458 ANNALS NEW YORK ACADEMY OF SCIENCES 



changes in the chemical constitution of the cell or axon. Thus, the in- 

 creased irritability that is induced by surrounding a nerve with a cal- 

 cium-deficient fluid follows a decrease of calcium within the nerve. 

 In order that the effects of such changes may be investigated, it is con- 

 venient to have available a solution which will maintain nerve in a 

 stable functional state for long periods of time, and to which the effects 

 of other environmental solutions may be referred. It is customary to 

 choose for reference a solution having a salt content and pH approxi- 

 mating that of the animal's body fluids. By direct test, it has been 

 found that a frog nerve can be kept in a solution at pH 7.2 (phosphate 

 buffer) containing sodium chloride (116 mM), potassium chloride (2.0 

 mM), and calcium chloride (1.8 mM) for many hours, with no sig- 

 nificant change in excitability or in rate of aerobic oxidation. Squid 

 nerve, which we have also employed, maintains a similar stable func- 

 tional state in sea water (Woods Hole) at pH 8.0 or in a solution con- 

 taining sodium chloride (405 mM), potassium chloride (11 mM), and 

 calcium chloride (70 mM). Modifications of these solutions have been 

 used as the experimental means of chemical activation. 



The calcium ion concentration of the environmental fluid is espe- 

 cially important in determining the excitability of nerve. This familiar 

 phenomenon (cf., e.g., MisskeM can be studied quantitatively and under 

 quickly reversible conditions in squid giant axons or in bundles of frog 

 axons from which the perineurium has been removed. Under those cir- 

 cumstances, diffusion equilibrium between the axons and the surround- 

 ing fluid is attained relatively quickly. In figure 1, the threshold 

 strength of direct current necessary to initiate an impulse, which is the 

 rheobase, is plotted as a function of the concentration of calcium 

 chloride in the fluid bathing a giant axon of the squid. A similar re- 

 lation is obtained for the a fibers in a frog sciatic nerve (figure 2). 



The increased excitability produced by the action of solutions having 

 a low concentration of calcium chloride is presumably due to the dif- 

 fusion of Ca^+ from the cell structure. Indeed, Tipton' has shown by 

 chemical analysis that as much as 40 per cent of the total calcium of 

 frog nerve is in diffusion equilibrium with the surrounding fluid, some 

 of this diffusible calcium being in the cell phase. His evidence for 

 intracellular precipitation of added calcium is a further indication that 

 changes in the calcium chloride content of the bathing fluids lead to 

 changes in the cellular content. The spatial distribution of these 

 changes in cellular calcium are unknown. 



When frog nerve is equilibrated with solutions containing from 1.0 

 mM to 0.3 mM calcium chloride, or when squid nerve is equilibrated 



