665 J. Physiol. ('959) I48, 665-670 ON INCREASING THE VELOCITY OF A NERVE IMPULSE BY J. DEL CASTILLO* AND J. W. MOORE From the National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland, U.S.A. (Received 29 May 1959) Various formulations of the local circuit theory of nerve impulse propagation (Offner, Weinberg & Young, 1940; Hodgkin, 1954) agree in suggesting that the velocity of the impulses in non-myelinated fibres is inversely proportional to the sum of the longitudinal resistances of the axoplasm and the external medium, and a number of experiments have been satisfactorily interpreted on this basis (Osterhout & Hill, 1930; Hodgkin, 1939; Katz, 1947). These experiments have been confined to variations of the resistivity of the extracellular solution; but since the major component of the total resistance is intracellular, much more marked effects are to be expected following a decrease of the longitudinal resistance of the axoplasm. In experiments using the 'voltage clamp' technique (Cole, 1949; Hodgkin, Huxley & Katz, 1952) for the investigation of the ionic currents flowing across the surface membrane of the giant nerve fibre of the squid, the internal longitudinal resistance of the axon is reduced by the presence of an axial current-passing wire electrode. It was, therefore, interesting to investigate the influence of such axial electrodes on the conduction velocity of nerve impulses initiated by external stimulation. METHODS All the experiments were performed with giant motor-nerve fibres dissected from the hindmost stellar nerve of the squid Loligo pealii and freed from the accompanying small nerve fibres. The cleaned axons were placed in a horizontal voltage clamp cell, just below the surface of oxygenated sea water that flowed at constant temperature. The axons lay on platinum stimulating electrodes near one or both ends. Three other external electrodes, extending over the experimental portion of the axon, were grounded. The conduction velocity of the nerve impulses generated by external stimulation of the fibre was measured by means of a pair of glass capillary electrodes inserted into the membrane about 18 mm apart. The micro-electrodes had an external tip diameter of about 1,u and a d.c. resistance of less than 1 MQ. Electrodes with these characteristics were selected in order to minimize distortion of the action potentials by high source impedance. In spite of the relatively large size of * Present address: University of Puerto Rico School of Medicine, San Juan 22, Puerto Rico.
666 J. DEL CASTILLO AND J. W. MOORE the tips no evidence of damage could be found in the axons after their use, and the impalements could be repeated several times without causing an appreciable decrease in resting potential. Each micro-electrode was connected via a calomel half-cell to a high input impedance preamplifier provided with input capacity neutralization. The pre-amplifier design was similar to that of MacNichol & Wagner (1954) but with increased gain and frequency response. The outputs of the two pre-amplifiers were fed into an electronic switch, and the action potentials picked up by the two micro-electrodes were displayed separately on the cathode ray oscilloscope screen. The interval between the peaks of the two action potentials in Fig. IA represents, therefore, the time taken by the nerve impulse to travel the distance between the two electrode tips. This distance was measured by means of a binocular microscope provided with an eye-piece scale, with an accuracy of about 2%. In order to be able to compare accurately the action potentials recorded at the two microelectrodes, the responses of the two input units were matched as closely as possible (see Fig. 2), with the micro-electrodes placed in sea water just outside the axon, and were readjusted again after the double impalement if the resistance of either of the micro-electrodes changed accidentally during the penetration of the membrane. After the conduction velocity of an axon had been measured in this way, the micro-electrodes were withdrawn and the axon was impaled with the axial wire electrode. These axial electrodes were 50L or 75iz platinum or silver wires extending 30 mm from glass shanks of 0-8 mm diameter and insulated except for a length of 16-18 mm at the end. Although the exposed portion was usually platinized by conventional procedures, a collodion coating or gelatin-coated Ag-AgCl was used in some experiments. The electrodes were tested by the potential response to a 50 c/s square wave of constant current, and the value at 0-5 msec or 1-0 msec was usually taken as the effective resistance and expressed in E. cm units. With the axial electrode properly sealed in position, the micro-electrodes were reinserted into the axon at approximately the same positions as before, and the conduction velocity was measured again. The tips of the micro-electrodes were placed in the axon within the region corresponding to the bared part of the wire, about 2 mm from each end. Full details about the axon chamber and the procedure used for the measurement of the resistance of the electrodes will be given in a future publication. EXPERIMENTAL RESULTS AND DISCUSSION The conduction velocities measured in the giant axons, before their impalement with the axial wire electrode, agreed well with those reported by other workers. A typical value, as seen in Fig. 1 A, was 21-5 m/sec at a temperature of 200 C. With an inter-electrode distance of somewhat under 2 cm, these velocities resulted in a delay of about 0-75 msec between the peaks of the action potentials recorded at the two micro-electrodes. The insertion of the axial wire electrode always resulted in a marked decrease of this interval, i.e. in a large increment in the rate of conduction of the nerve impulse; and, as expected, the velocity was related in an inverse fashion to the resistance of the wire. No attempt was made to establish the exact form of this relation, as axial wire electrodes of 'medium' and 'high' resistances are not easily reproducible, and we were not particularly interested in a systematic study of their properties. When the electrode resistance was as low as about 20. - cm (wire surface resistance x length), the increment in conduction velocity was so great that the temporal displacement between the action potentials recorded at the two
VELOCITY OF NERVE IMPULSE 667 micro-electrode tips became negligible, and the traces produced by the two recording channels on the oscilloscope screen appeared superimposed, as in Fig. 1 B. The gains of the two channels were made slightly unequal to show that there were, in fact, two inputs. When the site of stimulation was reversed from one end of the axon to the other, no appreciable changes were observed either of the velocity in nerves without internal wires, or in closeness of coincidence of action potentials with the wire in place. A few records, with conditions as in Fig. 1 B, were taken at high sweep speeds to try to set a lower limit on difference in time and, from that, the apparent 'velocity'. The temporal displacement between the rising phases of the two action potentials was.. msec Fig. 1. Effect of a low impedance axial electrode on the conduction velocity of impulses in the giant axon of the squid. In both A and B two capillary micro-electrodes were inserted into the axon at points about 16 mm apart. The action potentials picked up by each electrode were displayed separately on a cathode ray oscilloscope provided with an electronic switch. In A the recorded interval (0.75 msec) corresponds to a velocity of conduction of 21-5 m/sec. In B a wire electrode of low impedance has been inserted along the axis of the fibre, shortcircuiting the longitudinal resistance of the axoplasm; this increases the velocity of conduction several hundred times and makes the two recorded action potentials virtually coincide. The gains of the two channels were made slightly different to show that there really were two input signals. Temperature 20 C.
668 J. DEL CASTILLO AND J. W. MOORE about 1 p,sec. If the responses of the pre-amplifiers to a step of potential are essentially blunted exponentials, as in Fig. 2, their response to a voltage ramp will be linear with time after about 40 psec. The responses will lag the input ramp by 10 and 14,sec respectively; in other words, there should be a 4,usec displacement between them. Because the response from the micro-electrode nearer the site of stimulation was the slower of the two, and because the maximum rate of rise of the action potentials was maintained relatively constant for about 40,usec, it can be concluded that the maximum actual temporal difference between the spikes was about 3 psec. This corresponds to a velocity of 5300 m/sec or 250 times the velocity in the normal axon. 1 00 p SC( Fig. 2. Typical transient responses of the two micro-electrode pre-amplifiers were obtained by applying a voltage ramp to condensers connected to the input grids, introducing a current step to the grid (Lettvin, Howland & Gesteland, 1958). This is equivalent to the response to a voltage step at the tip of the micro-electrode. The time constants are 10 and 14 pssec respectively for the left and right traces. The limitation on the time resolution of the measuring system was the response of the micro-electrode-pre-amplifier system. As already noted, the micro-electrode time constant (lumped product of the tip resistance and capacitance across the capillary wall) was reduced by using larger tips of lower resistance than is common practice. When, as in this case, the micro-electrode time constant approaches that of the amplifier, input cable capacity limits the system response time. The cable lengths between the two micro-electrodes and their respective pre-amplifiers were different and account for most of the difference seen in Fig. 2. For more precise investigation of the difference in simultaneity of the action potentials, it would be preferable to mount the pre-amplifier inputs as close to the micro-electrodes as possible.
VELOCITY OF NERVE IMPULSE 669- These experiments show clearly that a reduction in the internal longitudinal resistance of a nerve fibre, produced by short-circuiting the electrolytic resistance of the axoplasm with a metal conductor, produces a marked increase in the rate of conduction of the action potential. As expected, this change is much greater than similar variations elicited by altering the specific resistivity of the external solution. In extreme cases, when the resistance of the axial wire is as low as 20 U. cm, the velocity of conduction becomes practically infinite over the short region occupied by the wire electrode, showing that the excitable membrane of the axon over the bare wire is behaving as a single electrical unit. The action potential is generated simultaneously over the entire electrode region, rather than being propagated from point to point with a resulting time delay. It is questionable whether or not one may use the term 'conduction velocity' under such conditions. The currents generated by the approaching action potential discharge simultaneously the entire region over the wire. This is indicated by the slower rise at the foot of the action potential (Fig. 1 B) and perhaps also by the longer delay after the stimulus artifact for the shortcircuited axon. The measurements presented in this paper are, therefore, not only relevant to the local circuit theory of nervous conduction, but they also provide a good example of the effectiveness of a 'space clamp'. A perfectly spaceclamped region may be defined as one over which potential changes may occur with time, but where at any instant the potential is the same at every point. Therefore, a propagated action potential crossing the clamped region should enter at one end and depart from the other without delay. This would only be possible if the longitudinal resistance of the clamped length of the fibre were negligible. The above experiments show that a fairly close approximation to this ideal condition can be obtained in practice by threading platinized platinum wires with an effective resistance of about 20 U. cm along the interior of nerve fibres about 500,u in diameter. SUMMARY 1. The conduction velocity of propagated action potentials in the squid giant axon is greatly increased if the internal longitudinal resistance of the axoplasm is short-circuited by an axial wire electrode. 2. The increase in the conduction velocity is related in an inverse fashion to the surface resistance of the electrodes, and with wires of a resistance as low as 20 U. cm the apparent conduction velocity over short lengths of axon may be 250 times greater than normal. 3. It is concluded that the space clamp achieved by using electrodes of the above mentioned resistance closely approaches, for practical applications, an ideally perfect space clamp.
670 J. DEL CASTILLO AND J. W. MOORE We should like to express our appreciation to Dr K. S. Cole for his participation in the planning, execution and reporting of this work, and to the Marine Biological Laboratory, Woods Hole, for the facilities which made this work possible. REFERENCES COLE, K. S. (1949). Dynamic electrical characteristics of the squid axon membrane. Arch. Sci. phy8iol. 3, 253-258. HODGKIN, A. L. (1939). The relation between conduction velocity and the electrical resistance outside a nerve fibre. J. Physiol. 94, 560-570. HODGKN, A. L. (1954). A note on conduction velocity. J. Physiol. 125, 221-224. HODGKIN, A. L., HuxLEY, A. F. & KATZ, B. (1952). Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J. Physiol. 116, 424 448. KATZ, B. (1947). The effect of electrolyte deficiency on the rate of conduction in a single nerve fibre. J. Physiol. 106, 411-417. LETTvm, J. Y., HOWLAND, B. & GESTELAND, R. C. (1958). Footnotes on a headstage. Inst. Radio Engrs. Trans. Med. Electronics, No. 10, 26-28. MAcNIcHOL, E. F., Jr. & WAGNER, H. G. (1954). A high-impedance input circuit suitable for electrophysiological recording from micropipette electrodes. Naval Med. Res. Inst. Rep. 12, 97-118. OGFNER, F., WEiNBERG, A. & YOUNG, G. (1940). Nerve conduction theory: Some mathematical consequences of Bernstein's model. Bull. math. Biophys. 2, 89-103. OSTERHOUT, W. J. V. & HIL, S. E. (1930). Salt-bridges and negative variations. J. gen. Physiol. 13, 547-552.