Abstract
Understanding the mechanisms guiding interneuron development is a central aspect of the current research on cortical/hippocampal interneurons, which is highly relevant to brain function and pathology. In this methodological study we have addressed the setup of protocols for the reproducible culture of dissociated cells from murine medial ganglionic eminences (MGEs), to provide a culture system for the analysis of interneurons in vitro. This study includes the detailed protocols for the preparation of the dissociated cells, and for their culture on optimal substrates for cell migration or differentiation. These cultures enriched in interneurons may allow the investigation of the migratory behavior of interneuron precursors and their differentiation in vitro, up to the formation of morphologically identifiable GABAergic synapses. Live imaging of MGE–derived cells plated on proper substrates shows that they are useful to study the migratory behavior of the precursors, as well as the behavior of growth cones during the development of neurites. Most MGE-derived precursors develop into polarized GABAergic interneurons as determined by axonal, dendritic, and GABAergic markers. We present also a comparison of cells from WT and mutant mice as a proof of principle for the use of these cultures for the analysis of the migration and differentiation of GABAergic cells with different genetic backgrounds. The culture enriched in interneurons described here represents a useful experimental system to examine in a relatively easy and fast way the morpho-functional properties of these cells under physiological or pathological conditions, providing a powerful tool to complement the studies in vivo.
Highlights
The γ-aminobutyric acid (GABA)-ergic interneurons are modulators of brain function essential to keep the balance between excitation and inhibition (Gelman and Marín, 2010)
We have setup and described in details the conditions for proper transfection of the cultured medial ganglionic eminences (MGEs)-derived cells. This is an important aspect of the study, since we found that the application of previously reported transfection procedures were inappropriate for the transfection of MGE-derived cells, due to either insufficient transfection efficiency, or to the heavy loss of cells following the transfection procedures that are in use with other types of cells and neurons
We first describe the protocol for the isolation of MGEs from E14.5 mouse embryos; present the detailed protocols required to set up the cultures on coverslips for morphological and functional analysis, or on tissue plastic for biochemical analysis; and we describe the conditions tested to optimize the culture of MGE-derived dissociated cells by using different extracellular substrates
Summary
The γ-aminobutyric acid (GABA)-ergic interneurons are modulators of brain function essential to keep the balance between excitation and inhibition (Gelman and Marín, 2010). Switching from tangential to radial migration, they reach and populate different layers within the cortex and the hippocampus (Guo and Anton, 2014) In their migration interneuron precursors are guided to reach their final destination by extracellular cues including motogenic factors and neurotrophins (Hernández-Miranda et al, 2010). Once they have reached their final destination, they make synaptic connections with excitatory and other inhibitory neurons. The differentiation and maturation of interneurons involve sequential processes including migration, the outgrowth, and branching of axons and dendrites, and the formation of inhibitory synapses
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