DISTRIBUTION OF NEUROTRANSMITTER SECRETION IN GROWING AXONS I. ANTONOV, S. CHANG, S. ZAKHARENKO and S. V. POPOV* Department of Physiology and Biophysics M/C 901, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612, U.S.A. Abstract —Neurotransmitter secretion from the nerve terminal is mediated by the fusion of synaptic vesicles with the plasma membrane. It is generally believed that neurotransmitter release in mature synapses is localized to the presynaptic nerve terminals. To probe the topology of neurotransmitter secretion along developing axons in culture, we recorded membrane currents from myocytes manipulated into contact with axons. At the early stages of growth, exocytotic events were detected along the axon as well as at the growth cone. At the later stages of growth, neurotransmitter secretion adopted the form of a smooth proximodistal gradient, with the highest level of activity at the growth cone region. Our results reveal the existence of a previously undetected early stage of axonal growth and suggest developmental regulation in the pattern of neurotransmitter secretion along the growing nerve processes.  1999 IBRO. Published by Elsevier Science Ltd. Key words: axonal differentiation, synaptic vesicles, neurotransmitter secretion, axonal growth. Neurons secrete neurotransmitters via a highly regu- lated process involving the fusion of synaptic vesi- cles with the plasma membrane. 4,33 Following exocytosis, the molecular components of synaptic vesicles are retrieved be endocytosis, and synaptic vesicles are recycled locally within the nerve term- inal. 17 This exo-endocytotic recycling pathway is orchestrated by a number of synaptic vesicle-speci- fic proteins. 13 In mature synapses, synaptic vesicles are clustered at the sites specialized for neurotrans- mitter secretion (“active zones”). The clustering of synaptic vesicles at the active zones is accompanied by accumulation of specific proteins at both the presynaptic nerve terminal and the postsynaptic membrane. 10,57 Synaptic vesicle exocytosis is tightly regulated by Ca 2+ entry during excitation of the presynaptic cell, 42 whereas fusion of synaptic vesicles in the resting state is inhibited. However, synaptic vesicle exocytosis and neurotransmitter secretion can also occur spontaneously. 36,66 The spontaneous neuro- transmitter release may be important in the develop- ment of initial synaptic contacts into mature and fully functional synapses. 3,61 It is generally believed that, in developing nerve processes free of contact with postsynaptic targets, spontaneous neurotransmitter secretion is localized primarily to the growth cone region. 36 In the major- ity of cases, insertion of newly synthesized plasma membrane and cytoskeletal components into the growing nerve processes also occurs primarily at the growth cone area. 16,18,63 Therefore, in addition to its function in neuronal pathfinding and naviga- tion, the growth cone serves as the major site for the assembly of new axonal structure, exo-endocytotic activity and neurotransmitter secretion. In this model of axonal development, the function of the axonal shaft is limited primarily to the delivery of cell body-synthesized material to the growth cone region. However, experimental evidence suggests that some newly synthesized membrane components may be inserted along the axon. 26,34,51 Moreover, constitutive endocytotic recycling of the plasma membrane along the axon has been reported in developing hippocampal 27,44 and Xenopus spinal cord 19 neurons. EXPERIMENTAL PROCEDURES Cell culture Cultured Xenopus (purchased from Xenopus Express, Homosassa, FL) spinal cord neurons were prepared accord- ing to previously reported methods. 1,59 The cells were plated on acid-washed cover glasses and grown in culture medium consisting of (v/v): 50% Leibovitz L-15 medium (Gibco), 49% Ringer solution (115 mM NaCl, 2 mM CaCl 2 , 2,5 mM KCl, 10 mM HEPES, pH 7.6) and 1% fetal bovine serum (Gibco). The cultures were used for experiments after one to four days incubation at 20°C. Xenopus myocytes were plated separately on Petri dishes, grown in a culture medium supplemented with 3% fetal bovine serum, and used for experiments after 24–48 h incubation at 20°C. In some experiments, the neurotrophic factor neurotrophin-3 (NT-3; Regeneron) was added to the culture medium during cell culture preparation at the final concentration of 50 ng/ml. 975 Neuroscience Vol. 90, No. 3, pp. 975–984, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(98)00497-7 Pergamon *To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; HEPES, N-2-hydroxyethyl- piperazine-N 0 -2-ethanesulfonic acid; MEPC, miniature endplate current; NT-3, neurotrophin-3; PBS, phosphate- buffered saline.