Optogenetic Monitoring of Norepinephrine Release

Carbon fiber microelectrodes (CFME, 75 μm in length) were lowered into 300 μM coronal slices and placed where the densest ChR2-eYFP expression was observed (CeA/BLA border) Th^IRES-Cre::LC-BLA:ChR2 mice. To detect NE, the CFME was ramped from −0.4 V to 1.3 V versus a Ag/AgCl reference electrode (in the bath) at a rate of 400 V/s at 10 Hz. Slices were stimulated with 30 pulses of a blue LED (Thorlabs, 473 nm, 5 ms pulse width, 1 mW) via a 40X objective at 10 Hz every 5 min to release NE. Electrochemical data was collected and analyzed using a combination of Tar Heel CV (ESA, Chelmsford, MA) (Robinson and Wightman, 2007 ), HDCV (http://www.chem.unc.edu/facilities/electronics_software.html) (Bucher et al., 2013 (link)), and Labview (RRID:SCR_014325). Following collection, background subtracted cyclic voltammograms (CVs) were smoothed one time with a Fast Fourier Transformation (Bucher et al., 2013 (link)). CVs had characteristic oxidation and reduction peaks coinciding with catecholamine detection (ox: 600–700 mV red: −200–300 mV). Oxidative currents were analyzed at the peak of the oxidative potential for individual experiments. Clearance half-life (t1/2) was measured in Clampfit 10.2 (Molecular devices) as previously described (McElligott et al., 2013 (link)).