Ricardo Miledi

The Effect of Calcium on Acetylcholine Release from Motor Nerve Terminals

The method of external focal recording from the neuromuscular junction has been used to locate the site of action of calcium ions in the transmission process. The muscle is placed in a calcium-deficient medium (which contains magnesium as a substitute), and the effect of localized calcium application from the recording micropipette is studied. Electrophoretic application of calcium is followed within less than 1 s by increased terminal release of acetylcholine, shown by a large increase in the number of quantal components of the end-plate potential. This effect is observed even under conditions when the terminal axon spike diminishes in size during the application of calcium. It is concluded that the action of calcium is concerned directly with the release of the transmitter, and not indirectly—as is sometimes suggested—by facilitating propagation or increasing the amplitude of the terminal nerve spike.

The measurement of synaptic delay, and the time course of acetylcholine release at the neuromuscular junction

Focal recording from active spots of a neuromuscular junction was used to measure the ‘synaptic delay’ between terminal axon spike and end-plate current (e.p.c.). Synaptic delay is defined as the time interval between peak of inward current through the presynaptic membrane and commencement of inward current through the postsynaptic membrane. By substituting magnesium for calcium in the medium, and by adjustable electrophoretic application of calcium from the recording electrode, the e.p.c. can be restricted to the small portion of a single junction which is in contact with the microelectrode, and the statistical average amplitude of the e.p.c. can be reduced to less than quantal unit size. Under these conditions, the latency of the unit components of the e.p.c. can be determined and its statistical fluctuations studied. The synaptic delay at a single end-plate spot has a minimum value, at 20 °C, of 0.4 to 0.5 ms and a modal value of about 0.75 ms. There is considerable fluctuation of the measured intervals during a series of nerve impulses; over 50 % occur within a range of 0.5 ms, the rest being spread out in declining fashion over a further 1 to 4 ms. These latency fluctuations are shown to be a statistical consequence of the quantal process of transmitter release. The contribution of various factors to the minimum synaptic delay are discussed. Terminal conduction time has been effectively eliminated by the method of focal recording. Diffusion of acetylcholine towards the receptors, and its reaction with them must cause delays whose exact values are uncertain, but whose extreme upper limits can be shown to make up only a small part of the observed minimum delay. It is concluded that the synaptic interval arises chiefly from a delay in the release of transmitter after the arrival of the nerve impulse.

Transmitter Leakage from Motor Nerve Endings

When endplates of anti-esterase treated frog muscle are subjected to a massive ionophoretic dose of curare, a small local hyperpolarization is recorded in many fibres, amounting on the average to about 40 μV. The origin of this effect may be attributed to leakage of cytoplasmic acetylcholine (ACh) from nerve terminals, building up an ACh concentration of the order of 10-8 M in the synaptic cleft and causing a minute steady depolarization of the endplate. It is calculated that such a steady leakage of ACh, although producing a barely detectable electrical effect, could exceed the efflux due to spontaneous quantal discharges by two orders of magnitude, and account for the assayed amounts of ACh release from resting muscle.

Transmitter release induced by injection of calcium ions into nerve terminals

Calcium ions injected into the presynaptic nerve terminal in the giant synapse of the squid, evoked transmitter release while similar doses of Mg and Mn were ineffective. The transmitter release induced by intracellular application was still observed when Ca was replaced in the external fluid by Mn, in spite of the fact that this abolished transmitter release in response to presynaptic depolarization.

A Calcium-Dependent Transient Outward Current in Xenopus laevis Oocytes

Membrane currents were investigated in Xenopus laevis oocytes under voltage clamp. Depolarizing pulses, given from a holding potential of about –100 mV, elicited a transient outward current when the membrane potential was made more positive than about –20 mV. As the potential was made increasingly positive the transient outward current first increased and then decreased. The amplitude of the transient current increased when the external Ca2+ concentration was raised; and the current was abolished by Mn2+. It appears that when the membrane is depolarized Ca2+ ions enter the oocyte and trigger an outward current, possibly by opening Cl– channels.

Chloride current induced by injection of calcium into Xenopus oocytes.

Membrane currents of Xenopus oocytes were studied with the membrane under voltage clamp. Intracellular injection of the calcium‐chelating agent EGTA reduced, or abolished, the transient outward chloride current normally activated by membrane depolarization. Intracellular injection of calcium ions evoked large membrane currents, which inverted direction close to the chloride equilibrium potential. Injections of strontium, or barium, were less effective than calcium, while magnesium was ineffective. Large chloride currents could be evoked by calcium injections in oocytes which showed only small or no transient outward currents. The current activated by calcium injection increased with increasing depolarization up to high (ca. +60 mV) positive potentials, even though the transient outward current was suppressed by strong depolarization. The results indicate that the transient outward current depends upon entry of calcium through voltage‐gated calcium ion channels and show that the oocyte membrane contains numerous chloride channels which are activated by intracellular calcium. Only a few of these chloride channels are activated by depolarization.

The Release of Acetylcholine from Nerve Endings by Graded Electric Pulses

1. The effect of brief depolarizations focally applied to a motor nerve ending was studied. Particular attention was paid to the relation between (i) strength and duration of the pulse and (ii) the size and latency of the resulting end-plate potential. 2. The release of acetylcholine lags behind the depolarization which causes it. If pulses of less than 4 ms duration are used (at 5 °C), the release starts after the end of the pulse. 3. Within a certain range, lengthening the pulse increases the rate of the ensuing transmitter release. 4. Unexpectedly, lengthening the depolarizing pulse also increases the latency of the transmitter release. This finding is discussed in detail. It is regarded as evidence suggesting that entry into the axon membrane of a positively charged substance (external Ca2+ ions or a calcium compound CaR+) is the first step leading to the release of acetylcholine packets from the terminal.

Propagation of Electric Activity in Motor Nerve Terminals

External micro-electrodes were used to stimulate non-myelinated motor nerve terminals and to record pre- and post-synaptic responses at the neuromuscular junction of the frog. The synaptic terminals of the motor axon are electrically excitable. Antidromic nerve impulses can be set up by local stimulation of terminals along the greater part of their length. Presynaptic spikes can be recorded from the non-myelinated terminal parts of motor axons. As the impulse proceeds towards the tip of the terminal branch, the shape of the spike changes from a predominantly negative to a predominantly positive-going wave. Similar changes occur in muscle fibres near their tendon junctions, and can be attributed to the special local-circuit conditions at the ‘closed end’ of a fibre. The velocity of impulse propagation in motor nerve endings was determined by three different methods: (a) from the latency of antidromic nerve spikes elicited at different points along terminals, (b) from two-point recording of spikes along a terminal, (c) from the differential latency of focal end-plate potentials recorded at two spots of a myoneural junction. The average velocity obtained by these methods was approximately 0.3 m/s at 20 °C. Extracellular muscle fibre spikes recorded at junctional spots showed no significant differences from those recorded elsewhere, provided the spikes were initiated by direct stimulation and did not coincide with transmitter action. Direct current polarization produces a graded increase in frequency of miniature end-plate potentials when the endings are being depolarized, and sudden high-frequency bursts during excessive hyperpolarization. External two-point recording shows that these bursts arise independently at different spots of the synaptic terminals.

Effect of Lanthanum Ions on Function and Structure of Frog Neuromuscular Junctions

1. Electrophysiological and electron-microscopic studies were made of the effect of lanthanum ions on frog neuromuscular junctions. 2. In the presence of 1 mM La2+, nerve impulses continued to invade the nerve terminals but ceased to release transmitter. 3. Lanthanum caused a rapid and large increase in the frequency of miniature end-plate potentials; presumably because La activates the mechanism of transmitter release without the usual prerequisite of presynaptic membrane depolarization. At 4 °C, La caused a 10000-fold, or even larger increase in the rate of leakage of transmitter quanta. Such high rate of transmitter release was not accompanied by obvious changes in electron-microscopic structure of the nerve terminals. 4. With continued La-treatment, the frequency of miniature end-plate potentials subsides slowly until they are no longer detectable at most end-plates. During this period the number of synaptic vesicles is reduced until practically all the endings become completely depleted of synaptic vesicles. In contrast, coated vesicles and membrane-bound tubes and cysternae become more numerous.

Tetrodotoxin and neuromuscular transmission

1. The puffer fish poison, tetrodotoxin (T. T.) was applied to eliminate impulse propagation in nerve and muscle fibre, and the physiological properties of the neuromuscular junction were studied under this condition. 2. Spontaneous miniature end-plate potentials of normal frequency and amplitude were recorded in the T. T.-paralysed muscle. 3. Depolarization of motor nerve endings by locally applied current still produces the usual increase in the frequency of miniature end-plate potentials (e. p. ps). 4. When brief current pulses are applied to the nerve endings e. p. ps can be evoked, whose size varies with the intensity of the current. The responses are composed, like normal e. p. ps, of a statistically varying number of miniature potentials. The response fails when calcium is removed from the bath. 5. When two identical pulses are applied at varying intervals, facilitation of the second e. p. p. occurs, similar to that observed normally with pairs of nerve impulses. 6. It is concluded that tetrodotoxin while blocking electric excitation in nerve and muscle does not interfere with the release of acetylcholine from nerve endings nor with its local action on the muscle fibre.