Sprouting by intact Helisoma neurons: role for glutamate

We examined the possible relationship between sprouting by intact neurons and the elevation of blood acidic amino acids that is reported to occur in Helisoma in response to osmotic stress. In the present study, buccal ganglion neurons 5 were examined following exposure of animals to conditions that induce estivation, a behavioral state exhibited by these freshwater snails in nature. Surprisingly, sprouting activity occurred in some neurons (31%) in normal animals, this being transiently elevated (to 71%) after 4 days of estivation. Blood acidic amino acids (i.e., glutamate and aspartate) were also elevated under this condition, their levels reaching a plateau on day 4. These observations give rise to two hypotheses: 1) the acidic amino acids can induce sprouting, or 2) these agents can release trophic factors, which, in turn, induce sprouting. The first hypothesis was tested by exposing semi-intact preparations in vitro to a range of concentrations of the acidic amino acids; no promotion of sprouting was observed by either glutamate or aspartate. The second hypothesis was supported, however, by the following observations. First, blood from estivated animals was tested for trophic activity. Such blood (but not that from control animals) could promote sprouting by intact neurons in vitro, a response that depended on factor(s) whose filtration behavior on Amicon membranes suggests an approximate molecular weight of 30-100 kD. Second, since medium conditioned by Helisoma ganglia is known to contain a protein(s) that is necessary for sprouting by isolated neurons, we tested the ability of such medium to promote sprouting by intact neurons. Conditioned medium promoted sprouting by intact neurons in vitro in a manner quantitatively and qualitatively similar to that observed in vivo during estivation. Additionally, sprouting by intact neurons in conditioned medium was also attributable to factor(s) that acted as 30-100 kD molecules on Amicon membranes. To test the ability of the acidic amino acids to release or activate these factors, medium was conditioned for short periods in the presence of glutamate or aspartate; glutamate, but not aspartate, was found to enhance conditioned medium. We conclude that glutamate can promote sprouting by intact neurons, but that its action is indirect and is mediated by macromolecular trophic factors.