Subtype-selective Electroporation of Cortical Interneurons

Summary

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Abstract

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex¹. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene^2,3, to drive the expression of specific genes through in utero electroporation.

Introduction

The bulk of cortical GABAergic interneurons originate from two transient embryonic structures named the medial and caudal ganglionic eminences (MGE and CGE respectively)⁴. Parvalbumin and somatostatin expressing interneurons originate in the MGE whereas Calretinin (Cr), Vasointestinal peptide (VIP) and Reelin (Re) expressing interneurons originate from the CGE. These interneuron subtypes can be distinguished by their birthdates. MGE derived subtypes are born between embryonic day 9.5 (e9.5) and e16.5^5,6. In contrast, CGE derived interneurons are born from e12.5 through e18.5 with their production peaking at e15.5⁶. The genetic targetin....

Protocol

All animals were treated in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee of the NYU School of Medicine.

Mouse Strains
Swiss Webster female mice provided by TACONIC were used for these experiments. In order to specifically target superficial layer interneurons, e15.5 embryos were used.

Note: The plasmid used in this work (Dlx5/6.eGFP plasmid 3 µg/µl) was generated using standard cloning techniques. The eGFP cDNA was cloned into a Dlx5/6-Pmin-polyA plasmid. This plasmid is available upon request (demarn02@nyumc.org....

Results

We adapted the in utero electroporation technique to achieve cell type specific targeting of maturing neurons. To drive the expression of eGFP in CGE-derived interneurons, we used the Dlx5/6 enhancer element and restricted our injections to e15.5, the stage when the majority of CGE-derived interneurons are generated. We carried out the analysis at P8 and P15 ¹¹(Figures 1 and 2). We confirmed the ventral origin of electroporated neurons by co electroporating a.......

Discussion

Limitations of the Technique

While this technique allows for cell autonomous analysis of cell processes, it is not suitable for population analysis. The electroporations are very sparse with less than a thousand cells electroporated per brain. As a consequence, the technique cannot be used to assess behavioral consequences arising from the genetic manipulation of CGE-derived interneurons.

While electroporations carried out at e13.5-e14.5 target MGE-derived subtypes,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electroporator  with pedal	Protech International	CUY21
5 mm paddle electrodes	Protech International	CUY650P5
Heating pad	Kent Scientific	DCT-15
Sutter Instruments P30 Puller	Sutter Instruments	3282322
Fluovac Anesthesia Systems	Harvard Apparatus	726425
Delicate Operating Scissors 4.75" Straight Sharp/Sharp	Roboz	RS-6702
5-0 Silk Black Braid 18" C-1 Box 36	Roboz	SUT-1073-21
Micro Clip Applying Forceps 5.5"	Roboz	RS-5410
2 Clamp scissors	Roboz	RC-4894
Holding forceps	Fine Science Tools	11031-15
Glass capillary tubing	FHC	27-30-0	Borosil 1.0 mm OD x 0.75 mm ID
Fast Green	Sigma-Aldrich	F7258
Sterile PBS	Life Technologies	20012-027