Figure 1

Automated GCaMP6f fluorescent image segregation for tracking neuronal calcium transients in high-density cultures. Representative immunostaining for (A) glial fibrillary acid protein (GFAP; astroglia), (B) ionized calcium binding adaptor molecule 1 (Iba1; microglia), (C) neuronal nuclei (NeuN; neurons), (D) oligodendrocyte lineage transcription factor 2 (Olig2; oligodendrocytes), (E,F) microtubule associated protein 2 (MAP2; neurons), (E) T-box brain 1 (TBR1; glutamatergic neurons), (F) glutamate decarboxylase molecular weight 67 kD (GAD67; GABA-ergic neurons). 10x fluorescent micrographs were acquired on DIV8, 12, and16 using an LSM 780 confocal microscope. Scale bar is 50 μm. (G) Quantification of immunostaining for cellular markers from DIV8 to DIV16. Positively labeled cells were manually counted in 4–10 optical fields. Error bars are mean +/− 95% confidence intervals (CI). (H) The relative expression of excitatory and inhibitory neurotransmitter receptors in the cultures was determined by RTPCR. (I–L) Neuronal cultures were infected at plating with 2000 MOI of AAV1.Syn.GCaMP6f.WPRE.SV40. At time of acquisition culture plates were transferred to the stage of a Zeiss 5 Live spinning disc confocal microscope with environmental chamber. After brief environmental equilibration, 250 frames were captured by time series acquisition at 5 frames per second. Maximum intensity projections were generated from the time series and imported into ImageJ for automated region of interest (ROI) generation. (I–J) Maximum intensity projection of raw time series acquisition with derived ROIs overlaid in red. (K) Binary image masks used for generating ROIs in (J,L) To determine correspondence between ROIs and individual cell bodies, cells were counterstained with DAPI immediately prior to imaging. ROIs were generated and overlaid on maximum intensity projections. The number of DAPI+ nuclei per ROI determined using ImageJ Cell Counter plugin for 494 ROIs. The percent of ROIs containing 1 or more nuclei is graphed. As shown, 90% of ROIs corresponded to 2 or fewer cell bodies. Findings are representative of two independent cultures.