ASSAY OF VESICLE MOTILITY IN SQUID AXOPLASM

This chapter describes assay of vesicle motility in squid axoplasm. The giant axon of the Atlantic squid Loligo pealeii is used routinely for preparation of isolated axoplasm (“extruded” axoplasm). Medium to large squid with translucent mantles (0.3-0.5 m long) are selected and decapitated. The mantle is split along the dorsal ridge and pinned to a dissection table with running seawater. The biomechanics of axoplasm requires that axons should be greater than 100 pm in diameter for routine mechanical extrusion. This represents a minimum diameter from which limited amounts of material can be obtained, so larger diameters are preferred. Choice of buffers for introduction of experimental agents is crucial, particularly for maintaining structural integrity of axoplasm. The axoplasm is highly sensitive to ionic strength and ionic composition. The buffers are distinguished by the use of organic anions (amino acids and related compounds), low levels of chloride and other halides, high osmotic strength, low levels of free Ca2+, and the presence of suitable reducing agents. An inverted Zeiss Axiamat with high-numerical-aperture differential interference contrast (DIC) optics, including a 100X planapochromatic objective (1.3NA) and matched condenser (1.4NA) with zoom set at 2.5X is used. © 1993 Elsevier Science Publishers, B.V.