We recorded from three different z planes separated by 10 μm, thus resulting in a temporal resolution of 10 Hz. To precisely register the electrophysiological with the optical recordings in time we recorded the trigger signal given by the camera at the beginning of every frame as a separate trace in the electrophysiological recording. Automated counting of trigger signals allowed then to determine exactly the beginning of every single frame of the calcium imaging in the electrophysiological trace. Image analysis was carried out automatically by custom made MATLAB software. As a first step in the analysis process, each set of three images from different z positions was collapsed into one maximum projection image. All maximum projection

images from one recording were collected in one stack. Next, an F0 image was generated in which learn more each pixel represented the median of all pixel values at this position throughout the stack. The F0 image was top-hat filtered using a disk-shaped structure (radius, three pixels) to correct for uneven background brightness. All areas brighter than two times the SD of the F0 pixel values and larger than 200 pixels

were detected as dendrites. The F0 image was also used to generate a ΔF/F0 stack by subtracting it from each frame of the stack and dividing the result by F0. Next, we calculated the derivative in time of the ΔF/F0 stack using a 3D convolution filter. The derivative was eroded with a disk-shaped structure (radius, two pixels) and subsequently binarized using a threshold of 15% ΔF/F0 s−1. A calcium transient was defined as a minimum of ten connected pixels in the binarized stack. The spatial Selleckchem Anticancer Compound Library center of individual calcium transients was defined as the pixel coordinates where the largest increase in fluorescence occurred within each signal. Where Bumetanide necessary, these positions were shift corrected across recordings. Next, calcium transients were assigned to specific dendritic sites whose locations along the dendrite were determined in an iterative process: in the first step the center of the first calcium transient in a given recording was estimated as described above. Subsequently, all transients that occurred within

4 μm distally or proximally from the center of the first transient along the dendrite were considered to be transients from the same site. The center coordinates of all these transients were averaged and this value was used as a refined locus for this site of activity for the second iteration. During the iterations, some transients were newly included or excluded from the population assigned to this particular site and its center location adapted slightly. The process was stopped after 20 iterations, when in fact no more changes occurred. Then the first transient in time that was not yet assigned to any site was used as a starting point for determining the next site of activity. Only sites where at least two transients occurred were considered for further analysis. On average 7.