Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor

Abraham G. Beyene; Kristen Delevich; Jackson Travis Del Bonis-O’Donnell; David J. Piekarski; Wan Chen Lin; A. Wren Thomas; Sarah J. Yang; Polina Kosillo; Darwin Yang; Linda Wilbrecht; Markita P. Landry

Nanosensor ΔF/F imaging of the same acute brain slice field of view upon electrical stimulation by 0.3 mA in standard ACSF (top row) and in ACSF plus 10 µM nomifensine (bottom, +Nomifensine). Three still frames are presented from each movie. "Pre" is a representative frame before electrical stimulation is applied, "Stim" represents frame corresponding to peak ∆F/F following stimulation, and "Post" is a representative frame after nIRCat fluorescence has returned to baseline. Scale bars = 10 µm. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/356543 doi: bioRxiv preprint entire field of view, overlaid with ROIs identified from an automated program from per-pixel ∆F/F stack projections of nIRCat fluorescence modulation (see Methods). Color bar represents nIRCat fluorescence intensity of labeled tissue. Scale bar = 20 µm. (e) Frequency histogram of ROI sizes depicted in (d), exhibiting a log-normal distribution with median ROI size of 2 µm. (f) ROI size distribution from three different fields of view (representing n = 3 biological replicates) in the dorsal striatum, each stimulated separately by 0.3 mA stimulation. In each of these fields of view, ROIs showed similar median size and size distribution even when compared across biological replicates. Box plot definitions: red-line = median, edges of box: 25 th and 75 th percentiles, top and bottom lines: minimum and maximum values of non-outlier data, red points: outlier data (g) A higher magnification view of an ROI with an effective radius of 5 µm. Maximum ∆F/F projection of the ROI shows presence of smaller fluorescence hotspots within the ROI. Scale bar = 5 µm. (h) Overlay of representative normalized FSCV (gray) and nIRCat (blue) traces showing that nIRCat ROI signals exhibit a wider diversity of decay kinetics. Inset: An example of nIRCat experimental data (blue dots) fitted to first order kinetics (red line) to compute decay time constants (). To identify nIRCat fluorescence change hotspots (i.e., regions of high ∆F/F), we analyzed our video-rate acquisitions using a custom-built program that accounted for background fluorescence and identified regions with fluctuations in fluorescence intensity in the poststimulation epoch (see Methods for details). We defined nIRCat ∆F/F hotspots as regions of interest (ROIs) based on a per-pixel stack projection of maximal ∆F/F in the imaging time series. The ROIs identified were found to vary in size from 1 µm to 15 µm ( Figure 3d , 3e). We found that ∆F/F hotspots do not necessarily correspond to high nIRCat labeling of the brain tissue, suggesting that the hotspot is a consequence of variation in dopamine release and not nanosensor loading in the tissue (Figure 3d , Figure S3 a-c). Using data from single pulse electrical stimulation experiments, the program identified ROIs whose size distribution was observed to exhibit a log-normal distribution with median ROI size of 2 µm (Figure 3e ). Repeat stimulations with the same stimulation amplitude in fields of view of the dorsomedial striatum across biological replicates generated similar size distributions (Figure 3f ). Closer examination of several larger ROIs (> 5µm) suggested these may be comprised of smaller hot-spots in close proximity (Figure 3g , Figure S3 d-e).