Methods for perfusing the giant axon of Loligo pealii.

It was possible to perfuse the interior of the giant axon of Loligo pealii or forbesi, [C. S. Spyropoulos, I. Tasaki and T. Teorell, National Institutes of Health Annual Report, NINDB, 1959; Baker, P. F., A. L. Hodgkin and T. D. Shaw, Preprint of Proc. Physiol. SOC. (1961); also cited in preprint, Baker, P. F. and T. D. Shaw (1961), Plymouth Marine Biological Laboratory report for 1960-1961 (in press)] with an artificial solution and maintain the resting potential and the ability of the axon to produce normal action potentials. The technique employed (Diagram A) in the first series of experiments was as follows: Using an axon of Loligo pealii about 25-35 mm in length, a column of axoplasm was removed by introducing a capillary (300 p in diameter). Subsequently each end of the axon was cannulated using glass pipettes (approx. 200-300 ,/A in diameter) and fine nylon thread. The perfusing fluid was introduced through one cannula and drained through the other. The rate of perfusion varied; in most experiments it was of the order of 0.01 ml/sec. The perfusion rate was usually but not always controlled with the Phipps and Bird Syringe-Driver. This rapid rate effected a rapid equilibration of the diffusible components of the intracellular space. A pair of silver wires was introduced through the cannula for stimulation and recording (under space clamp conditions). This electrode assembly facilitated drainage by guiding the perfusate from the ejecting to the withdrawing cannula. In another series of experiments, this was accomplished by means of a calomel micropipette which was also used as a potential recording electrode. In later experiments, simpler methods were used and longer axons (35-50 mm). In the method illustrated by diagram B a long glass capillary ( 3 5 0 , ~ in diameter) with a long slit (20 mm long and 100 p in width) on one side was employed.