Figure 4

Measurement of axonal and dendritic outgrowth from primary neurons. Dissociated hippocampal neurons were cultured in 384-well plates and incubated with the indicated compounds for 3 days, starting one day after plating. a) Heatmaps were automatically generated by NeuriteQuant to summarize the main measurements: neurite length (red), neuronal cell body area (green) and mean neuronal marker signal intensity (blue). The left heatmap shows these measurements for dendrites only, detected using MAP2, and the right heatmap shows the corresponding measurements for total neurites (axons plus dendrites) using the general neurite marker TuJ1. The shift in colour hue from green to red with increasing cytochalasin D concentrations in the left heatmap indicates a dose-dependent shift in the ratio between neurite length and neuronal cell body area. b) Representative automatically acquired images of neurons incubated with either vehicle (DMSO) or cytochalasin D prior to staining with TuJ1 (axons plus dendrites; red) and MAP2 (dendrites only, green); yellow indicates regions of overlap in the merged image. c) Quantitative analysis of cytochalasin D dose response curves demonstrates a >50% increase in the normalized neurite length (the ratio of total neurite length to total neuronal cell body area per field), as based on the dendritic marker MAP2. In contrast, analysis based on the general neurite marker TuJ1 shows a slightly opposite trend, because axon length decreased in response to cytochalasin D (not shown). Corresponding amounts of vehicle had no significant effect on neuronal morphology as detected using either marker. Data represent mean ± standard error of 9 image fields from three independent repetitions per condition.