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Discrete Threshold and Repetitive Responses in the Squid Axon Under 'Voltage-Clamp'

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Discrete Threshold and Repetitive Responses in the Squid Axon Under 'Voltage-Clamp' Discrete Threshold and Repetitive Responses in the Squid Axon Under ‘Voltage-Clamp” I. TASAKI AND A. F. BAK From the Laboratory of Neurophysiology, National Institute of Neurological Diseases and Blindness, National Institutes of Wealth, Bethesda, Maryland ABSTRACT TASAKI, I. AND A. F. BAK. Discrete threshold and repetitive responses in the squid axon under ‘voltage-claw@ Am. J. Physiol. 193(z): 301-308. 1958.- By using the so-called voltage-clamp technique, records were obtained indicating the presence of undulatory or oscillatory membrane current under fixed potential difference between the internal and external elec- trodes. This oscillatory membrane current was observed only when rectangu- lar membrane depolarization was in the range between 15 and about 35 mv. The amplitude of the oscillatory membrane current was of the order of I ma/cm2. The oscillatory membrane current disappeared suddenly when the depolarizing clamping pulse was reduced below the ‘threshold.’ Various sources of artefact were examined, INTRODUCTION INCE Hodgkin, Huxley and Katz (I) membrane of the toad nerve fiber under volt- published the results of their voltage- age-clamp (3). Encouraged by the similarity Sclamp experiments, it has been widely between the toad nodal membrane and the accepted that, under the voltage-clamp condi- squid axon membrane under TEA, we ex- tions, the membrane current takes smooth, panded our observation on this discontinuous well-defined time courses,showing no sign of phenomenon to normal squid axons with an discontinuous threshold phenomena. During improved experimental setup. We found that the courseof a seriesof experiments designedto this phenomenoncould be demonstrated in the study the properties of the squid axon mem- majority of the fresh squid axons available at brane treated with tetraethylammonium Woods Hole. chloride (TEA), one of us (I. T.) working in The phenomenon in question can be stated collaboration with S. Hagiwara, encountered a as follows: when the ‘membrane potential’ of strange phenomenon which appeared to the squid giant axon is raised above its resting conflict with the widely accepted view as to level by 15-25 mv and is kept at this level for a the behavior of the axon under voltage-clamp. period of about IO msec. or longer, the mem- The phenomenon observed could be taken as brane current required to maintain the new signs of the existence of a discrete threshold potential level undergoes rapid undulating or and of repetitive firing of responsesunder the ‘oscillatory’ changes which resemble the so-calledvoltage-clamp conditions. Since such phenomenon of repetitive firing of impulses a phenomenon was entirely unexpected and under ordinary experimental conditions. When appeared to be inconsistent with the basic the size of the clamping rectangular pulse is concept of voltage-clamping, no explicit reduced by small steps, this phenomenon dis- statements were made of the phenomenon in appears suddenly at a certain intensity level, our previouspublications on the squid axon (2). indicating the existence of a ‘threshold.’ When Recently, we could demonstrate the the level of depolarization by the clamping existence of a similar phenomenonin the nodal pulse is about 40 mv or higher, the time course of the membrane current observed always Received for .publication November 7, 1957. resembles that published by the British in- 1 The work presented in this article was carried out at the Marine Biological Laboratory, Woods Hole, vestigators (I). Mass. In pursuing this phenomenon,we were fully 302 I. TASAKI AND A. F. BAK used most frequently. The arrangement of diagram B is similar to that used by the n British investigators (I) and was used mainly in the early stage of the present investigation. The arrangement of diagram C is a combina- tion of the two, A and B. In the arrangement of diagram A, the internal electrode-set consisted of three silver wires of about 50~ in diameter. The potential- electrode, E,, had an exposed surface of 3-6 km mm in the middle of the current-electrodes. /////,II’// ///,II/// I/ EP ///~~pwqw~//, E p The main current-electrode, Em, had a 6 mm ’ f E; p fE; (sometimes 3 or 8 mm) long uninsulated FIG. I. Diagram A: experimental setup used for the surface, and the lateral current-electrode, El, experiments of figs. 2,3 and4. The drawings of the axon had two bare portions of 4 (sometimes3 or 8) and the internal electrodes are disproportionately short mm length adjacent to the bare portion of the and thick. The rectangular voltage pulse at the input of the high-gain differential amplifier, AZ, is used to main current-electrode. These internal silver clamp the membrane potential; the other rectangular wire electrodes were used both with and with- pulse is used to obtain records of ordinary action poten- out chloriding the surface electrolytically. The tials. Further details are in the text. Diagram B: ar- external potential-electrode, Ei, was a large rangement used for the experiment of fig. 7. Partitions, silver wire covered by a layer of gauze and P, are made with glass-plates (I mm thick) and with a small amount of Vaseline. Diagram C: arrangement agar gel. The ground electrode, which was used for the experiment of fig. 5. Partitions (made with similarly covered with gauze and agar gel, had Lucite plate and Vaseline) are watertight. a resistanceof about IO ohms to current pulses used in the present experiments. The sig- aware of the possibility that there might have nificance and the effect of the lateral current- been somekind of artefact in our experiments. electrode will be discussedunder RESULTS. We made therefore a number of control experi- The time course of the potential difference ments designedto explore such a possibility. between the two potential electrodes, E, and We try in the present paper to present the Ei, was recorded with a unity-gain differential results of these experiments as objectively as preamplifier, A 1 in figure I, diagram A (cf. possible. fig. 2 in ref. 2) ; the recording was as a rule A preliminary report of this work has been d.c.-coupled. The membrane current, 1%, was published elsewhere(4). determined by recording the IR-drop across METHODS resistance Ye which was generally between 2 and IO ohms. The high-gain differential The material, the experimental setup and the amplifier for negative feed-back, AZ in diagram procedures employed in the present experi- A, was a Tektronix amplifier of type I 12 (in- ments were similar to those described in a stead of type I 2 2 in the previous experiments), previous paper (2). Giant axons, taken from modified to give an output impedance of the squid available at Woods Hole, were used, about 250 ohms. This was achieved by adding in many caseswithout extensive cleaning of paired cathode-follower stages (two 6CL6 the surrounding tissues. In some cases, they tubes) at the output of the commercially were used after extensive cleaning under dark- available amplifier. The over-all voltage field illumination. The axon was mounted amplification, a, by this amplifier was ap- horizontally on a glass-plate of about 42 mm width, and a set of internal electrodes, con- proximately 2,300. The resistance connected sisting of two or three twisted silver wires was between the output of this amplifier and the inserted along the axis of the axon. current-electrodes, r in the diagram, was varied The arrangements of the internal and between 500 and 25,000 ohms. The smallest external electrodes employed in the present value of r/n (cf. p. 864 in ref. 2) available was experiments are illustrated by three diagrams therefore about 0.3 ohm. in-figure I. The arrangement of diagram A was As in the previous experiments (2), negative DISCRETE THRESHOLD AND REPETITIVE RESPONSES IN SQUID AXON 303 feed-back was accomplished by condenser- coupling. This did not give any limitation to our observation, since the duration of rec- tangular clamping pulses was in most cases shorter than 20 msec. and furthermore the time courseof the membrane potential, V, was always monitored by the second beam of a dual-beam oscillograph (DuMont type 322). Precautions were taken to eliminate all the causesfor a shift in the membrane potential resulting from closureof the manually operated key between the feed-back amplifier and the FIG. a. Records of membrane current associated with internal current-electrodes. The feed-back rectangular depolarization of a normal axon taken with circuit was closed immediately before the start the arrangement of fig. IA. Dotted lines in records A to E show the potential difference between the internal of a sweepof the oscillograph beams and was and external potential-electrodes controlled by the opened immediately after a photograph of the method of voltage-clamp. Record F was taken by potential and current traces was taken. A passing a short pulse of outward current through the period of at least IO seconds(often 30 sec. or membrane and later keeping the membrane current at more) was allowed before another clamping practically zero. Blanking, 0.1 msec. interval. The effec- tive length of the main current-electrode (Em), was 6 pulse was delivered. Most of the axons ex- mm, that of the lateral current-electrode 3 mm each amined showedno sign of progressivedeteriora- and the axon diameter 4oo p. Temperature, 22°C. tion during the course of experiments which sometimeslasted more than 3 hours. inward surge which has slow rising and falling Most of the experiments described in this phases. However, we rarely obtained oscillo- paper were carried out at room temperature graph records similar to those reported by the which was between 20 and 23°C. British investigators. Two most common RESULTS examples of our records are reproduced in Membrane Currents Caused by Clamping figure 2 and figure 3, illustrating the difference Pulses Near Threshold.
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