Melatonin: neuritogenesis and neuroprotective effects in crustacean x-organ cells

Abstract

Melatonin has both neuritogenic and neuroprotective effects in mammalian cell lines such as neuroblastoma cells. The mechanisms of action include receptor-coupled processes, direct binding and modulation of calmodulin and protein kinase C, and direct scavenging of free radicals. While melatonin is produced in invertebrates and has influences on their physiology and behavior, little is known about its mechanisms of action. We studied the influence of melatonin on neuritogenesis in well-differentiated, extensively-arborized crustacean x-organ neurosecretory neurons. Melatonin significantly increased neurite area in the first 24h of culture. The more physiological concentrations, 1 nM and 1 pM, increased area at 48 h also, whereas the pharmacological 1 μM concentration appeared to have desensitizing effects by this time. Luzindole, a vertebrate melatonin receptor antagonist, had surprising and significant agonist-like effects in these invertebrate cells. Melatonin receptors have not yet been studied in invertebrates. However, the presence of membrane-bound receptors in this population of crustacean neurons is indicated by this study. Melatonin also has significant neuroprotective effects, reversing the inhibition of neuritogenesis by 200 and 500 μM hydrogen peroxide. Because this is at least in part a direct action not requiring a receptor, melatonin's protection from oxidative stress is not surprisingly phylogenetically-conserved.

Figure 1

Cultured crustacean (Uca pugilator) x-organ cell at 400× oil immersion with phase contrast (micron bar applies to all 3 images). Images taken A) within 1 h of culture, B) within 3 h of culture, and C) at 24 h in culture.

Figure 2

Neurite area (microns²) in control versus 1 μM melatonin-treated crustacean x-organ cells measured every 24 h over 96 h. Bars represent means ± SEM of 40 cells.

Figure 3

Dose-response influence of melatonin on neurite area in crustacean x-organ cells measured at 24 and 48 h. Bars represent means ± SEM of 40 cells.

Figure 4

Influence of melatonin or melatonin + luzindole on neurite area in crustacean x-organ cells measured at 24 and 48 h. Bars represent means ± SEM of 40 cells.

Figure 5

Influence of peroxide or peroxide and 1 μM melatonin on neurite area in crustacean x-organ cells measured at 60 h. Bars represent means ± SEM of 40 cells.