Analysis of P2X7-Induced Neuronal Branching

Abstract

P2X7 receptors regulate different aspects of neuronal development, including neurogenesis, dendritic outgrowth, and axonal elongation. Primary neuronal culture is a widely used model system in neuroscience as it enables to study molecular and cellular events caused by the activation of different ion channels, receptors, and transporters under controlled conditions. Primary neuronal cultures derived from normal and genetically modified mouse models can be used with a wide array of molecular biological, anatomical, and functional techniques such as RNA sequencing, western blots, immunostaining, Ca2+ imaging, and electrophysiology. In addition, they can also be genetically manipulated relatively easily. Moreover, cells can survive for multiple weeks if they are properly maintained and thus the development and maturation of individual neurons and their morphological properties can be studied under different conditions. Here, we present a protocol for the isolation and culturing of primary hippocampal cells from embryonic mouse hippocampal tissue (embryonic days 17.5–18.5). The neurons are plated in poly-l-lysine/laminin coated coverslips, where astroglia proliferation is controlled for the proper study of individual primary neurons. To investigate the development of dendrites and axons, as a good correlate of neuron morphology, we present a transfection protocol, which allows us to fill the whole neuron with a fluorescent protein. Subsequently, we perform tracing and analysis of dendritic branching by Sholl analysis using Neurolucida tracing Software (MBF Bioscience).