RELEASE OF NEURITE OUTGROWTH PROMOTING FACTORS BY HELISOMA CENTRAL GANGLIA DEPENDS ON NEURAL ACTIVITY

Identified buccal neurons B5 and B19 from the molluse, Helisoma trivolvis, were plated into cell culture in order to assay for neurite outgrowth promoting factors released from central ring ganglia. The release and attachment of neurite promoting factors to the substratum of poly-lysine coated dishes could be inhibited by blocking spontaneous bioelectric activity in central ring ganglia used to condition the medium and dishes. Bioelectric activity within neurons in central ring ganglia was assayed by intracellular recording and found to be inhibited by exposure to the sodium channel blocker, tetrodotxin (TTX; 2 x 10(-5)M), or CoCl2 (10 mM). Neither of these agents appeared to be toxic over a three day period since activity within neurons in central ring ganglia was restored following superfusion with saline. To examine the effect of blocking neural activity on the ability of central ring ganglia to release neurite outgrowth promoting factors, we compared the percentage of neurons that extended processes under 5 different conditions: (1) dishes containing conditioned medium and substrate attached growth factors (Super SAM); (2) dishes with substrate attached growth factors only and defined medium (SAM); (3) dishes containing substrate attached growth factors prepared in the presence of TTX; or (4) CoCl2; and (5) dishes containing unconditioned defined medium. The percentage of neurons extending processes under the 5 conditions were: (1) 71% (n = 32); (2) 51% (n = 33); (3) 14% (n = 37); (4) 15% (n = 47); (5) 0% (n = 40), respectively. The addition of TTX to dishes containing neurons in conditioned medium (super SAM) did not inhibit neurite outgrowth, but even brief applications of CoCl2 to neurons under the same conditions inhibited neurite outgrowth in a non-reversible manner. Analysis of concentrated conditioned medium by sodium dodecyl-sulphate polyacrylamide gel electrophoresis and silver staining revealed that a number of proteins having apparent molecular weights of 44, 29, 26, 23, 17, 15, and 14.5 kDa were significantly reduced in quantity in conditioned medium produced in the presence of TTX or CoCl2. Taken together, these observations indicate that neurite outgrowth promoting factors were released from central ring ganglia in a manner dependent on neural activity.