Neurite Branch Development of an Identified Serotonergic Neuron from Embryonic Helisoma: Evidence for Autoregulation by Serotonin

Abstract

Previous studies have shown that in select neurons, neurite outgrowth can be regulated by the same neurotransmitter that is synthesized and released by those neurons. However, it is not known whether such an autoregulatory mechanism is utilized during the normal course of nervous system development in either invertebrates or vertebrates. In the present study, we tested this hypothesis on the first pair of identified serotonergic neurons to be expressed in embryos of the pulmonate gastropod, Helisoma trivolvis. Embryonic neurons C1 (ENC1) elaborate a stereotyped pattern of neurite outgrowth prior to the differentiation of subsequent serotonergic neurons. Embryos were treated with either p-chlorophenylalanine (pCPA) or 5-hydroxytryptophan (5-HTP) to lower or raise embryonic serotonin content, respectively. High-performance liquid chromatography with electrochemical detection was used to measure the effects of these treatments on serotonin content, and serotonin immunohistochemistry was carried out to quantify the extent of neurite outgrowth of ENC1. Embryonic serotonin content was significantly reduced at both 24 and 48 hr after treatment with 0.02% pCPA, whereas dopamine levels were unchanged. Although the proximal neurite outgrowth of ENC1 appeared unaffected by the pCPA treatment at both of these time points, the distal outgrowth in the target cell region appeared more profuse. This effect on outgrowth was quantified by counting the number of neurite branch points, which was significantly increased both 24 and 48 hr after pCPA treatment. In contrast, 5-HTP treatment resulted in an increase in embryonic serotonin content and a significant decrease in the number of ENC1 branch points. Treatment with dopamine had no effect on the pattern of ENC1 neurite outgrowth. Together, these results support the hypothesis that a neuron my utilize its own transmitter in an autoregulatory fashion to regulate neurite formation during embryonic development.