by examining the decay phase of spontaneous miniature end-plate currents (m.e.p.c.s) recorded from toad sartorius muscle fibres. Following exposure to procaine (0.05-0.1 mM) at pH 7.4, the decay of m.e.p.c.s rapidly became biphasic, and could be described as the sum of two exponential components. When the same concentrations of procaine were applied at pH 9.9, the development of biphasic m.e.p.c.s took much longer. Reversal of the effect upon washing out the procaine was much slower at pH 9.9 than at pH 7.4. A rapid change in pH from 7.4 to 9.9 during exposure to a constant concentration of procaine quickly reduced the effect of procaine on m.e.p.c.s. The effect gradually returned after prolonged exposure to procaine at pH 9.9. These results suggest that procaine applied at high pH, where it is predominantly in uncharged form, may be diffusing to a site of action which is not directly accessible from the external surface of the membrane. The voltage-dependence of procaine action was similar whether it was applied at pH 7.4 or 9.9. Intracellular injection of procaine rapidly produced biphasic m.e.p.c.s, whether the extracellular pH was 7.4 or 9.9. The effect of membrane potential on these m.e.p.c.s was similar to that seen for biphasic m.e.p.c.s produced by extracellular application of procaine. The results indicate that procaine can affect end-plate channels when applied to either surface of the muscle membrane, and that the voltage-dependence of procaine action does not arise from the influence of membrane field on the movement of charged procaine molecules into open channels.

Temperature and end-plate currents in rat diaphragm.

1. Spontaneous miniature end-plate currents (m.e.p.c.s.) were recorded in rat diaphragm at 7, 22 and 37 degrees C at -80 mV. The onset rate, measured as 20-80% rise time, was sensitive to temperature with activation energy 14 kcal mol-1 deg-1, and was not sensitive to membrane voltage between -60 and -130 mV. 2. The rise time recorded by external electrodes was 144 microseconds at 37 degrees C (6) and was similar to that found by internal electrodes. 3. The fall time was temperature-sensitive with activation 18 kcal, and was prolonged when the end-plate was hyperpolarized. 4. With acetylcholine (10 microM) the current increased to a peak and then fell within 30 s to a value which declined slowly. From fluctuation analysis the channel open time of 237 microseconds (7) at 37 degrees C was estimated. External recording gave comparable values (4). Comparison of the initial estimates with those obtained after 3-6 min of continued application showed no consistent change. The channel conductance was 26 pS at 37 degrees C. 5. The time constant of m.e.p.c. decay was consistently longer than the channel open time obtained from noise analysis. 6. With carbachol (40 microM) the current increased to a peak and then declined to a steady value. Fluctuation analysis by internal and external recording gave an increase of 5% in root mean square current with channel open time of 83 microseconds (6) at 37 degrees C, and channel conductance 17 pS.

Electrophysiological and chemical determination of acetylcholine release at the frog neuromuscular junction.

1. Mass fragmentography was used to measure the tissue content and release of acetylcholine (ACh) by frog sartorius muscles, which had been previously treated with an irreversible cholinesterase inhibitor. The frequency of miniature end-plate currents (m.e.p.c.s) was also measured. 2. Exposure of muscles for 15 min to 2 mM-LaCl3 resulted in a large release of ACh which subsided to low levels after 1 h. About 4 h later treatment with 50 mM-KCl, or with the calcium ionophore A 23187, or with a second dose of LaCl3, all failed to augment ACh release, notwithstanding the fact that the ACh content of La3+-treated muscles was about the same as that of controls. 3. Hypertonic NaCl or raised KCl concentrations were used to increase m.e.p.c.s and this also increased ACh release; it was estimated that each quantum corresponded to the release of 12 000 molecules of ACh. 4. ACh release by nerve stimulation was greatly potentiated by 10 mM-tetraethylammonium chloride, and this enabled the ACh released by ten, and even single, stimuli to be detected; it was calculated from the ACh released and the quantal content that each quantum contained on the average 13 000 molecules. 5. ACh released by nerve stimulation at 0.2/s in the absence of tetraethylammonium was about half that expected on the basis of previous estimates of quantal content; it was increased about two-fold by alpha-bungarotoxin. 6. It is concluded that chemical and electrical stimulation of the nerve evoked quantal ACh release, without influencing non-quantal ACh leakage. The results are consistent with the view that ACh quanta are derived from synaptic vesicles. They also show that resting ACh release is not due to leakage of ACh ions along an electrochemical gradient in the membrane.

Properties of junctional and extrajunctional acetylcholine-receptor channels in organ cultured human muscle fibres.

1. Current noise, obtained during steady ionophoretic application of acetylcholine (ACh) to voltage-clamped human fibres has been analysed to derive properties of end-plate channels and also extrajunctional ACh-activated channels which are present at the muscle-tendon junction of normal fibres. In addition, ACh-receptor channels present at the end-plate and tendon region in organ cultured muscles have been compared with those in fresh muscles.2. Extrajunctional channels in the tendon region of fresh fibres have a longer mean life-time, tau, and a smaller single channel conductance, gamma, than the junctional channels. tau was 1.71 +/-0.11 msec and gamma was 25.05 +/- 1.18 pS for junctional channels; tau was 3.16 +/- 0.33 msec and gamma was 12.76 +/- 1.29 pS for extrajunctional channels.3. Properties of channels in the end-plate and tendon region were unchanged during short-term (< 7 days) organ culture at 23 or 36 degrees C. The voltage sensitivity of the mean channel life-time was similar at junctional and extrajunctional sites.4. In muscles organ cultured for 7 days at 36 degrees C, double component noise spectra were obtained at some end-plates. The fast and slow time constants underlying the noise appeared to correspond to simultaneous activation of junctional and extrajunctional channels.5. After organ culture for 3-4 weeks at 23 degrees C the mean life-time of the end-plate channels was prolonged while their single channel conductance was unchanged, tau = 3.58 +/- 0.16 msec; gamma = 22.11 +/- 0.83 pS (V(m) = -80 mV, T = 21 degrees C).6. As end-plate channel properties were unchanged in short-term cultures it was possible to assess post-junctional sensitivity by comparing miniature end-plate current (m.e.p.c.) amplitudes in both normal and myasthenia gravis affected muscles. No evidence was found for a change in post-synaptic sensitivity during organ culture at 23 degrees C of normal or myasthenia gravis muscle fibres, which seems to rule out reversible block of receptors by anti-receptor antibody as playing an important role in myasthenia gravis.

The effects of hydrostatic pressure on the spontaneous release of transmitter at the frog neuromuscular junction.

1. The effects of hydrostatic pressure (0.1-15.55 MPa) on the spontaneous release of transmitter at the frog neuromuscular junction were investigated. 2. The major effect of high pressure is on the release mechanism, pressure (0.1-10.40 MPa) producing an exponential decrease in frequency of the miniature end-plate currents in normal Ringer solution. The frequency decreases to 0.52 and 0.24 of the control value at 5.25 and 10.40 MPa respectively. This effect is reversible on decompression. 3. The sensitivity of the release process to high pressure is unaltered in 10 mM-K+, 6 mM- and 10 mM-Ca2+ and hypertonic (165 mM-NaCl) Ringer solution, although the high Ca2+ media shift the threshold for the pressure effect to higher pressures. 4. Higher pressure (10.40-15.55 MPa) produces a small increase in the time constant of decay (tau D) of m.e.p.c.s with no effect on the growth phase. A pressure of 15.55 MPa increases tau D to 1.35 of the control value. 5. The possible actions of high pressure on both the pre- and post-synaptic processes are briefly discussed.

FAST DESENSITIZATION OF THE NICOTINIC RECEPTOR AT THE MOUSE NEUROMUSCULAR JUNCTION

1 When low concentrations of carbachol (2-20 muM) were applied by local superfusion to mouse diaphragm endplates, there occurred a rapid decrease (within seconds) in the height of miniature endplate currents (m.e.p.cs) in addition to the increase of muscle membrane conductance.2 With 2, 5, 10 and 20 muM carbachol, m.e.p.c. heights were diminished by 5, 10, 30 and 50% respectively. A subsequent slow decrease in height took place at a rate corresponding to that reported for the slow desensitization produced by bath-applied carbachol (see Adams, 1975).3 The effect of carbachol on m.e.p.c. height was not affected by poisoning of acetylcholinesterase (AChE). After poisoning of AChE, 4 muM acetylcholine (ACh) depressed m.e.p.c. height by 23%.4 At 20 muM carbachol, both the onset and offset of the effect on m.e.p.c. height lagged behind the subsynaptic conductance change, and the calculated change of subsynaptic agonist concentration, by about 3 s; the onset rate was at least ten times faster than expected for slow desensitization.5 When the conductance responses produced by carbachol were corrected for fast desensitization, the slope of the log-response log-dose line (Hill coefficient) was increased from 1.7 to 2.0.6 The Hill coefficient for fast desensitization was 1.4. The data were compatible with a cyclic model for fast desensitization, with receptor activation not a prerequisite for desensitization of receptors.7 The failure of AChE poisoning to affect m.e.p.c. height during desensitization suggests that desensitized receptor associated with exogenous agonist can continue to bind quantal ACh.

Effects of extracellular sodium concentration on null potential, conductance and open time of endplate channels.

(i) Effects of extracellular sodium concentration, [Na]o, on endplate channel characteristics were investigated in voltage-clamped, glycerol-treated toad sartorius fibres. (ii) The relation between [Na]o (and [K]o) and acetylcholine null potential could be reasonably well fitted by the Goldman-Hodgkin-Katz type of equation, except when [Na]o was higher than normal. Anions had no significant effect on the null potential. (iii) Endplate channel open time (phi), whether measured from miniature endplate currents or from current fluctuations induced by iontophoresis of acetylcholine, varied inversely with [Na]o. The relation between phi-1(= alpha) and [Na]0 could be fitted by alpha = alpha max [Na]o/(Km + [Na]o) with a Km of 92 mM. (iv) Endplate conductance, measured at the peak of endplate currents or at the peak of spontaneous miniature endplate currents, increased nonlinearly with [Na]o. (v) Single channel conductance, gamma, also increased nonlinearly with [Na]o. Experimental observations at -90 mV could be fitted by the relation gamma = gamma max [Na]o/(Km + [Na]o), giving values for gamma max and Km of 47 pS and 146 mM respectively. Correcting channel conductance for the contribution from potassium ions gave values of gamma max and Km of 78 pS and 423 mM respectively. (vi) The results are consistent with the hypothesis that binding sites for Na ions can modulate both channel lifetime and conductance and that these sites become saturated at higher sodium concentrations.

Postsynaptic currents in different types of frog muscle fibre.

Miniature end-plate currents (mepcs) were recorded from fast and slow muscle fibres in two muscles, m. cruralis and m. ileofibularis, under voltage clamp or extracellularly. Both fibre types showed an identical sensitivity to α-bungarotoxin, and an exponential dependence of mepc decay time-constant upon membrane potential, but there were some important differences: i) the rise time and half-time of decay were approximately 3 times longer in slow junctions than in fast ones: ii) inhibition of AChE had a more prominent effect on mepcs recorded in fast fibres while preserving the significant difference in mepc time course between fast and slow fibres; iii) treatment by α-bungarotoxin had a greater effect on mepc time course in slow junctions even in the absence of anticholinesterase; iv) a more prominent variability of mepc time course was observed in slow junctions, especially after treatment by anticholinesterase drugs.

The interaction of extracellular H +, Na +, Ca 2+ and Sr 2+ on the decay of miniature end-plate currents

We suggested previously that increases in the intracellular [Ca 2+] or [Mg 2+] lengthen miniature end-plate currents (m.e.p.c.s.) because the divalent cations decrease the negative surface potential on the end-plate, and thereby increase the voltage drop within the membrane. The increased voltage drop would lengthen the open time of the end-plate channels. Increased extracellular [H +] is also known to lengthen m.e.p.c.s. 15; perhaps also acting by changing the surface potential. There has been dispute about the observations and their interpretation. Reducing the [Na +] 0 atpH= 7.4 from 120 to 12 mM decreased the half-time of m.e.p.c. decay by nearly a factor of two. When the same changes in [Na +] 0 were performed atpH= 5.2, no change in m.e.p.c. decay was detected. This suggests that the H + titrates the negative surface charges, so they no longer can be affected by [Na +] 0. The data do not fit the hypothesis that lifetime is determined by the presence of permeant ions in the channel 4,16. AtpH= 7.4 replacing the 2.5 mM CaCl 2 by SrCl 2 decreased the m.e.p.c. decay rate by 60% 17. This effect is not seen atpH= 5.2. We suggest that atpH= 7.4, Sr 2+ binds to fixed negative charges on the end-plate and thereby slows the decay. H + titrates the fixed charges and decreases the Sr 2+ effect. In low [Na +] saline, raising [Ca 2+] 0 shortened m.e.p.c. decay — an effect exactly opposite to that expected from the change in surface potential. Under these conditions, increasing the [Ca 2+] decreases the m.e.p.c. decay rate by some other mechanism; this second effect of Ca 2+ on m.e.p.c. decay may account for some of the different results reported.

Noise analysis of acetylcholine induced current at neuromuscular junctions of the mouse diaphragm

Miniature end-plate currents (mepcs) and membrane noise elicited by acetylcholine (ACh) iontophoresis were investigated at neuromuscular junctions of the mouse diaphragm. All the experiments were performed at a holding potential of −70 mV at a temperature of 19°C. The equilibrium potential of the ACh response was estimated to be near zero; the mepcs displayed a peak amplitude of 2.46 ± 0.13 nA (mean ± S.E.) and relaxed exponentially with a time constant of 1.63 ± 0.11 msec. Single ACh-activated channels had a conductance of 26.5 ± 1.5 pS and a mean life time of 1.69 ± 0.13 msec.

Postsynaptic effects of some central stimulants at the neuromuscular junction.

1 Miniature endplate currents (m.e.p.cs) were recorded with extracellular electrodes from sartorius muscles of toads. 2 Central excitant analogues of amylobarbitone (3M2B) and halothane (DBE) decreased the amplitude and time constant of decay of m.e.p.cs and hence reduced the amplitude of miniature endplate potentials. The decay remained exponential with single time constant. 3 A central excitant analogue of ether (indoklon) reduced the amplitude of m.e.p.cs and made their decay biphasic. The decay could be fitted by the sum of two exponentials. 4 Bemegride, a central excitant, prolonged m.e.p.cs. Their decay remained exponential with single time constant. The effect was not due to inhibition of acetylcholinesterase. 5 All of the drugs tested, including amylobarbitone, reduced the temperature-sensitivity of the decay of m.e.p.cs. 6 The biphasic decay of m.e.p.cs caused by indoklon could not be explained simply by supposing that the drug blocked open endplate channels unless it was assumed that the normal rate of channel closing also increased and became much less temperature-sensitive than normal.

THE KINETICS OF LOCAL ANESTHETIC BLOCKADE OF END-PLATE CHANNELS

The effect of the local anesthetic QX222 on the kinetics of miniature end-plate currents (MEPC)and acetylcholine-induced end-plate current fluctuations was studied in voltage-clamped frog cutaneous pectoris neuromuscular junctions. The rate constants for a kinetic scheme of local anesthetic blockage of end-plate channels were calculated from the MEPC decay parameters. At 18 degrees C the blocking rate constant was 1.1 +/- 0.3 x 10(7) exp (-0.009 +/- 0.003 x V)s -1M-1, and the unblocking rate constant was 5.7 +/- 0.6 exp (0.011 +/- 0.002 x V)s -1. The dissociation constant was close to 10 microM at -80 mV. End-plate fluctuations indicated that the local anesthetic QX222 lowered the effective single-channel conductance, suggesting a finite blocked state conductance that was calculated to be 1.6 pS. The apparent differences between QX222 interaction with end-plate and extrajunctional channels are discussed.

Effects of divalent cations on toad end-plate channels.

Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltage-clamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20 mM Ca or Mg, MEPCs had time constants of decay (tau D) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both tau D and channel lifetime were seen; the null potential was -34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1-5 mM, were much more effective in increasing tau D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of tau was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in tau, especially when taken together with changes in channel conductance.

Effect of carbachol on miniature end-plate potentials and currents of rat skeletal muscle

Experiments on rats showed that carbachol (10 µM) appreciably quickens miniature end-plate potentials in the soleus muscle kept in medium with potassium chloride concentration increased to 13 mM. Potentiation of spontaneous mediator release also took place when the membrane potential of the muscle fibers was clamped at the level of the potassium equilibrium potential. It is concluded that the presynaptic action of carbachol on synaptic liberation of acetylcholine from motor nerve endings is unconnected with depolarization of the postsynaptic membrane but takes place as a result of the direct effect of the mimetic on motor nerve endings.

Changes in neuromuscular junction endplate current time constants produced by sulfhydryl reagents.

The acetylcholine receptor is a protein that contains certain critical disulfide bonds. Experiments were designed to determine the role such bonds might play in the physiological activity of the receptor. Modification of the receptor with sodium bisulfite and diamide produced an increase in the time constants of the miniature endplate current without changes in the single-channel properties of the receptor. Controls were done to determine that this change in the miniature endplate current was not due to an effect on acetylcholinesterase at the endplate. These data are interpreted to mean that the reagents increase the time acetylcholine is bound to the receptor before the channel opens and is most probably due to a change in receptor affinity brought about by chemical modification of the receptor protein.

Kinetic parameters for acetylcholine interaction in intact neuromuscular junction.

The dependency of miniature endplate current (mepc) rise time upon mepc amplitude and acetylcholine receptor site density was measured in lizard intercostal muscles and used to fit the rate constants in a simple kinetic scheme. The kinetic scheme included diffusion, two-step sequential binding of acetylcholine to receptor, and opening of the ion channel. Numerical simulation of the observed mepc behavior yielded the following kinetic constants; (i) diffusion constant, 4 X 10(-6) cm2 sec-1; (ii) forward binding rates, 4.7 X 10(7) M-1 sec-1; (iii) channel relaxation rate, 25 msec-1. The value above for the forward binding rates assumed both rates to be equal. If they are different, the slower of the two is in the range of 2-5 X 10(7) M-1 sec-1. A radial profile of bound receptor indicated that activation of the receptor was very local, occurring essentially within a radius of about 0.3 micrometers from the point of acetylcholine release.

The timing of channel opening during miniature end-plate currents

Many chemical transmitters act by opening channels with exponentially distributed life-times. We present a way to analyze a synaptic current in terms of its component channels. We can estimate the numbers and times of channel opening within the synaptic current. This approach is used to study miniature end-plate currents (m.e.p.c.s) at the frog neuromuscular junction. The results support the idea that some transmitter rebinds after dissociation from post-synaptic receptors, and suggest that the time of channel closing is related to the time at which acetylcholine leaves the receptor.

Relation between subsynaptic receptor blockade and response to quantal transmitter at the mouse neuromuscular junction.

When a quantum of transmitter is released into a synaptic cleft, the magnitude of the subsynaptic response depends upon how much transmitter becomes bound to receptors. Theoretical considerations lead to the conclusion that if receptor density is normally high enough that most of the quantal transmitter is captured, subsynaptic quantal responses may be insensitive to receptor blockade. The effectiveness of receptor blockers in depressing the subsynaptic response should be diminished by interference with processes that normally dispose of transmitter, but increased if receptor density is reduced. In conformity with equations derived from a simple mathematical model, the apparent potency of (+)-tubocurarine (dTC) to depress the peak height of miniature end-plate currents (MEPCs) in mouse diaphragm was substantially reduced by poisoning of acetylcholinesterase (AChE) and increased by partial blockade of receptors by immunoglobulin G from patients with myasthenia gravis or alpha-bungarotoxin. We calculated from the data that normally capture of quantal acetylcholine (ACh) by receptors is approximately 75% of what it would be if there were no loss of ACh by hydrolysis or diffusion of ACh form the synaptic cleft. This fraction is increased to approximately 90% by poisoning of AChE. Conversely, it normally requires blockade of approximately 80% of receptors-and after AChE poisoning, approximately 90% of receptors-to reduce ACh capture (and MEPC height) by 50%. The apparent potency of dTC to alter MEPC time-course (after AChE poisoning) and to depress responses to superperfused carbachol was much greater than its apparent potency to depress MEPC height, but corresponded closely with the potency of dTC to block receptors as calculated from the action of dTC on MEPC height. These results indicate that the amplitude of the response to nerve-applied acetylcholine does not give a direct measure of receptor blockade; it is, in general, to be expected that an alteration of subsynaptic receptor density may not be equally manifest in responses to exogenous and endogenous neurotransmitter.

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 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 e- 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.

Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena

The triangularis sterni muscle (TS) of the mouse is a thin trapezoidal sheet of fibres in which individual neuromuscular junctions are easily observed with Nomarski optics. Thus, microelectrodes are readily positioned to accurately record various synaptic phenomena. For example, miniature end-plate currents were easily recorded with a focally positioned extracellular electrode and the end-plate sensitivity to acetylcholine averaged 2062 mV/ nC. In addition, the intercostal nerves segmentally innervate the TS. Electrophysiologic and histologic analysis showed that each nerve innervates a sharply defined territory of the muscle surface. These preliminary observations suggest that the TS may be ideal for studies of synaptic function and the processes underlying synapse stabilization in the mammal.