Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Summary

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Abstract

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

Introduction

The hindbrain represents a key relay hub of the nervous system by communicating between the central and peripheral nervous systems via ascending and descending neuronal networks. It regulates basic functions including respiration, consciousness, hearing, and motor coordination ^1-3. During early embryonic development, the vertebrate hindbrain is transiently subdivided along its anterior-posterior (AP) axis into repetitive rhombomeres, in which distinct neuronal cell types are formed and generate multiple brainstem nuclei centers ⁴. The hindbrain is also divided along its dorsal-ventral (DV) axis into a basal and alar plate, at which discrete neuro....

Protocol

1. Hindbrain Electroporation

1.1 Egg handling

1. Place the eggs horizontally in a humidified incubator (37-38.5 °C). Embryos are electroporated after 65-70 hr of incubation, when they reach 16-17 (HH) stage (25-30 somites).
2. Remove the eggs from the incubator; they remain in the horizontal position.

1.2 Preparations

1. Pull glass capillaries (0.5 mm diameter).
2. Connect bent L-shaped gold Genetrodes electrodes (3 mm diameter) with an adaptor holder to the pulse generator. Electroporation parameters include 25 volts, 5 pulse numbers, 45 msec pulse length, 300 msec pulse in....

Results

This protocol was recently used to uncover the axonal patterns and projection sites of dA1 subgroup of interneurons in the chick hindbrain ¹⁰. To specifically label these axons, an enhancer element (Atoh1), that has previously been characterized as specific for spinal dI1 neurons ^8,12,13, was confirmed to be expressed in hindbrain dA1 cells ¹⁰. The element was cloned upstream to Cre recombinase and co-electroporated at E2.75 along with a Cre dependent cytoplasmic GFP reporter plasmid (pCA.......

Discussion

In ovo electroporation is a feasible, reliable, and effective tool to examine cell specification and axonal guidance during chick nervous system development ²⁰. In this protocol we describe a mode of electroporation in the chick hindbrain at E2.75 using enhancer elements which enable the conditional labeling of specific interneurons. This strategy is combined with the PiggyBac-mediated transposition system to insert foreign genes into the chick genome, which enables tracking of axonal routes, projecti.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Equipment	Company	Catalogue Number
L-shaped gold Genetrodes 3 mm electrodes	BTX, Harvard Apparatus	45-0162
pulse generator, ECM 830	BTX, Harvard Apparatus	45-0002
OCT (Optimal Cutting Temperature) Compound	Tissue-Tek Sakura	4583 O.C.T. Compound
Nail Polish	From Any Commercial Supplier