Raw movie files were processed using a custom-built MATLAB program (https://github.com/jtdbod/Nanosensor-Brain-Imaging). Briefly, for each raw movie stack (600 frames), a per-pixel ∆F/F defined as (F − F0)/F0 was calculated using the average intensity for the first 5% of frames as F0, and F represents the dynamic fluorescence intensity at each pixel. ROIs were identified by calculating a median filter convolution and then performing thresholding using Otsu’s method to identify ROIs with strong fluorescence modulation over background, followed by a morphological dilation operation. ∆F/F traces were then calculated for each generated ROI by averaging pixel values over the ROI. ROI sizes were computed using the measured pixel area and by approximating each as a circle to calculate an equivalent radius.

To compare responses across stimulation amplitudes and bath application of nomifensine, mean results were obtained as follows: First, all identified ROIs from a field of imaging were averaged. Mean traces were further averaged over different fields of view within the same slice and across slices (1 to 2 fields of view per slice and 1 to 2 slices per animal) and then averaged over experimental animals. Decay time constants (τ) were computed by fitting ∆F/F time traces to a first-order decay process on an ROI basis or field-of-view average basis. Latency to peak was computed as tpeak − tstim, where tpeak is the time at which peak signal is attained and tstim is time of stimulation. All statistical tests of significance (P values) were computed and reported from unpaired two-tailed t test.

ROI-level analysis of drug washes were computed by generating an ROI mask from one of the triplicate stimulations and then computing ∆F/F traces using the same ROI mask for all experimental runs (before and after application of drug). Each raw movie file was corrected for translational and rotational drift using StackReg plugin in FIJI and then processed using our custom MATLAB script. We removed all ROIs that were greater than 4 μm in size for subsequent analysis. For each ROI, post-to-pre drug ratios were computed as (∆F/F)max-post-drug/(∆F/F)max-pre-drug. Here, we define (∆F/F)max as the amplitude of nIRCat response in the post-stimulation epoch. Mean values from triplicate stimulation were used to evaluate the ratio.

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