Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes.

ABSTRACT

During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.