VOL. 4 (1950) PERMEABILITY AND NERVE FUNCTION, II 97 



( AEC Catalog Item # S-5)*. Aliquots of the Ca'*^ solution were pipetted into the appropriate volumes 

 of Ca-free artificial sea water to give the correct Ca concentration (0.012 M). 



Preparation of biological material. The last stellar nerves (containing a giant axon) were excised 

 from specimens of Loligo peallii, after first tying both ends of the portion desired. Nerve sections were 

 then kept in fresh natural sea water for i^ to 2 hours before use. The results of Steinbach and 

 Spiegelman^ had indicated that during the first 2 hours after excision of stellar nerves, the chemically- 

 determined values for Na vary considerably and it is only after this time has elapsed that the axoplasm 

 comes into equiUbrium with its outer environment. The value for Na reaches its maximum value of 

 10 meq. per cent within this period. 



The nerves were then exposed to artificial sea water prepared according to Pantin^ in which all 

 or part of a given ion species had been replaced in isomolar concentration with radioactive material. 

 The sea water contained 0.52 M NaCI, 0.013 ^ KCl, 0.012 M CaClj, and 0.024 M MgClj. The pn was 

 adjusted to 7.7-8.0 by the addition of a small volume of bicarbonate or NaOH, the latter in those 

 cases where the adjustment required considerable amounts of alkali. After the desired period of 

 exposure, the nerves were removed and rinsed several times in a few changes of fresh natural sea 

 water. After blotting of filter paper, the proximal end was cut off. The axoplasm (nerve cytoplasm) 

 was extruded by the application of gentle but gradually increasing pressure with a pair of forceps 

 in the direction of the cut end. The extruded axoplasm was collected on a tared aluminum planchet 

 (130-150 mg each and about one inch in diameter) and weighed quickly with a torsion balance. One 

 ml of distilled water was then added to each planchet to insure even distribution of the radioactive 

 substance over the entire area of the planchet. 



Determination of radioactivity. Samples were then evaporated to dryness under infra-red lamps 

 and the radioactivity measured with a Tracerlab 64 Scaler**. Measured radioactivities were recal- 

 culated to zero time from the decay curve of the individual ion under investigation in order to correct 

 for the decomposition which occurred during the measurement of sample activities. This correction 

 becomes appreciably large, when using Na^* and K^^ which have half-lives of 14.8 and 12.4 hours 

 respectivelv. Comparison of the activities of the samples with standards prepared from aliquots of 

 the radioactive artificial sea water (and analysed at the same level in the counting chamber) enabled 

 the calculation of the ion content of the axoplasm samples. 



The method of preparation of the standards for Tables I, II, and III are given at the top of 

 each of these tables. The Na standards for the data given in Tables IV, VI, IX, and X were prepared 

 by diluting the sea water (containing 0.39 M Na^^Cl -f 0.13 M Na^^Cl) 250 times with distilled water. 

 0.5 ml aliquots were then evaporated to dryness in duplicate on aluminum planchets (1.04 micromoles 

 Na/0.5 ml). For Tables VII and VIII, Na standards were prepared by this same method. However, 

 since a reduction in the total NaCl concentration had been made in order to maintain the isotonicity 

 in the presence of added inhibitors of cholinesterase, the 0.5 ml aliquots contained only i.oo micro- 

 mole Na/0.5 ml. The K standards for the data given in Tables V and VII were prepared by diluting 

 the sea water (containing 0.013 M K^^Cl) 100 times and then evaporating i.o ml aliquots in duphcate 

 as above (0.13 micromole K/i.o ml). Radioactivities recorded in Tables IV through X have all been 

 corrected to zero time. 



Electrical. Nerves were tested for normality of conduction both before and after exposure to 

 radioisotope containing sea water. The nerves were stimulated through a pair of silver wire electrodes 

 by condenser discharge shocks of a time constant less than 0.2 milliseconds. Action potentials were 

 led off by means of a second pair of silver wire electrodes to a condenser coupled amplifier of a modified 

 Toeney differential type circuit and then recorded on a DuMont No. 279 Dual Beam Oscilloscope. 

 Only those nerves were used which still exhibited normal conduction at the end of the experiment. 

 Studies of the rates of ion exchange during electrical activity of the nerves were carried out in 

 the following manner: Nerve chambers were used of narrow bore polystyrene tubing (2 mm i.d.) into 

 which were sealed, at right angles to the length and at 5 mm intervals, 0.0156" diameter Pt wire as 

 described previously (II). Nerves were mounted in the chamber by threading a long thin wire through 

 the polystyrene tube (one end of the wire having previously been tied to the thread attached to the 

 nerve). The nerve was then carefully drawn into the tube. By slipping a piece of narrow bore rubber 

 tubing over that end of the polystyrene tube from which the thread issued, the thread — and thereby 

 the nerve — was fixed in position. The rubber tubing was then connected to a perfusion bottle filled 

 with sea water containing the radioactive ions. Perfusion of the nerve preparation was carried out by 

 means of gravity. The diameter of the plastic tubing chosen was such that only a very thin layer of 

 sea water remained between the nerve and the wall of the polystyrene tube. Thus, the difficulty of 

 excessive shunting by the sea water was largely eliminated and stimulation of, and recording from, 

 the nerve was possible throughout the period of exposure to the isotope containing sea water. 



* We are indebted to Dr G. Failla and Dr P. Aebersold for making the high specific activity 

 Ca^^ (carrier free) available to us. 



** We are indebted to Dr G. Failla and the Marine Biological Laboratory, Woods Hole, 

 Mass., for making the Scaler available to us. 



References p. 114. 

 7 



