Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Abstract

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Figure 1:. Schema for a typical survival experiment.

Rat cortical neurons are transfected at DIV4 using the procedure outlined in this article. Beginning 24 h post-transfection, cells are imaged at regularly spaced intervals in accordance with the specific requirements of the experiment. Images are stitched and stacked before cell death is scored, and Cox proportional hazard analysis is used to compare the risk of death between populations.

Figure 2:. Required file structure.

The provided FIJI macro requires that the raw data are formatted in a specific way. To utilize Image_Processing, organize the raw data as shown on the left. An example experiment and accompanying file structure is shown on the right.

Figure 3:. Scoring cell death in transfected rat cortical neurons.

Using the methods described in this article, rat cortical neurons were transfected with a plasmid encoding the fluorescent protein mApple. Cells were then imaged approximately every 24 h, the images were stitched and stacked, and cell death scored using the criteria provided. Cell death is indicated for Neuron 1 at 69 h, as evidenced by loss of fluorescence. Neuron 2 dies at 188 h, as indicated by fragmentation of the processes and rounding of the cell body. Neuron 3 survives for the duration of the experiment. Note that some cells become visible only late in the experiment, as evidenced by the appearance of a new cell at 235 h. Only cells that are visible at the initial time of imaging are included within subsequent analyses. Scale bar = 50 μm.

Figure 4:. Interpretation of Cox proportional hazard analysis.

(A) The output summary includes four important statistics that are highlighted in this figure. Box 1 includes the hazard ratio of the experimental group relative to the control group, while Box 2 and Box 3 show the p-values and 95% confidence interval for each hazard ratio, respectively. Box 4 highlights the results of the log-rank test. These data are also depicted via a Kaplan-Meier curve (B) and a cumulative risk of death plot (C).