Figure 1.
Domain structures of the GCaMP-family of genetically encoded calcium indicators (GECIs) and fusion constructs.
A, Domain comparisons of GCaMP2 and GCaMP1.6; red labels indicate the differences. B, Constructs for subcellular targeting of the GECIs.
Figure 2.
Recording backpropagating action potential responses from GECIs in hippocampal pyramidal cells.
A, Schematic showing the linescan location at the base of the apical dendrite. B, Raw linescan images (top row) showing a dark period prior to shutter opening, followed by a shutter-open fluorescence baseline and action-potential (bottom row) evoked responses (left, 3 action potentials at 83 Hz; right, 10 action potentials at 83 Hz). Fluorescence time series (middle row) were obtained by averaging over the spatial extent of the dendrite (indicated by vertical white lines).
Figure 3.
Action-potential evoked responses in GCaMP-based GECIs.
A, Amplitudes of GCaMP2 responses for individual hippocampal pyramidal cells (thin lines, left) in response to trains of action potentials given at 83 Hz, and the mean across cells (thick gray line). Dashed lines show perforated-patch recordings. Inset shows same data for 1–10 action potentials on a linear x-axis. Example single-trial responses (right) from four cells to 1, 5, 10 and 40 action potentials at 83 Hz (indicated by horizontal black lines). B, Responses of individual cortical layer 2/3 pyramidal cells (thin lines) and the group mean (thick gray line) expressing GCaMP2 after in utero electroporation (see Materials and Methods). C, GCaMP2 responses from hippocampal pyramidal cells at 34.5–35.5°C. D,E, Responses of previous versions of GCaMP family GECIs. B–E, Same conventions as in A.
Figure 4.
Action-potential evoked responses in GECIs targeted to subcellular locations.
A, Amplitudes of the response to action potential trains at 83 Hz for the membrane-targeted GECI hCD4-GCaMP2 (left), for individual cells (thin black lines) and for the group mean (thick gray line). Insets show same data for 1–10 action potentials on a linear x-axis. Example traces (right) show single-trial responses to trains of action potentials at 83 Hz (indicated by horizontal black lines). B, Responses from the membrane-targeted construct MARCKS-GCaMP2. C, Actin-potential evoked responses from the GCaMP2-actin fusion. Conventions as in A. D–F, Action-potential responses measured simultaneously with GECIs (green curves, traces) and with the synthetic dye X-Rhod-5F (500 µM; red curves, traces). Cells were loaded with X-Rhod-5F for ≥20 min prior to data collection. Example traces show single-trial responses measured simultaneously from the green and red channels. Cells shown in D–F are different from those in Figure 3A and in panels A–C.
Figure 5.
Accuracy of action potential detection.
Results of simulations (see Materials and Methods) giving percentage of action potential trains (for the indicated numbers of action potentials at 83 Hz) that can be detected at a 5% false positive rate for 1 second of data (i.e., such that when a time series is divided up into subsequent intervals of 1 second length, 5% of these intervals will contain a false positive). Thin dotted lines show results for individual cells and the thick black line gives the group mean.
Figure 6.
GCaMP2 and GCaMP2-actin uncaging responses in spines.
A, Apical dendrite of a CA1 pyramidal cell (left) expressing EGFP-actin (green) and mCherry (red), showing an enrichment of EGFP-actin at spines (predominantly green) compared to dendrite (predominantly red). A GCaMP2-actin fusion (right) shows a similar spine enrichment in the apical dendrite of a different CA1 cell. B, Example images showing spine and dendrite fluorescence for GCaMP2 (left) and GCaMP2-actin (right). Each image shows the baseline frame prior to uncaging. Images are median filtered in a 3×3 pixel neighborhood. C, Traces show uncaging-evoked ΔF/F fluorescence signals for ROIs covering the spine (gray) and dendrite (black) for GCaMP2 (left) and GCaMP2-actin (right). Uncaging occurs at the start of the third frame. Traces correspond to the spines/dendrites shown in B. D, Traces showing NMDA-receptor currents in response to glutamate uncaging at the spines shown in B. Each trace shows an average of 6 trials. E, ΔF/F fluorescence change in spines versus peak NMDA-receptor current versus for GCaMP2 (gray circles) and GCaMP2-actin (black circles). Lines indicate measurements taken from the same spine at different uncaging powers.
Figure 7.
Brightness of the GECIs compared.
GECIs were expressed (34–48 hours) in pyramidal cells and brightness was measured as the mean fluorescence intensity collected in linescan mode across the base of the apical dendrite. Each plot symbol shows a single cell, except for the FRET probes in which symbols connected by a dashed line indicate cyan and yellow channel measurements from the same cell. Illumination was 7 mW at the objective back aperture (see Materials and Methods for details).