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

For nanosensor labeling, slices were transferred into a small volume brain slice incubation chamber (Scientific Systems Design, Inc., AutoMate Scientific) and kept under oxygen saturated ACSF (total 5 mL volume). 100 µL of 100 mg/L nIRCat nanosensor was added to the 5mL volume and the slice was incubated in this solution for 15 minutes. The slice was subsequently recovered and rinsed in oxygen saturated ACSF to wash off nIRCats that did not localize into the brain tissue. The rinsing step was performed by transferring the slice through 3 wells of a 24 well plate (5 seconds in each well) followed by transfer to the recording chamber with ACSF perfusion for a 15-minute equilibration period before starting the imaging experimentation. All imaging experiments were performed at 32°C. Acute slices were prepared as described previously. Extracellular dopamine concentration evoked by local electrical stimulation was monitored with FSCV at carbon-fiber microelectrodes (CFMs) using Millar voltammeter. CFMs were ~ 7 µm in diameter encased in glass capillary pulled to form a seal with the fiber and cut to final tip length of 70-120 µm. The CFM was positioned ~100 µm below the tissue surface at a 45-degree angle. A triangular waveform was applied to the CFM scanning from -0.7 V to +1.3 V and back, against Ag/AgCl reference electrode at a rate of 800 V/s. Evoked dopamine transients were sampled at 8 Hz, and data were acquired at 50 kHz using AxoScope 10.5 (Molecular Devices). Oxidation currents evoked by electrical stimulation were converted to dopamine concentration from post-experimental calibrations. Recorded FSCV signals were identified as dopamine by comparing oxidation (+0.6 V) and reduction (-0.2 V) potential peaks from experimental voltammograms with currents recorded during calibration with 2 µM dopamine dissolved in ACSF. For stimulation, a bipolar stimulation electrode (FHC CBAEC75) was positioned on top of the brain slice and approximately 100 µm away from the CFM. Following 30-minute slice equilibration in the recording chamber, dopamine release was evoked using a square pulse (0.3 mA pulse amplitude, 3 ms pulse duration) controlled by Isoflex stimulus isolator (A.M.P.I) and delivered out of phase with the voltammetric scans. Stimulation was repeated 3 times. To compare FSCV and nIRcat data, each signal was normalized against its peak value ([DA]max or (∆F/F)max) and co-aligned at stimulation time. Latency to peak were computed as tpeak -tstim where tpeak is the time at which peak signal is attained and tstim is time of stimulation. Decay time constants () were computed from model fits to a first order decay process.