In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Summary

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

Abstract

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.

In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

Introduction

In the mammalian brain, the generation of new neurons (neurogenesis) occurs after birth mainly in two regions, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus¹. Considerable evidence gathered in recent years supports a critical role for postnatal neurogenesis in hippocampal and olfactory bulb memory functions^1-3. Importantly, postnatal neurogenesis also holds therapeutic potential because of its relationship with degenerative neurological disorders, and the ability of neuroblasts to migrate to injured sites in the brain^4-6.

The subventr....

Protocol

This procedure is in accordance with the UK Home Office Regulations (Animal Scientific Procedures Act, 1986). Scientists should follow the guidelines established and approved by their institutional and national animal regulatory organizations.

1. Postnatal Electroporation

1.1. Preparation of Glass Capillaries, DNA Solution and Electroporator

1. Prepare pulled glass capillaries (O.D.: 1.5 mm, I.D.: 0.86 mm) for DNA injection. (Indicative settings for a Sutter P-97 capillary puller are: Heat 283; Pull 50; Velocity 90; Time 50). Make a mark on the capillary corresponding to a volume of approximately 2 µl.....

Results

Labeling of SVZ-derived migratory neuroblasts can be observed along the RMS, usually 4-8 days after a successful electroporation (Figure 1B). Longer time points can also be chosen, but less cells will be found in the RMS since most of them will have entered the OB. Neuroblasts start acquiring typical morphology and features of mature granule cells in the OB around 2-3 weeks after electroporation (not shown). After culturing for ~1 hr, brain slices from electroporated mouse pups can be reliably imaged for.......

Discussion

Efficient migration of neural progenitors along the RMS ensures their subsequent maturation into functional neurons¹⁰. Prominent streams of neural progenitors directed towards the OB are visible in human infancy and are likely to play an important role in early postnatal human brain development²⁷. Moreover, these cells are able to target sites of the brain affected by injury and neurodegeneration^4,28. Being able to monitor in real time the effect of gene manipulation on neuroblast dynamic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Millicell	Millipore	PICM0RG50
35 mm Glass bottom culture dish	MatTek	P35G-0-14-C
Gey's Balanced media	Sigma	G9779-500ML
Glucose, 45%	Sigma	G8769-100ML
HEPES	Sigma	H3375-25G
Pen/Strep	GIBCO	15140-122
FCS	GIBCO	10109-163
B27 supplement	Invitrogen Life Technologies	17504044
L-Glutamine	Invitrogen Life Technologies	25030-081
DMEM (phenol red-free)	GIBCO	31053-028
Fast Green	Sigma	F7252-5G
Glass capillaries for injection	Harvard Apparatus	30-0057
Aspirator tube	Sigma	A5177
Sutter P-97 capillary puller	Sutter Instrument	P-97
ECM830 Square Wave Electroporator	Harvard Apparatus	45-0052
Platinum Tweezertrodes 7 mm	Harvard Apparatus	45-0488
Footswitch Model 1250F	Harvard Apparatus	45-0211
Gel for electrodes	Cefar Compex	6602048
Isoflurane	Merial	AP/DRUGS/220/96
Vibratome	Leica	VT1000S
Glue	Roti coll	Roti coll 1
UltraViEW VoX spinning disk system	Perkin Elmer		Customized setup (multiple laser sources can be used) equipped with Hamamatsu ORCA R2 C10600-10B CCD camera
Volocity software	Perkin Elmer		Acquisition, Quantitation, Visualization Modules
Environmental chamber for microscopy	Solent Scientific		Custom-made
Ti-E inverted microscope	Nikon		CFI Super Plan Fluor ELWD 20X/0.45 NA objective is recommended for the application described in this paper