Stampfii6 also reported that during the relatively refractory period the action potential

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1 ON THE EFFECT OF ANESTHETICS UPON ISOLATED, SINGLE FROG NERVE FIBERS* By V. HONRUBIAt AND R. LORENTE DE NO THE ROCKEFELLER INSTITUTE Communicated October 16, 1962 Experimental work with isolated single nerve fibers was begun in Professor Kato's laboratory in the early 30's and has later been continued in a number of laboratories. Kato" 2 advanced two concepts which have received wide acceptance in the literature. (1) The nerve fibers obey an extended form of the all-or-nothing law, i.e., they produce either the maximal response which they are capable of producing or none at all. (2) Anesthetics act upon nerve fibers instantly, as soon as they reach the surface of the fibers at a certain critical concentration. Kato also concluded that the major part of the time of action observed with nerve trunks was in fact diffusion time through the connective tissue sheath. Kato's observations were extended by Tasaki,3 who while working in Professor Kato's laboratory created his well-known bridge insulator method to record the action potential of isolated single nerve fibers.4 In agreement with Kato's concepts, Tasaki found that anesthetics act upon the nerve fibers instantly. Weak concentrations of anesthetics reduce the height of the action potential, but this potential retains its all-or-nothing character. Higher concentrations, but still concentrations that are weak in relation to those needed to block conduction in nerve trunks with intact sheath, abolish the action potential instantly. Tasaki also reported that during the refractory period the action potential has a subnormal height, but nevertheless it retains its all-or-nothing character. Tasaki supported Kato's belief that the connective tissue sheath effectively hinders diffusion of substances into the nerve trunk. The observations of Kato and Tasaki were extended by Huxley and Stampfli5 who reported that sodium-free solutions abolish the action potential instantly. Stampfii6 also reported that during the relatively refractory period the action potential retains its all-or-nothing character. The observations made by Kato, Tasaki, and Huxley and Stampfli are in sharpest disagreement with the observations made in this laboratory with frog sciatic nerves with intact external sheath, with normally sheathless nerves (bullfrog spinal roots), and with desheathed sciatic nerves. The observations made in this laboratory led to these conclusions:7'-" (1) The external connective tissue sheath of frog nerve is perfectly permeable to all solutes, be they ionized or not. (2) The action of a substance, be it an anesthetic like cocaine, procaine, or xylocaine, or be it a substance of a different kind such as, for example, an excess of potassium ions, depends upon two factors, (a) concentration and (b) time of action. (3) The presence of sodium ions outside the nerve fibers is not necessary for the production of the action potential. Later, when the doctrine of decremental conduction was reinstated in this laboratory,12 it appeared that with nerve fibers submitted to the action of depressant agents such as anesthetics, sodium-free media, and an excess of potassium ions the all-or-nothing law is not valid. Contrary to what this law demands, the magnitude 2065

2 2066 PHYSIOLOGY: HONRUBIA AND LORENTE DE N6 PROC. N. A. S. of the action potential increases with the magnitude of the applied stimulus. This situation becomes exaggerated during the refractory period, since, as had been originally demonstrated by Adrian"3 and Lucas,14 the decrement during conduction increases during the refractory period. Under conditions such as these, two alternatives must be considered: (1) Dissection of single nerve fibers out of a nerve trunk radically alters their properties. (2) Kato, Tasaki, and Huxley and Stampfli used inappropriate techniques and drew incorrect conclusions from incomplete observations. The second alternative has proved to be true. When the proper technique is used, it is found that isolated single nerve fibers behave in essentially the same manner as the undissected fibers of nerve trunks have been observed to behave relatively recently in this laboratory and, much earlier, in the laboratories of the classical authors who founded the doctrine of decremental conduction. Consequently, much of the work done in the past with single fibers by Kato, Tasaki, and Huxley and Stampfli, and other authors stands in need of radical revision. In this laboratory, the observations have been made with single nerve fibers dissected from bullfrog nerve trunks, using Tasaki's4 bridge insulator method, which with minor modifications is the method universally used to obtain electrical recordings of the activity of single nerve fibers. As a rule, in using this method, the segments of the internodes enclosed in the air gaps are allowed to undergo a certain amount of drying in the atmosphere of the laboratory. This procedure seems to have the advantage of increasing the height of the recorded potential, but we have found that it is highly harmful to the isolated nerve fiber. As soon as the internodes begin to dry, the nerve fiber begins to deteriorate and usually within a short time it dies. For this reason, we have enclosed the preparation in a box filled with humidified air. The results obtained with the use of anesthetics are presented in this communication. Those obtained with the use of sodium-free media will be presented in a following communication. Figure 1 illustrates the results obtained with the use of xylocaine at two concentrations, 0.25 mm and 3 mm. Xylocaine hydrochloride was added to normal Ringer's and the ph was adjusted to 7.3 by adding a few drops of dilute NaOH. Throughout the experiment, two stimuli of fixed strength were used. The first stimulus was barely sufficient to initiate an action potential and, since the untreated fiber was more or less accurately obeying the all-or-nothing law, this near-threshold stimulus also was a maximal stimulus. The second stimulus was approximately three times greater than the first. After the application of the anesthetic, the behavior of the response to the first stimulus would disclose the changes both in the stimulation threshold and in the height of the action potential, while the response to the second stimulus would give, at least for a certain length of time, the height of the maximal action potential that the nerve fiber still was capable of producing. To be sure, with the untreated nerve fiber the second stimulus elicited an action potential of subnormal height because this potential was elicited during the refractory period left by the first response; but after the action of the anesthetic had prevented the fiber from responding to the first stimulus, the response to the second stimulus was subject only to the effect of the subliminal catelectrotonus created by the first stimulus.

3 VOL. 48, 1962 PHYSIOLOGY: HONRUBIA AND LORENTE DE NO 2067 The series of records 2 to 6 (Fig. 1) show that the development of the action of the drug (0.25 mm xylocaine) was gradual. The first action potential decreased in height before the raise in the threshold of stimulation had prevented the fiber from producing it, but the second action potential only decreased in height. Record 5 shows that during anesthesia the all-or-nothing law was not even approximately valid. Applied currents of progressively increasing magnitude produced action potentials of progressively increasing height. The recovery in Ringer's solution also was a slow process (Fig. 1, records 7 to FIG. 1.-Tracings of oscillograph records obtained with an isolated, single frog nerve fiber. The experimental arrangement is indicated in the diagram at the bottom, center. Segments of the fiber, each including 2M an exposed node of Ranvier, are immersed in 2 5 three chambers or pools separated by two air gaps. Nodes N1 and N2 are surrounded by a 5 mm solution of xylocaine, which raises the threshold of stimulation and pre- 4 5 vents initiation of action potentials. Node N, is immersed first in Ringer's solution and later in the anesthetic solutions or Ringer's solution. The times of action of each I solution are given on the right hand side of 7 each record. The time calibration (5 msec) applies to all records except record 18. The potential I' 3\M Z2 calibration (50 mv) applies to all records. A 2* The current calibration (4 X 10-9 amp) measures the stimulating current. Unless 5* s otherwise indicated (record 18), the current used to elicit the first action potential was 13 14P 15, f_ kept constant. It is given by the vertical bar at the left of record 1. In the case of 4 1 a\ 24 records 5 and 13 the response to the second stimulus was elicited when the first stimulus 1G J 17 Is lo was subliminal. The action potentials of progressively increasing height were elicited 50 v by single stimuli of progressively increasing.v 14lo0Amr. magnitude. The magnitudes of those currents which elicited the largest action potentials are given at the left of records 5 and 13. 9). The first action potential appeared 1.5 min after the anesthetic solution had been replaced by normal Ringer's solutions, at which time the second action potential still had a markedly subnormal height. With advancing time, the first action potential progressively increased in height, but this height still was slightly subnormal after 6 min recovery. Thirty seconds later, the first action potential had regained practically its initial height. The fiber was then submitted to the action of a 3 mm solution of xylocaine (Fig. 1, records 10 to 14). This solution acted, of course, faster, but nevertheless it acted at a remarkably low rate since the first action potential was still present after 30 see of action of the drug. (The time is counted from the instant at which the renewal of the solution in the central pool had been completed, i.e., about 2 to 3 see after the anesthetic solution had come in contact with the nerve fiber.) As a matter of fact, the rate of action of the 3 mm xylocaine solution upon the isolated single fiber was not significantly greater than the rate at which a slightly stronger solution has been

4 2068 PH YSIOLOG Y: HONR UBIA A ND LORENTE DE Ax6 PROC. N. A. S. observed countless times in this laboratory to act upon superficial fibers of nerve trunks with intact sheath. After 5 min, even the second action potential had almost disappeared. Nevertheless, as is shown by record 13, action potentials of progressively increasing height could be elicited by progressively increasing the magnitude of the stimulating current. Recovery from anesthesia in Ringer's solution was a slow process indeed. After 7 min, the action potential still was subnormal in height (Fig. 1, record 17). The recovery was approaching completion after 24 min, at which time the action potential had regained its approximately all-or-nothing character (Fig. 1, record 18). It is important to note that the action potentials reproduced in Figure 1 are monophasic because the threshold of stimulation of node N2 had been markedly raised by 5 mm xylocaine. In comparable experiments, in which node N2 was kept immersed in Ringer's solution, it has been repeatedly observed that when the applied current is sufficiently large to initiate a large action potential at the anesthetized node N1, this action potential initiates an action potential at node N2, whereby the recorded response becomes diphasic, much as if no anesthetic at all had been applied to node N1. Since the pka of xylocaine (7.85) is lower than the PKa of cocaine (8.41) and since the free bases of all the anesthetics of the type of cocaine have approximately equal anesthetic potencies, a 3 mm1 solution of xylocaine at ph 7.3 has a considerably greater anesthetic potency than a 3 mm (0.1 per cent) solution of cocaine at the same ph. Consequently, the statement made by Tasaki,3 that a 0.1 per cent solution of cocaine, or even weaker solutions, instantly abolishes the action potential, is incorrect. Because the free base of cocaine is only slightly soluble in water, cocaine solutions (annot have high anesthetic potency. Far more potent solutions can be prepared with xylocaine, the free base of which is readily soluble in water. A number of experiments were done with 5 and 7 mm xylocaine and two experiments were done in which the isolated single frog nerve fiber was submitted to the effect of 10 ml\i xylocaine at ph 7.3. After more than 10 min, the nerve fiber still was capable of producing action potentials. This result agrees with the observation repeatedly made in this laboratory with the naturally sheathless spinal roots of bullfrogs that at any concentration anesthetics such as cocaine, procaine, xylocaine, or phenyl urethane do not prevent the establishment of the action potential even after they have begun to cause irreversible deterioration of the nerve fibers. The reason why the observations made by Kato and Tasaki were incomplete is easy to find. These authors used very brief shocks to stimulate the nerve fibers to produce action potentials. Owing to the peculiar shape of the so-called strengthduration curve, after the anesthetic has raised the threshold of stimulation, brief shocks cannot reach the stimulation threshold unless such tremendous voltages are used that the membrane of the nerve fiber is destroyed by the stimulating current before the action potential can be initiated. A very slight improvement of their technique, namely a lengthening of the pulses of current they used to stimulate the nerve fibers, would have enabled Kato and Tasaki to obtain complete results. At this point, the following should be stated. When it is stimulated with single pulses of current, an isolated single nerve fiber may obey the all-or-nothing law more

5 VOL. 48, 1962 PHYSIOLOGY: HONRUBIA AND LORENTE DE N or less closely. But during the refractory period that follows after a first response, contrary to categoric statements made by Tasaki and Stampfli, the all-or-nothing law ceases to be even approximately valid. All through the refractory period the height of the action potential depends upon the magnitude of the stimulus, and therefore the height of the action potential increases with the magnitude of the stimulus in the same manner as it does with fibers in a state of anesthesia (Fig. 1) or with fibers in a state of sodium deficiency. (The properties of nerve fibers in a state of sodium deficiency are analyzed in a following communication.) From the observations made in this laboratory, it follows that dissection of single fibers adds little to the damage that removal of the external connective tissue sheath has already done to the nerve fibers. In regard to the action of anesthetics, removal of the external connective tissue sheath produces two changes in the nerve fibers. It increases their sensitivity to the action of anesthetics and markedly reduces the ability of the nerve fibers to recover from anesthesia. For example, the following observations can be made using a pair of sciatic nerves, one with intact sheath and the other desheathed. A 7 mm solution of xylocaine (ph 7.3) applied to a 15-mm-long segment of the first nerve and a 3 mm solution of xylocaine at the same ph applied to a 15-mm-long segment of the second nerve produce total block of conduction in the two nerves in the same length of time, about 10 to 15 min. The block begins to be established practically instantaneously in the two nerves. Immediately after the conduction block has become total, the treated segments are immersed in Ringer's solution. In the nerve with intact sheath, conduction begins to be re-established within less than one minute and thereafter the recovery of conduction proceeds at a high rate. In the desheathed nerve, conduction does not begin to be re-established until several minutes have elapsed. Thereafter, the recovery proceeds at a much lower rate than in the nerve with intact sheath. If we were to follow the line of argument used by Kato and Tasaki, we would have to draw these two conclusions. The presence of the sheath delays the inward diffusion of the anesthetic into the nerve, since, in order to obtain the same degree of anesthesia, 7 mm xylocaine must be used with the intact nerve, while 3 mm xylocaine is sufficient for the desheathed nerve. But the presence of the sheath greatly enhances the outwards diffusion of the anesthetic since the intact nerve recovers from the state of anesthesia much more rapidly than the desheathed nerve. Of course, to draw two such contradictory conclusions would be absurd. The truth is that the external sheath is perfectly permeable to xylocaine (as it is permeable to any other solute) and offers no hindrance to its diffusion either inwards or outwards. Removal of the sheath, however, alters the properties of the nerve fibers. Since in what concerns the function of the external sheath of frog nerve (the lamellated sheath of Ranvier) considerable confusion exists in the literature, it is advisable to add a few explanatory words to summarize the results of work done in this laboratory. Proof of the statements to be made and references to the literature must be left for a future publication. In the same way as there is a symbiosis between the axon and its satellite cells, the Schwann cells, there is a symbiosis between the nerve fibers and the connective tissue of nerve, which constitutes the external milieu of the nerve fibers. Removal

6 2070 PHYSIOLOGY: HONRUBIA AND LORENTE DE Nd PROC. N. A. S. of the lamellated sheath alters the properties of the nerve fibers because it alters the properties of their external milieu. Strange as this may seem to some nerve physiologists, the external sheath of frog nerve (the lamellated sheath of Ranvier) plays a role similar to that of the cellulose wall of plant cells. The sheath is needed for the maintenance of water equilibrium in the nerve. The sheath contains a circular system of elastic fibers which enables it to exert a radial pressure upon the nerve trunk. This pressure is of course small since necessarily it must be smaller than the pressure inside the blood capillaries. Nevertheless, it plays an exceedingly important role in the regulation of water equilibrium in the interstitial connective tissue and in the nerve fibers. Upon removal of the lamellated sheath, the interstitial connective tissue swells up, almost instantly, and becomes edematous. In the nerve fibers, a movement of water from the axon into the myelin layer takes place: the volume of the axon decreases, while that of the myelin layer increases.11 Summary.-Anesthetics of the type of cocaine do not abolish the ability of isolated, single frog nerve fibers to produce action potentials. They only increase the threshold of stimulation and reduce the height of the maximal action potential that the nerve fiber is capable of producing. During anesthesia, the all-or-nothing law is not even approximately valid. The height of the action potential increases with the magnitude of the stimulating current. Neither the establishment of nor the recovery from anesthesia is instantaneous. They are time-dependent processes. * This research was supported in part by a grant (B-2650) from the U.S. Public Health Service. t Postdoctoral Research Fellow of the U.S. Public Health Service. 1 Kato, G., The Microphysiology of Nerve (Tokyo: Maruzen, 1934). 2 Kato, G., in Excitation Phenomena, Cold Spring Harbor Symposia on Quantitative Biology, vol. 4 (1936), p Tasaki, I., Nervous Transmission (Springfield, Ill.: Charles C Thomas, 1953). 4 Tasaki, I., quoted in reference 1. 5Huxley, A. F., and R. Stampfli, J. Physiol., 112, 496 (1951). 6 Stampfli, R., J. de Physiulogie, 48, 710 (1956). 7 Lorente de No, R., A Study of Nerve Physiology, Studies from the Rockefeller Institute, vols. 131, 132 (1947). 8 Lorente de No, R., J. Cell. Comp. Physiol., 33, Suppl. (1949). 9 Lorente de N6, R., J. Cell. Comp. Physiol., 35, 195 (1950). 10 Lorente de N6, R., J. Gen. Physiol., 35, 145, 203 (1951). '1 Lorente, de No, R., in The Neuron, Cold Spring Harbor Symposia on Quantitative Biology, vol. 17 (1952), p Lorente de N6, R., and Condouris, G. A., these PROCEEDINGS, 45, 592 (1959). 13Adrian, E. D., J. Physiol., 46, 384 (1913). "Lucas, K., J. Physiol., 46, 470 (1913).