cephalopods. Flaig (1947) reported that stimulation appeared to increase the

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1 234 J. Physiol. (I95I) II4, THE TURGIDITY OF GIANT AXONS BY B. G. CRAGG From the Laboratory of the Marine Biological Association, Plymouth, and the Neuropsychiatric Research Centre, Whitchurch Hospital, Cardiff (Received 6 December 1950) This paper describes an objective method of recording changes in the turgidity of biological structures, which has been applied to large axons dissected from cephalopods. Flaig (1947) reported that stimulation appeared to increase the turgidity of turgid specimens of axons from Loligo pealii, but the method of observation was subjective: the axons were rolled between the fingers. This author also found that stimulation caused a slight tremor and dilatation in the axon, and delayed the extrusion of axoplasm from a cut end in sea water. Smitten (1946) observed a gelation in neurones of the interauricular septum of the frog-heart when these were stimulated, and reviewed similar earlier work most of which depended on micromanipulation to assess the turgidity. The method here described is analogous to that used by Buchthal, Kaiser & Knappeis (1944) to investigate the 'viscosity' of muscle fibres during contraction. It is found that although the turgidity of giant axons is not usually affected by stimulation, in some cases characteristic changes do occur. METHOD DiMsectian. Large axons from the hindmost stellar nerves were dissected from Loligo forbesi or Sepia officinalis, usually in sea water collected with the animals, though some Loligo had been in the internal sea water at the Plymouth laboratory and were dissected in this. The axons used ranged in diameter from 100 to 800/i. and varied considerably in turgidity when dissected. The animals varied in condition from very active to moribund. A few were given an anaesthetic of 1 % urethane in sea water before dissection, and in a few experiments novocaine was applied to the ends of the nerves before ligaturing and cutting. These procedures, however, did not appear to affect the turgidity of the axons. Apparatu8. A small oscillating force was applied to a balanced glass prod resting, initially without pressure, on the axon in such a way that the resistance to distortion by the axon was the major factor limiting the prod's movement. The latter was recorded, using either an ink-writer or an oscilloscope and camera, by amplifying voltage pulses from a photo-cell which became illuminated at the limit of the prod's movement. As the oscillating force causing movement was of constant amplitude a decrease in the movement of the prod indicated an increase in the resistance of the axon and vice versa. To detect a dilatation, the oscillator was turned off and the micrometer set in the most sensitive position to record any movement of the prod. The apparatus is sketched in Fig. 1.

2 TURGIDITY OF AXONS 235 Water circulated through the Perspex trough A to control the temperatures of the trough B in the range 1018' C. This was measured by the thermocouple M. The axon lay in sea water in the waxed Perspex trough B with its ends raised up in the air over the pairs of silver wire electrodes N and P. The glass prod D was moved by the magnetic force between the attached piece of soft iron E and the pole pieces G of the electromagnet F. The micrometer movement K was adjusted so that, at the limit of the downward movement of the prod, the screen L allowed light to pass from the lamp H to the photocell J. In some experiments the longitudinal extension modulus was determined by hanging the axon in ligatures between the hooks at Q and E. The whole transducer was mounted in a light-excluding box on paving stones supported by sorbo rubber blocks to reduce Fig. 1. Plan and elevation of transducer. A and B, Perspex troughs; C, axon; D, glass prod; E, soft iron; F, electromagnet with G, pole pieces; H and J, lamp and photo-cell; K micrometer; L, black screen; M, thermocouple; N and P, stimulating and pick-up electrodes; Q, hook of second micrometer. interference from external vibration. The current in the electromagnet was obtained from a phaseshift oscillator covering the range 1-50 cyc./sec. Stimulation consisted of pulses at /sec. (sometimes pulses of alternate polarity were used so that a turgidity change could not be attributed to a net current flow) and was recorded on a second channel of the ink-writer or oscilloscope. The period of stimulation used was 1-10 sec. A second oscilloscope was connected to the electrodes P to display the action potentials. The photo-cell was connected to the recording device through an amplifier which was either direct coupled, or used with a time constant of 10 sec. (for some of the dilatation experiments) or 1 sec. (for the experiments using the mechanical vibration.) Experiment. The axon in the trough B was set under the prod in the field of a dissecting microscope: An amplitude of movement of the prod of between 1 and 100l. was used. With the micro-

3 236 B. G. CRA(GG meter K in the best setting, a movement of 0.1 F. produced a deflexion several times larger than the noise level. The monitoring oscilloscope was used to ensure that the axon was not stimulated by the mechanical vibration at the amplitude and frequency used. RESULTS Lateral turgidity The great majority of records-20 stimulations of 10 Sepia axons and 96 stimulations of 21 Loligo axons-showed no alteration of turgidity. These records were obtained in the range of conditions mentioned under 'Method' (temperature, stimulation, vibration, etc.). Typical ones are reproduced in Fig. 2. Each upward pulse in these records represents a downward movement of the prod on the nerve. The stimulation is A. Displacement 1 3 s I indicated on the lower channel. Force Stimulation Isec. B. Displacement i-e Stimulatiora Fig. 2. Turgidity unchanged by stimulation. A. Sepia axon, of 100g. diameter and average turgidity, at 170 C. in sea water. Stimulation: 50 pulses/sec. of alternate polarity; amplitude of vibration 10. approx. The animal was in good condition and had been given 10 urethane in sea water. B. Loligo axon of 700. diameter and average turgidity, at 12 C. in sea water. Stimulation: 100 pulses/sec. of same polarity; amplitude of vibration 10. approx. The animal was in fairly active condition, but had not been anaesthetized. The records of this kind which are free from external vibrational interference show no change of amplitude greater than about 1% of the total movement of the prod. When a small load was applied to the prod it was found to depress the axon about 5yt./dyne with wide variations. In a few records-4 stimulations of 3 Sepia axons and 5 stimulations of 2 Loligo axons-the-amplitude of movement of the prod showed a statistically significant decrease during the stimulation of the axon, indicating an increased turgidity. (Student's t test was applied to groups of waves before and during stimulation, and when the probability of obtaining so large a change from the same population was less than 0.05 the change was counted as significant.) The time course of the effect was similar in all cases though the extent varied, and the records reproduced in Fig. 3 show the effect in its most marked form. In record A of this figure the second channel displays a voltage proportional

4 TURGIDITY OF AXONS 237 to the current through the magnet to check the constancy of the oscillator. The heights of successive waves of the first two channels were measured afterwards with a travelling microscope and it was found that while the average height of the second channel waves (current in magnet) had increased by 8 % (last 10 waves before stimulation and first 10 during stimulation), the average amplitude of the first channel waves (movement of prod) had decreased by 21 % in the same period. As the photo-cell only became illuminated during the last half of the prod's movement (in this record), this implies a reduced movement of the prod of about 10 % over 10 waves lasting 4 sec. The smallest displacement wave during stimulation occurred 2 sec. after the beginning and A. Displacement 1 3 sc I Force Stirniulatlon_ B. Displacement Is Stimulation Fig. 3. Turgidity increases with stimulation. A. Sepia axon, of 100ju. diameter and fairly high turgidity, at 160 C. in sea water. Stimulation: 50 pulses/sec. of alternate polarity; amplitude of vibration 10i. approx. The animal was in excellent condition and had been given 1 % urethane in sea water. B. Loligo axon, of 500,u. diameter and average turgidity, at 15-4 C. in sea water. Stimulation; 100 pulses/sec. of the same polarity; amplitude of vibration lo,u. approx. The animal was in moderately active condition and was not anaesthetized. was only about 70% of the pre-stimulation average. It should be noted that there is a delay of about 1 sec. before the effect becomes noticeable, and that it passes off after about 6 sec. and before the end of stimulation. This is typical of the records showing an increased turgidity. Other records showing this effect occurred with axons in sea water when high rates of stimulation were used, and a low frequency of measuring vibration of sufficient amplitude to test the properties of the axoplasm, though all these variables were within the range described above in which negative results were also obtained, and no specific factor determining the result has been found. However, an axon which increased in turgidity in one stimulation would do so again when stimulated 10 sec. later. With one single Loligo axon two successive stimulations caused a decrease of turgidity as shown in Fig. 4. This effect is of comparable magnitude to that described above, but of different time course, developing after about 0 5 sec.

5 238 B. GB. CRAGG and not passing off until the end of stimulation. No unusual conditions were noticed in these experiments showing changes of turgidity. Lateral dilatation With the time constants used, a movement of the prod of about Ol,p. in 10 sec. or l1o,u. in 1 min. could be detected. It is necessary to show that no detectable dilatation of the axon occurs on stimulation, as this would itself cause a change in the amplitude of the prod movement record in the lateral turgidity experiments. No such movement was found in 5 stimulations of 2 Sepia axons or 27 stimulations of 8 Loligo axons for 10 sec., or in 2 Loligo axons stimulated for 1 min. each. The dilatation found by Hill (1950) is too small to be detected by this method. Displacement sec.- Stimulation Fig. 4. Turgidity decreases with stimulation. Loligo axon, of 600p. diameter and less than average turgidity, at C. in sea water. Stimulation: 100 pules/sec. of same polarity; amplitude of vibration 10. approx. The animal was in bad condition and was not anaesthetized. Longitudinal contraction Three Loligo axons of about 3 cm. length were suspended in ligatures and stimulated on 8 occasions but no contraction greater than O.l,u. in 10 sec. was detected. In view of Hill's results (1950) the effect of stimulation would appear to depend on the tension on the axof, which in this case is small and uncertain. Longitudinal extension modulus This was found to have a value of about 0 3 mm./dyne in an axon of length 3 cm. and diameter 500,u., and appeared to be unchanged in the course of 5 stimulations of 2 Loligo axons using a vibrational test similar to that described above. Sectioning under sea water Some axons were cut under sea water and prompt extrusion of the axoplasm was seen as described by Flaig (1947) and by Hodgkin & Katz (1949). When the axons were stimulated there were considerable variations in the extent and time course of extrusion, which often took the form of a fairly compact cylinder of axoplasm, but stimulation seemed to delay extrusion from most of the 10 axons used, in accordance with the observations of Flaig (1947).

6 TURGIDITY OF AXONS DISCUSSION 239 It seems possible that improvement of apparatus and selection of axons would lead to a smaller proportion of records showing unchanged turgidity. In three of the records classed as negative indications of an increased turgidity appeared during stimulation but were not statistically significant, owing to an increased variance caused by extraneous vibration. Unfavourable conditions for the axon or excessive amplitude or frequency of the measuring vibration must lead to a negative result, since any damage would interfere with conduction in response to stimulation. Flaig (1947) reported that an increase of turgidity occurred in turgid axons, but did not say what proportion of axons showed this effect. Since both an increase and a decrease of turgidity can occur, there must be an undetermined biological variable as well. The internal fluid of cephalopods is somewhat different from sea water (Robertson, 1949) though Flaig (1947) found the increased turgidity in sea water. The osmotic pressure of the sea water bathing the nerve was not deliberately varied. The time course of the effect coincides roughly with the preliminary period of shrinkage found by Hill (1950), which is however too small to be detected with this apparatus and so could not be the direct cause of the change in the deflexion record of the prod. A possible mechanism for a sol-gel transformation is suggested by the observation of Hodgkin & Katz (1949) that the calcium ion has a specific effect in dispersing extruded axoplasm. The same ion is believed to have a fairly specific acceleratory effect on the action of adenosinetriphosphatase (e.g. DuBois & Potter, 1943). A movement of the calcium ion during conduction or a change in the ATP/ATPase balance might produce the effect observed. SUMMARY 1. A transducer designed to record changes in the turgidity of biological structures is described. 2. It has been found that a small proportion of giant axons of Sepia and Loligo show characteristic changes of turgidity when stimulated. 3. No dilatation or longitudinal contraction was detected in these axons on stimulation. The author is indebted to Dr D. Richter for suggesting the problem and for his continued interest, to Mr J. A. Reynolds for technical assistance, to the Director and Staff of the Marine Biological Association Laboratory, Plymouth, for their generous hospitality, and to the Rockefeller Foundation for a personal grant. REFERENCES Buchthal, F., Kaiser, E. & Knappeis, G. G. (1944). Acta phy8il. Scand. 8, 16. DuBois, K. P. & Potter, V. R. (1943). J. biol. Chem. 150, 185. Flaig, J. V. (1947). J. Neurophyil. 10, 211. Hill, D. K. (1950). J. Phyeio. 111, 304. Hodgkin, A. L. & Katz, B. (1949). J. exp. Bil. 26, 292. Robertson, J. D. (1949). J. exp. Bil. 26, 182. Smitten, N. A. (1946). Amer. Rev. Svie Med. 3, 414.