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.

Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS

“…Statistical analyses were performed in R or Graphpad Prism 7. For primary neuron survival analysis, the open-source R survival package was used to determine hazard ratios describing the relative survival between conditions through Cox proportional hazards analysis (8). Significance determined via the two-tailed t-test was used to assess differences between treatment groups for neuronal activity and transcript abundance via RT-PCR.…”

“…Cortices from embryonic day (E)19-20 Long-Evans rat embryos were dissected and disassociated, and primary neurons were plated at a density of 6x10 5 cells/ml in 96-well plates, as described previously (6). At in vitro day (DIV) 4, neurons were transfected with 100 ng pGW1-mApple to mark cell bodies and 50-100 ng of an experimental construct using Lipofectamine 2000 (Invitrogen 52887), as previously described (5,7,8). Following transfection, cells were placed in either Neurobasal Complete Media (Neurobasal (Gibco 21103-049), 1x B27, 1x Glutamax, 100 units/mL Pen Strep (Gibco 15140-122)) or NEUMO photostable medium with SOS (Cell Guidance Systems M07-500) and incubated at 37°C in 5% CO2.…”

Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS

“…We therefore utilized automated microscopy in conjunction with survival analysis to track individual neurons prospectively over time and determine their risk of death in an unbiased and highthroughput manner (14,15,59,69,70). Rodent primary mixed cortical neurons were transfected with mApple and EGFP-tagged TDP43 isoforms and imaged by fluorescence microscopy at 24h intervals for 10d (71). Custom scripts were used to automatically generate ROIs corresponding to each cell and determine time of death based on rounding of the soma, retraction of neurites, or loss of fluorescence ( Figure 5A).…”

“…Custom scripts were used to automatically generate ROIs corresponding to each cell and determine time of death based on rounding of the soma, retraction of neurites, or loss of fluorescence ( Figure 5A). The time of death for individual neurons was used to calculate the risk of death in each population relative to a reference group, in this case neurons expressing EGFP 70,71 . In keeping with the results of previous studies, flTDP43 overexpression resulted in a significant increase in the risk of death in comparison to EGFP alone (HR=2.22 p<2x10 -16 ).…”

“…Cortices from embryonic day (E)19-20 Long-Evans rat embryos were dissected and disassociated, and primary neurons were plated at a density of 6x10 5 cells/ml in 96-well plates, 27 as described previously 131 . At in vitro day (DIV) 4, neurons were transfected with 100 ng pGW1-mApple to mark cell bodies and 50-100 ng of an experimental construct using Lipofectamine 2000 (Invitrogen 52887), as previously described 14,70,130 . Following transfection, cells were placed in either Neurobasal Complete Media (Neurobasal (Gibco 21103-049), 1x B27, 1x Glutamax, 100 units/mL Pen Strep (Gibco 15140-122)) or NEUMO photostable medium with SOS (Cell Guidance Systems M07-500) and incubated at 37°C in 5% CO2.…”