Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Summary

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Abstract

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Introduction

The cerebellum is an excellent system to study mechanisms of axon growth and migration. The cerebellum has been the subject of anatomical studies since the dawn of neuroscience¹. Modern microscopy and immunohistochemical techniques have significantly expanded and refined the initial discoveries by Santiago, Ramon, and Cajal^2-4. Mouse genetics and molecular studies uncovered essential growth and transcription factors in the control of cerebellar development, which led to greater understanding of crucial events required for proper wiring of different types of neurons including cerebellar granule neurons (CGNs)^5-7.

Protocol

CGNs can be prepared either from postnatal day (P) 5 mouse pups or P6 rat pups. We follow a protocol, described by Bilimoria and colleagues, which uses a mitotic inhibitor to select for postmitotic CGNs¹³.

Ethics statement:
All experiments involving live animals have been conducted according to the animal protocol approved by the "Verbraucherschutz und Lebensmittelsicherheit" of Lower Saxony, Germany.

In vitro assay:

1. Preparation of DNA Plasmid, Media, and Buffers for the Calcium Phosphate Transfection Method

1. Dissolve plasmid DNA in sterile, endotoxin....

Results

To analyze the morphology of CGNs in response to different culturing conditions, we transfected the neurons on DIV 0 as described above. After transfection, we placed one set of neurons into full medium (BME, 10% calf serum, 2 mM PSG, 25 mM KCl) and another set into minimal medium containing insulin (BME, 25 mM glucose, 2 mM PSG, 10 μg/ml insulin). We subjected the neurons to immunocytochemistry using the GFP antibody at DIV 1, 2, and 3, followed by measuring axons and dendrites for set 1 and axons only for set 2. Owing .......

Discussion

Advantages and limitations of the described in vitro and in vivo methods:

Cultured CGNs from mouse and rats are equally well suited for morphological analyses. Owing to the bigger size of a rat cerebellum, the yield of CNGs from rat pups exceeds that of mouse pups 3-4x. Aside from CGNs, cortical and hippocampal neurons can be used as culture system as well. The calcium phosphate method results in a low (0.01-5%) transfection efficiency, which is desired to analyze the morphol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Gibco	11960-044
BME	Gibco	41010-026
Insulin	Sigma-Aldrich	Si-1-4011
Poly-L-Ornithine	Sigma-Aldrich	P-2533
CaCl2	Appli-Chem	A3652
Isoflurane	Actavis Deutschland
Tissue-Tek OCT	Sakura
ECM 830 and tweezertrodes	Harvard Apparatus
Epifluorescence microscope and camera	Nikon
SP2 confocal microscope	Leica
ImageJ	NIH
Imaris 7.4.2	Bitplane, Inc.
GraphPad Prism	GraphPad Software, Inc.
MS Excel	Microsoft
Loading tip 1-200 µl	Costar	4853
Pipette tip 200 µl	Sarstedt	70.760.502
Microlance 3 needle, 30 G	BD	302200
50 µl gastight Syringe 1705	Hamilton
Glass coverslips	Thermo Scientific Menzel Glaeser	CB00120RA1