Abstract
Neuron differentiation includes formation and outgrowth of neurites that differentiate into axons or dendrites. Directed neurite outgrowth is controlled by growth cones that protrude and retract actin-rich structures to sense environmental cues. These cues control local actin filament dynamics, steer growth cones towards attractants and away from repellents and navigate neurites through the developing brain. Rodent hippocampal neurons are widely used to study the mechanisms underlying neuron differentiation. Genetic manipulation of isolated neurons including gene inactivation or reporter gene expression can be achieved by classical transfections methods, but these methods are restricted to neurons cultured for several days, after neurite formation or outgrowth. Instead, electroporation allows gene manipulation prior to seeding. However, reporter gene expression usually takes up to 24 hours and time course of gene inactivation depends on the half live of the targeted mRNA and gene product. Hence, these methods do not allow to study early aspects of neuron differentiation. In the present study, we provide a detailed protocol in which we combined electroporation-based gene manipulation of mouse hippocampal neurons prior to initial seeding with a replating step after two days in vitro that resets neurons into an undifferentiated stage. By categorizing neurons according to their differentiation stage, thorough morphometric analyses, live imaging of actin dynamics in growth cones as well as guidance cue-mediated growth cone morphological changes, we demonstrate that differentiation and function of replated neurons did not differ from non-replated neurons. In summary, we provide a protocol that allows to thoroughly characterize differentiation of mouse primary hippocampal neurons.
Significance statement
Unraveling the molecular mechanisms that control neuron differentiation requires reporter gene expression or gene inactivation. In mouse primary hippocampal neurons, a widely used cellular system to study neuron differentiation, classical transfection methods are restricted to later stages of differentiation. Instead, electroporation allows genetic manipulation prior to seeding. However, time course of reporter gene expression or gene inactivation frequently hinders a full characterization of neuron differentiation, specifically of early stages. To circumvent this limitation, we combined electroporation-based genetic manipulation prior to initial seeding with a replating step after two days in vitro, which reset neurons into an undifferentiated stage. We show that replated neurons differentiated similar to non-replated neurons. We provide a detailed protocol that allows to comprehensively characterize the molecular mechanisms underlying neuron differentiation.
Footnotes
Authors report no conflict of interest.
This work was supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG) to MBR (RU 1232/7-1). FS was funded by the DFG Research Training Group ‘Membrane Plasticity in Tissue Development and Remodeling’ (GRK 2213).
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
Jump to comment: