Oregon green bapta 1

For calcium imaging experiments, 250 μM Oregon Green BAPTA-1 (OGB-1, Invitrogen) or 500 μM Fluo-4 (Invitrogen) were added to the pipette solution. After whole-cell access, the dye was allowed to diffuse for 10–15 min before performing experiments. The basal OGB-1 fluorescence was used to acquire the neuronal morphology by confocal stack acquisition. For high-speed imaging, the cell was illuminated in wide field with a 470 nm LED system (CoolLed, Roper Scientific) via a 63× 1.0 N.A. water-immersion objective (Zeiss). Collected fluorescence was long-pass filtered at 500 nm and projected onto an EMCCD camera (Evolve 128, Photometrics). APs were induced in the soma by injecting a 10 ms current pulse, synchronized with LED illumination and 500 Hz EMCCD acquisition for 40 ms. This protoc ol was repeated 10–15 times for OGB-1, and 70–80 times for Fluo-4, each sweep separated by 10 s. Electrophysiological signals recorded from the soma and CCD Frame Read-out signals were sampled and acquired at 40 kHz.

For intracellular Ca²⁺ imaging in PCs, a calcium-sensitive dye, Oregon Green BAPTA-1 (100 μM; Thermo Fisher Scientific), was dissolved in the intracellular solution and introduced into the cells using a patch pipette, and the concentration of EGTA in the intracellular solution was decreased to 0.5 mM. The patched cells were examined with an upright microscope (BX51WI, Olympus) equipped with a confocal scanning unit and an argon laser (FV300, Olympus) at an excitation wavelength of 488 nm, and 5–9 sequential confocal images obtained at 3–4 μm intervals (z-axis) were acquired every 0.8 s and were projected onto a plane to obtain images of dendrites at 10-s intervals [26 ,27 (link)].

For calcium imaging experiments, 50 μM Oregon Green BAPTA 1 (Life Technologies) was added to the patch pipette. Fluorescence signal, acquired using excitation at 480 nm and emission at 535 nm, was recorded at 25–100 Hz using a high speed camera (MiCAM Ultima, Brainvisions) while using a high power LED (Prizmatix, Givat-Shmuel, Israel) as an excitation light source.

The neurons were stained with Oregon green BAPTA1 (Life Technologies) for 1 hour and washed three times with the medium. The entire network was observed for more than 10 minutes on a Nikon microscope with spinning disc fluorescence. Acquisition time was 500 ms every 500 ms (2 Hz sampling), which is sufficient to determine functional architecture11 (link)50 (link)51 (link)52 (link)53 (link). The images were then parsed with the Matlab code provided by50 (link) where network structures are analyzed by applying cross-covariance signal processing and graph theory to single-cell recordings, according to cross-covariance analysis54 and graph theory applied to small world networks55 (link). Neurons were first segregated by size (10 μm) over the entire experiment duration. For all localizations, Δf/f₀ was monitored. Based on each spontaneous spike, a global threshold (where for each pixel, Δf/f₀ was larger than the average pixel value for each image) was applied to all four populations and the inter-population activity was plotted over time with a randomized number so that the cross correlation process would not be biased by proximity effects. The inter population correlation matrix was extracted, which allowed computation of the adjacency matrix for each motif and the correlation coefficient spatial distribution (Fig. S8).