An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Summary

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Abstract

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

Introduction

Neurites, including both axons and dendrites, are the projections from neurons involved in the establishment of the neural networks. The dynamic outgrowth of neurites is essential for neurodevelopment. However, the underlying regulatory mechanisms underneath remain unclear. In particular, neurite damage is often observed in various neurodegenerative diseases and after brain injuries¹. Therefore, investigation of the roles of putative molecules in various neurite outgrowth regulatory pathways would improve our understanding of the process. Moreover, it may reveal novel therapeutic targets for various neurological disorders. Neuronal cell lines a....

Protocol

All procedures followed were in accordance with the ethical standards of the animal experimentation ethics committee of the Chinese University of Hong Kong.

1. Preparation of Coverslips

1. Place a sterile 18 mm circular coverslip into each well of a 12-well tissue culture plate.
2. Coat the coverslip with 5 µg/mL poly-D-lysine solution in a humidified 37 °C incubator for at least 1 h.
3. Aspirate the poly-D-lysine solution from the tissue culture plate and rinse the coated coverslips once with sterile water.

2. Rat Embryonic Neuron Dissection

1. Sacrifice a timed-p....

Results

To test this methodology, we used Cyto D and nerve growth factor NGF, which have been shown to inhibit and stimulate neurite outgrowth respectively^14,15,16. The neurite length of neurons transfected with EGFP were measured after treatment with Cyto D or NGF. The transfection efficiency of EGFP to the neurons was 2.7% (1,068 neurons counted). As shown in Figure 1

Discussion

As stated before, PC12 and its subclones are widely employed for studying neurite extension because they have excellent transfection efficiency^2,3. In contrast, primary neurons have a low transfection rate, which is a major obstacle for studying neurite outgrowth regulators by transfection⁷. Here, we describe a convenient protocol for quantifying neurite outgrowth in primary neurons. Despite the low overall transfection efficiency, more th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#5 tweezers	Regine	5-COB
18 mm Circle Cover Slips	Thermo Scientific	CB00180RA	Sterilize before use
B27 Supplement	Gibco	17504044
Cytochalasin D	Invitrogen	PHZ1063	Dissolved in DMSO
D-(+)-Glucose	Sigma-Aldrich	G8270
Dimethyl Sulfoxide	Sigma-Aldrich	D2650
Dissecting Scissors, 10 cm	World Precision Instruments	14393
Dissecting Scissors, 12.5 cm	World Precision Instruments	15922
EndoFree Plasmid Maxi Kit	QIAGEN	12362
Fluorescence Mounting Medium	Dako	S302380
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668019
Neurobasal Medium	Gibco	21103049
NGF 2.5S Native Mouse Protein	Gibco	13257019
Nugent Utility Forceps, 10 mm, Straight Tip	World Precision Instruments	504489
Paraformaldehyde	Sigma-Aldrich	P6148
pEGFP-C1	Clontech	#6084-1
pCI FE65			Please see references 8 and 15
PBS Tablets	Gibco	18912014
Penicillin-Streptomycin	Gibco	15140122
Poly-D-lysine hydrobromide	Sigma-Aldrich	P7280
Spatula	Sigma-Aldrich	S4147
Trypsin-EDTA (0.05%), phenol red	Gibco	25300062
Trypan Blue Solution, 0.4%	Gibco	15250061