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

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 toxicity^1,2,3,4. 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 solutions^4,5,6. 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 neurotoxicity^7,8,9. 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 workflow^8,9. 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.