Selective axonal transport in a single cholinergic axon of Aplysia--role of colchicine-resistant microtubules

Abstract

Substance-specific selective axonal transport was examined in a single axon by injecting [3H]leucine and [14C]acetylcholine simultaneously into the cell body of a giant cholinergic neuron (R2) in the abdominal ganglion of Aplysia kurodai. The ganglion and attached nerves were cultured for several hours after the injection and the migration of radioactive substances along the axons of the injected neuron was examined. The substances examined were 3H labeled membrane proteins and soluble proteins synthesized in the cell body, 14C labeled bound acetylcholine formed in the cell, injected [3H]leucine and soluble [14C]acetylcholine. Membrane proteins and bound acetylcholine (plus a part of soluble acetylcholine) moved along the axon somatofugally at maximum velocities of 2.4 and 1.7 mm/h, respectively, at 25 degrees C. Soluble proteins, free leucine and most of the soluble acetylcholine did not move by fast axonal transport but diffused inside the axon of the neuron R2 at rates predicted from their expected diffusion constants in the axoplasm [Koike H. and Nagata Y. (1979) J. Physiol. 295, 397-417]. The diffusion kinetics of these substances were analysed and used for determination of true axon length, and to separate axonal transport components from diffusing components. An antimitotic drug, colchicine, selectively suppressed the axonal transport of membrane proteins but not of acetylcholine at 1-5 mM concentration, though it finally blocked the axonal transport of acetylcholine at 20 mM. When 1-5 mM colchicine was separately perfused only to the distal axon of the neuron R2, the migration of membrane proteins was stopped just proximal to the colchicine perfusion zone but acetylcholine migration was not disturbed by the drug. The moving component of acetylcholine was recovered by sucrose density centrifugation from a compartment previously reported as that of vesicular acetylcholine. As a possible mechanism of this selective axonal transport, it is proposed that there are two groups of microtubules: a colchicine-sensitive group of microtubules which may transport membrane proteins, and a colchicine-resistant group which may preferentially transport the transmitter substance acetylcholine at a slower rate.