Summary

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Published: July 26, 2021
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Summary

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Abstract

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer’s disease, and Parkinson’s disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival.

Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Introduction

The study of neuronal disorders requires experimental models that are amenable to genetic, molecular, and cellular analyses. Primary cortical neurons are a very potent model for studying neuronal survival and toxicity1,2,3,4. Under the appropriate conditions, primary neurons will progressively develop synaptic contacts and present hallmarks of mature neurons. Therefore, this model is more reliable than immortalized cell lines in modeling the physiology of the neurons and more amenable to manipulations than animal models. However, in comparison to cell lines, primary cortical neurons are difficult to transfect and relatively fragile. Moreover, the intricate morphology of cortical neurons may complicate the imaging and analysis in high-density cultures. Conversely, low-density primary neuronal cultures are easier to analyze but tend to be more fragile.

Taking all of this into account, this paper presents an affordable, versatile, and scalable in vitro workflow to model and investigate neuronal survival (Figure 1). Key points of this protocol are i) the method of in vitro electroporation of the neurons with a fluorescent marker to allow sparse labeling and imaging of live cells; ii) its versatility in different models of cytotoxicity; and iii) the semi-automated or automated analysis.

The electroporation system (see the Table of Materials) provides an open and inexpensive procedure that does not involve kits and specific solutions4,5,6. Moreover, this method can be easily adapted to obtain an optimal transformation efficiency and density of transfected neurons, mixing electroporated with non-electroporated cells. The co-culture with naïve (non-electroporated) neurons significantly improves the viability of the electroporated cells compared to low-density cultures. Moreover, it enables sparse labeling with an adjustable density of the electroporated neurons, maintaining a consistent level of gene expression. It is important to aim for 3-5% of electroporated neurons in survival assays.

Having sparse labeling essentially facilitates the automation of the image analysis because the cells are well-separated and easily distinguishable. Notably, this experimental paradigm may be adapted to test multiple genes of interest by simultaneously co-culturing neurons co-electroporated with different cytosolic fluorescent markers (e.g., cyan fluorescent protein, red fluorescent protein (RFP), green fluorescent protein (GFP)) in the same well. Similar to other cytotoxicity assays (e.g., propidium iodide or lactate dehydrogenase), the assay described herein is based on the fact that neuronal death is accompanied by rupturing of the cell membrane. This provokes the release of cytosolic proteins by diffusion and, consequently, the loss of GFP fluorescence.

An in vitro model of stroke, namely the OGD model, is presented as an example of neurotoxicity7,8,9. This protocol entails exposing the primary neurons to a salt buffer similar to an artificial cerebrospinal fluid but deprived of oxygen and glucose. Although this model has been presented as an example of neurotoxic stress, different cytotoxic conditions can be tested with the same workflow8,9. Finally, sparse labeling easily enables the development of automated imaging analysis. Here, a protocol was established based on standard immunofluorescence and ImageJ analysis in a smaller setup. Next, this workflow was adapted and scaled up using a cell imaging system that allows an automatized analysis in live imaging in mid- and high-throughput modes. In conclusion, this paper presents a flexible, affordable, and scalable methodology to study neuronal survival in different experimental models of toxicity using live imaging and automated quantification.

Protocol

All procedures using animals should be supervised by the bioethical animal committee of the institute and performed in compliance with local regulations. The procedures presented herein were approved by the delegated authority and comply with the regulations in Spain and Europe. 1. Primary neuronal culture NOTE: All the steps are performed inside the culture hood, using sterile materials and solutions to maintain sterile conditions. Poly-L-lysine (PLL) c…

Representative Results

This protocol aims to establish an in vitro model of stroke. It is important to obtain an adequate neuronal density, which will allow the recognition of individual electroporated neurons to analyze them individually. The stage of the neuronal culture after plating is also crucial. The maturation of neurons in culture is progressive. The dependence on growth factors, neurite outgrowth, connectivity, and electrophysiological activity will vary greatly depending on the stage. In these specific conditions at 4-6 day…

Discussion

This protocol shows an effective way of modeling a stroke in vitro. To achieve this goal, we proposed sparse labeling of cortical neurons using the electroporation system NEPA215. This is an open system that allows customization of the protocol with minimal operative cost compared to other systems that employ kits or specific devices. Mixed culture of naïve and electroporated neurons allows more flexibility and robustness as compared to low-density neuronal culture. This allows the s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We would like to thank Carlos Dotti for sharing his expertise in neuronal culture. We also thank Alicia Martínez-Romero from the Cytomics Core Facility of the Centro de Investigación Principe Felipe (CIPF), which is supported by European FEDERER funding. The project is supported by the Spanish Ministry of Economy and Competitiveness for (SAF2017-89020-R) reagents, materials, and the salaries of YDC and PF. PF is also supported by the grant RyC-2014-16410. CGN and PF are supported by Conselleria de Sanitat of the Generalitat Valenciana, as well as AGM (ACIF/2019/015). Ángela Rodríguez Prieto is supported by the Spanish Ministry of Science, Innovation and Universities, with the grant PRE2018-083562.

Materials

10 cm petri dishes FISHER SCIENTIFIC, S.L. 1130-9283
3.5 cm petri dishes Sterilin
75 cm2 flask Corning 430641U
B-27 Life Technologies 17504-044
Cell culture plates Corning incorporation Costar® 3513
Cell incubator Thermo Electron Corporation Model 371
Cell strainer 70 µm Falcon 352350
Cold lights Schott 223488 KL 1500 LCD
Coverslips (15 mm) Marienfeld 111530
CU500 Cuvette Chamber Nepa Gene
CU600 Cuvette Stand Holder Nepa Gene
DAPI Sigma-Aldrich Quimica, S. L. D9542-10MG 1:2000
DMSO Panreac A3672
Dumont Fine Forceps FST 11254-20
Dumont Fine Forceps FST 11252-00
EC-002S NEPA Electroporation Cuvettes, 2mm gap Nepa Gene
Filter strainer Falcon 352350
Fine Scissors-Sharp-Blunt FST 14028-10
Fine Scissors-ToughCut r FST 14058-09
GFP chicken IgY Aves Labs GFP-1010 1:600
Glucose Sigma 68769-100ml
GlutaMAX-I Supplement 200 mM 100 mL Fisher Scientific 35050-061
HBSS Thermofisher 14175-095 https://www.thermofisher.com/es/es/home/technical-resources/media-formulation.156.html
Hepes 1 M ThermoFisher 15630-080
Horse Serum Invitrogen 26050088
MEM Thermofisher 11095080 https://www.thermofisher.com/order/catalog/product/11095080#/11095080
Microscope slide (polilysine) VWR 631-0107 Dimension: 25 x 75 x 1 mm
Mowiol 4-88 Sigma-Aldrich Quimica, S. L. 81381-250G
Needles yellow, 30 gauge BD Microlance™ 3 304000
NEPA21 electroporator Nepa Gene
Neubauer chamber Blau Brand 717805
Neurobasal Medium ThermoFisher 21103-49
Opti-MEM Invitrogen 31985-062
Parafilm Cole-Parmer PM996
Paraformaldehyde (PFA), 95% Sigma-Aldrich Quimica, S. L. 158127-500G Use solution: 4%
PEI Polysciences 23966-1
Plasmid for GFP pCMV-GFP-ires-Cre, described in Fazzari et al., Nature, 2010
Poly-L-Lysine SIGMA P2636
PS ( Penicillin, Streptomycin) ThermoFisher 15140122
Serrate forceps FST 11152-10
Stereomicroscope WORLD PRECISION INSTRUMENTS
Syringes BD Plastipak 1ml 303176
Triton X-100 Sigma-Aldrich Quimica, S. L. MDL number: MFCD00128254 Non-ionic
Trypsin-EDTA ThermoFisher 25300054
Tubes 15 mL Fisher 05-539-4
Tubes 50 mL VWR 21008-242
Tupperware Hermetic tupperware with screw lid. SP Berner – Taper 1 L Redondo con Rosca. Any equivalent hermetic Tupperware may be purchased in any supermarket.
Water bath SHELDON LABORATORY MODEL 1224 1641951

References

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Cite This Article
Rodríguez-Prieto, Á., González-Manteiga, A., Domínguez-Canterla, Y., Navarro-González, C., Fazzari, P. A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution. J. Vis. Exp. (173), e62759, doi:10.3791/62759 (2021).

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