Development of image analysis system for quantitative evaluation of neurite degeneration

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We constructed an experimental system for quantitative evaluation of neuronal morphology by on-line image analysis of central neurons in dissociated or organotypic cultures, thereby investigated neuronal degeneration and neuroprotection. (1) We prepared retinal tissue cultures from neonatal rats that received DiI injection into the superior colliculus, and observed morphology of retinal ganglion cells (RGCs). RGCs were identified as bright fluorescent spots aligned in one focal plane within the retina. The number and size of fluorescent spots gradually decreased during the course of cultivation. Cycloheximide or actinomycin D markedly inhibited the decrease in the number of viable RGCs. DEVD, a selective caspase-3 inhibitor, also significantly inhibited the decrease in RGC viability, and DNA fragmentation was found to be restricted in the ganglion cell layer. These results indicate that the gradual decrease in RGC viability results from induction of apoptosis. Moreover, nitric oxide de … More rived from neuronal nitric oxide synthase (NOS) was shown to play an important role in induction of apoptosis in RGCs, because an NOS inhibitor L-NAME as well as a relatively selective neuronal NOS inhibitor 7-nitroindazole markedly inhibited the decrease in RGC viability. (2) We found that cultivation of midbrain slice cultures with medium containing high concentrations of Mg^<2+> or that containing NMDA receptor antagonists resulted in a drastic decrease in the number of dopaminergic neurons. There were also prominent atrophic changes in neurites of dopaminergic neurons remaining after these treatments. Co-application of an adenylyl cyclase activator or a cyclic AMP analog abolished the effects of chronic Mg^<2+> and NMDA antagonists. These results suggest that moderate activation of NMDA receptors associated with spontaneous neuronal activity makes significant contribution to early development and maintenance of midbrain dopaminergic neurons, where cyclic AMP-related intracellular signaling plays a key role. Less