Ca2+ measurement
- Cell population: live Ca2+ fluctuations were
recorded at 37°C on ST-hmGlu5 GcaMP6s-P2A-Scarlet HEK293T cells, or when
specified on GcaMP6s-P2A-Scarlet HEK293T cells transfected with
pRK-SNAPTag-AT1 (25 ng/well), seeded at 5 x 104 cells
per well in 96-wells plates with transparent bottom, with the FDSS/µCell
plate reader (Hamamatsu®) – Acquisitions of 1Hz; Exc:
480 nm – Em: 540 nm; high-speed digital EM CCD camera. Each independent
experiment was performed in triplicate, which average corresponds to n =
1. Data were normalized to the maximum response at Vrestin each independent experiment. Basic calculations from the raw data
files were performed on Microsoft Excel (Office 2016), statistical
analyses and graphics on GraphPad Prism 8.1.
- Single cell imaging: GcaMP6s fluorescence fluctuations were recorded
on ST-hmGlu5 GcaMP6s-P2A-Scarlet HEK293T cells, using an Axio Observer 7
KMAT fluorescence microscope (Carl Zeiss), equipped with a Plan-Neofluar
40x/1.30 EC oil objective (M27, ZEISS®), Exc 470/40 nm – Em 525/50 nm
filters and ORCA-Quest qCMOS camera (Hamamatsu); all controlled with
Metamorph software. Images on HEK cells were acquired at 3Hz and
analysed with Fiji using a custom code computing the average
fluorescence intensity of the regions of interest. For the frequency
analysis, MATLAB was used to automatically detect the oscillations
occurrences and compute the instantaneous and global oscillation
frequencies. First, baselines were computed as the
10th percentile of the trace using a sliding window of
size 400 frames. Signal was then divided by the baseline. Oscillations
were detected using the function findpeaks with a minimal peak
prominence of 0.01. Instantaneous frequency was computed as the number
of oscillations minus one divided by the time between first and last
oscillation. Global frequency was computed as the number of oscillations
divided by the total duration. Spontaneous Ca2+transients in neurons infected with the pWPT-CAMKIIa-GcaMP6s-P2A-Scarlet
lentivirus were acquired at 10 Hz, for series of 2 min and 15 sec. Image
stacks were then analyzed as follow: images were filtered with a 5*5
median kernel. Each pixel was divided by its average value over the
whole stack. Each pixel was gaussian filtered along the time axis with a
sigma of 2 frames. Time derivative was applied to the stack with a step
of 6 frames. Images were thresholded with a value of image mean + 6 *
image standard deviation. A filter was then applied to keep only voxels
with at least 10 thresholded voxels in their 3*3*3 neighborhood, to
remove isolated voxels. Thresholded voxels spatially connected were
assembled as events. Maximal time projection of those events gave their
spatial footprint: the list of pixels in the image. Events with lower
than 16 pixels were rejected. Events were classified in different
categories based on two criterions: a threshold of 1000 pixels
separating big events from small events, a threshold of 0.8 px of
propagation distance separating events propagating or not. Propagation
distance was measured as the difference of position between the centroid
of the pixels thresholded at the beginning and at the end of the onset.
Both thresholds were applied to focus on “small, non-propagating
calcium transients” at synapses. Projection images were obtained by
accumulation of the events footprint. Fluorescence time traces were
extracted for each event as the average fluorescence over the event
pixels and normalized by the fluorescence just before the onset of the
event. Area under curve was computed between the onset and 4 seconds
later.