All image processing was performed using ImageJ (NIH). Mean pixel values were measured from ROIs were taken of a dendritic segment within the perfusion channel and also within the soma. The mean pixel values were normalized to the initial measurement taken at the start of the recording. For the fluorescence difference images, Fluo-4 frames before vehicle and glutamate perfusions were subtracted from frames following perfusions. We used the TurboReg plugin to register the fluorescence difference images with the anti-MAP2 immunostained image. We then used the lowest pixel value calculated by comparing each fluorescence difference image with the MAP2 immunostained image, resulting in the detection of fluorescent intensity changes in MAP2-positive neurons.
