Fluorescence-Activated Cell Sorting in the Early Identification of Brain Serotonin Neurons In Vitro
All mammalian brains contain a dense meshwork of axons that release serotonin, a major neurotransmitter involved in numerous neural processes. In contrast to many other axons that connect specific brain regions, these serotonergic axons move through most adult neural tissue in random walk-like trajectories. Additionally, these axons can regenerate after being severed and produce new trajectories. In virtually all brain regions, serotonergic axons accumulate at very high densities and are often referred to as fibers. Learning how these fibers develop in space and time is crucial to our fundamental understanding of the self-organization of the serotonergic system and will provide insights into neural axon development and regeneration, all of which require the examination of individual axons in controlled environments. Here, we present a method for producing neuronal cultures with serotonergic cells using a protocol developed by our lab and observations we have seen regarding serotonergic axon dynamics as well as a method for the identification and sorting of live serotonergic neurons in vitro for single-axon live-imaging analysis. This method utilizes fluorescence-activated cell sorting (FACS) on primary cells of transgenic mice constitutively expressing an EYFP fluorophore under the promoter for Tph2, an enzyme unique to serotonergic neurons. This will enable serotonergic cells to be identified and isolated in live, mixed cell cultures. Additionally, it will facilitate in-depth, real-time analyses of axon growth, branching, and interactions with each other and their environment, eventually supporting advanced modeling of the self-organization of serotonergic fibers. Furthermore, this protocol may assist the development of methods for isolating other neuronal populations, such as dopaminergic neurons, for various in vitro manipulations.