We thank Dr. Yuval Gottlieb-Dror for the electroporation illustration. This work was supported by grants to DSD from The National Institute for Psychobiology in Israel and from the Niedersachsen-Israel Research cooperation program and by grants to AK from The Israel Science Foundation, The Israel ministry of health, and The Center of Excellence-Legacy Heritage Biomedical Science partnership.

1. Hindbrain Electroporation
1.1 Egg handling
<ol>
	<li>Place the eggs horizontally in a humidified incubator (37-38.5 &deg;C). Embryos are electroporated after 65-70 hr of incubation, when they reach 16-17 (HH) stage (25-30 somites).</li>
	<li>Remove the eggs from the incubator; they remain in the horizontal position.</li>
</ol>
1.2 Preparations
<ol>
	<li>Pull glass capillaries (0.5 mm diameter).</li>
	<li>Connect bent L-shaped gold Genetrodes electrodes (3 mm diameter...

<ol>
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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...

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 neuronal progenitors become specified and differentiate in distinct DV locations 3,5,6. How the early AP and DV-specific neuronal patterns are governing the establishment of functional brainstem circuitries is largely unknown.
To gain knowledge on this fundamental question, tools are required in order to label specific subsets of neurons in the early hindbrain and to trace their axonal trajectories and connectivity at more advanced stages. We have previously utilized specific enhancer elements, and a Cre/LoxP-based conditional expression system for tracking axonal trajectory of dorsal spinal interneurons in the early chick embryo 7-9. In the current manuscript we have targeted the hindbrain and upgraded the experimental paradigm for labeling late embryonic hindbrain interneurons, axons and their synaptic targets, using a modified electroporation strategy and the PiggyBac - mediated DNA transposition. Our new strategy allows the tagging of distinct neuronal subtypes in one side of the hindbrain and the tracking of their axonal projections and synaptic sites at various embryonic stages, from 2 up to 12 days following electroporation. Based on this method, we labeled the dorsal-most subgroup of hindbrain interneurons (dA1/Atoh1+ cells) and revealed two contralateral ascending axonal projection patterns, each derives from a different AP location and elongates in a distinct funiculus. dA1 axons were found to project and form synapses in the auditory nuclei, midbrain and in multiple layers of the cerebellum 10.
The combination of chick electroporation, genetic tracing of neurons and analysis of projection sites at much advanced stages of development provides a unique platform to study the formation of neuronal networks in the brain and to elucidate molecular mechanisms that govern circuit formation.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

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.

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...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

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

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&#x2013;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.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K visualizações.  The Hebrew University of Jerusalem. Como as redes neuronais são estabelecidas no cérebro embrionário é uma questão fundamental no desenvolvimento da neurobiologia. Aqui nós combinamos uma técnica de eletroporação com novas ferramentas genéticas, como Cre / Lox-plasmídeos e sistema de transposição de DNA PiggyBac mediada no hindbrain aviária para rotular interneurons dorsal e rastrear suas projeções axonais e metas sinápticas em diferentes estágios de desenvolvimento.