Bernard Katz

Quantal components of the end-plate potential

In this paper a further study is made of the spontaneous synaptic potentials in frog muscle (Fatt & Katz, 1952a), and their relation to the end-plate response. It has been suggested that the end-plate potential (e.p.p.) at a single nerve-muscle junction is built up statistically of small all-or-none units which are identical in size with the spontaneous miniature e.p.p.s. The latter, therefore, could be regarded as the least unit, or the quantum, of end-plate response. A convenient picture of how hundreds of such quanta, each capable of producing a miniature potential of 0 5-1 0 mV, can build up an e.p.p. of, say, 70-80 mV is provided by the hypothesis that separate parcels of acetylcholine (ACh), released from discrete spots of the nerve endings, short-circuit the muscle membrane. The unit changes of membrane conductance produced at many parallel spots summate and lead to an intense depolarization of the muscle fibre. Although this is a plausible view, there is no direct proof that the normal e.p.p. is made up in this quantal fashion. The evidence comes from experiments in which the quantum content of the e.p.p. had been reduced to a small number by lowering the external calcium concentration (Fatt & Katz, 1952 a). It was then found that the size of the end-plate response approached that of the spontaneous potential and at the same time exhibited large random fluctuations, apparently involving steps of unit size. Similar observations were made by Castillo & Engbaek (1954) on muscles treated with Mg-rich solutions. The statistical behaviour of the end-plate response under these conditions has been investigated in more detail and subjected to a quantitative analysis.

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.

Statistical factors involved in neuromuscular facilitation and depression

When a series of impulses arrive at the nerve-muscle junction, the end-plate potentials (e.p.p.) which they produce are not constant but vary in size, depending on the number and frequency of the stimuli. Two main types of phenomena have been observed: (a) a progressive increase of the e.p.p. (facilitation, post-tetanic potentiation) and (b) a phase of depression (Wedenski-inlilbition, junctional fatigue). The present paper is concerned with the stage of neuromuscular transmission at which facilitation and depression occur and with the question whether quantal changes of e.p.p. amplitude are involved. It has been suggested that the progressive synaptic changes during repetitive stimulation are due to a variation in the output of acetylcholine (ACh) from the motor nerve endings rather than to post-synaptic events (e.g. Feng, 1941 b; Hutter, 1952; Eccles, 1953, p. 89 seq.). In view of the recent evidence indicating that ACh release occurs in discrete quanta, it is of interest to inquire whether a functional change of ACh output takes place at quantal or molecular level, involving either the number or the size of the miniature units of which the end-plate response is composed.

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.

On the localization of acetylcholine receptors

The presence of special chemoreceptor substances at the motor end-plate was recognized when Langley (1907) showed that the junctional region of a muscle is highly sensitive to cerain chemical stimulants. With the use of single fibres, Buchthal & Lindhard (1937) and Kuffler (1943) demonstrated that the stimulating action of acetyl4holine (ACh) is restricted to the synaptic area of the muscle membrane, or at least that the potency of ACh at the end-plate greatly exceeds that at nerve-free regions. More recently, it became possible to use micropipettes for very localized applications of ACh+ ions to single endplates. This method was employed by Nastuk (1951, 1953, 1954) who showed that a pipette of less than 0-5 , tip diameter can be used, either as a continuous point source from which ACh is allowed to diffuse at constant rate towards a nearby end-plate, or, even more effectively, as a momentary source from which ACh+ ions are released electrophoretically, the quantity of the discharge being controlled by the strength and duration of an outward-passing current pulse. This method offers further advantages, for the pipette is small enough to be inserted into the interior of the muscle fibre. Thus, given amounts of ACh can be applied to both sides of the end-plate membrane either in the conventional manner, to the outer surface of the membrane which faces the nerve endings, or to the inner surface which faces the sarcoplasm. Experiments will be described which show that intracellular application of ACh is, relatively or completely, ineffective (see also Castillo & Katz, 1954c). The evidence supports the view that specific chemoreceptors are located on the external surface of the end-plate, that is on the side of the muscle membrane which makes contact with the nerve endings, while the internal surface of that membrane is insensitive to applied ACh.

Interaction at end-plate receptors between different choline derivatives.

Interaction between different choline derivatives has been studied by applying them simultaneously to a motor end-plate and recording the resulting changes in the membrane potential of the muscle fibre. Choline potentiates the depolarizing effect of acetylcholine (Ach) when applied in normal Ringer. Decamethonium has a ‘diphasic’ action, initial depression of the Ach effect being followed by more prolonged potentiation. When these experiments are made after treating the muscle with an esterase inhibitor (prostigmine 10-6 w/v), the potentiation of the Ach effect, by decamethonium or choline, is absent and replaced by simple ‘curare-like’ inhibition. When decamethonium is allowed to interact with a rapidly acting stable ester (carbaminoylcholine or succinylcholine), it produces simple curare-like’ inhibition. The triple effects of choline and decamethonium, i. e. (i) weak depolarization, (ii) potentiation of Ach in normal Ringer solution, (iii) inhibition of Ach in the presence of prostigmine, can be explained by competitive reactions between the drugs and receptor as well as Ach-esterase molecules. It is suggested that the first step in a depolarizing end-plate reaction is the formation of an intermediate, inactive, compound between drug and receptor.

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.

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.