Tracking Mitochondrial Density and Positioning along a Growing Neuronal Process in Individual C. elegans Neuron Using a Long-Term Growth and Imaging Microfluidic Device.

Abstract

The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and subcellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show interanimal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in subcellular events such as organelle positions, we developed a microfluidic device to feed and immobilize Caenorhabditis elegans for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process in vivo Subcellular imaging of an individual neuron in multiple animals, over 36 h in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles (SVs) at synapses. Long-term imaging of individual C. elegans touch receptor neurons (TRNs) shows that the addition of new mitochondria takes place along the entire neuronal process length at a rate of ∼0.6 mitochondria/h. The threshold for the addition of a new mitochondrion occurs when the average separation between the two preexisting mitochondria exceeds 24 μm. Our assay provides a new opportunity to move beyond simple observations obtained from in vitro assays to allow the discovery of genes that regulate positioning of mitochondria in neurons.