In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Carlos G. Briz; Marta Navarrete; José A. Esteban; Marta Nieto

doi:10.3791/55139

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Method Article

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

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DOI:

10.3791/55139

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February 15th, 2017

Carlos G. Briz¹ , Marta Navarrete² , José A. Esteban² , Marta Nieto¹

¹Department for Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), ²Molecular Neurobiology Department, Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas (CSIC-UAM)

Summary

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

Abstract

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.

Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

Introduction

The development of dendritic and axonal structures is an important facet of circuit regulation in the nervous system, including in the cerebral cortex. It plays a critical role during the selective wiring of the diverse neuronal subpopulations. A number of recent reports have shown that, in addition to connectivity, the molecular diversity of neurons is reflected by the acquisition of highly specific modes of firing. However, the mechanisms determining the excitability and connectivity of the distinct neuronal subtypes during development, as well as their degree of coordination, are still poorly understood¹^,².....