Optogenetic Strategy

For optical activation or silencing of presumed glutamatergic BLA axon terminals, a viral vector (rAAV5–CaMKII–hChR2(H134R)–eYFP or rAAV5–CaMKII–eNpHR_3.0–eYFP) (1µL, 10¹² units/100 µL) was injected into the BLA using a 5 µL Hamilton syringe (33 gauge) to express ChR2 or eNpHR_3.0 in BLA terminals of SD rats. For control experiments, rAAV5–CaMKII–eYFP was used. The coordinates for the injections into BLA were as follows: 2.3 mm caudal to bregma, 4.5–4.8 mm lateral to midline, and 8.0–8.5 mm deep. For optical activation or silencing of CRF neurons, a viral vector (rAAV5/EF1a–DIO–hChR2–eYFP or rAAV5/EF1a–DIO–eNpHR_3.0–eYFP; 1 μL, 10¹² units/100 µL) was injected into the CeA using a 5 µL Hamilton syringe to express ChR2 or eNpHR_3.0 in CRF neurons of transgenic Wistar rats. For control experiments, viral vectors were injected in wild type rats. The coordinates for the injections into CeA were as follows: 2.5 mm caudal to bregma, 4.0–4.3 mm lateral to midline, and 7.3–7.6 mm deep. After injection, we waited 10 min for the virus to diffuse into the tissue before retracting the injection needle. All viral vectors were purchased from the vector core facility at the University of North Carolina, Chapel Hill, NC, aliquoted upon arrival, stored at –80°C and thawed before use. To activate light sensitive molecules with blue (473 nm) or yellow (590 nm) light, we used a head-mounted wireless system delivering LED light pulses (20 Hz, 5 mW, 5–10 min; Teleopto, Amuza, San Diego, CA, United States) through an optical fiber (200 μm diameter) stereotaxically implanted into the CeA two days before testing, using a small drill hole made in the anesthetized rat (isoflurane, 2–3%, precision vaporizer). Implanted fibers were held in place with dental acrylic as previously described (Thompson et al., 2015; Kiritoshi et al., 2016; Kim et al., 2017; Mazzitelli and Neugebauer, 2019; Ji and Neugebauer, 2020). Prior to surgery, function of each optic fiber was tested. The optical stimulation started 5 min before the evaluation of the electrophysiological and behavioral responses and continued during testing period (5–10 min). Each test stimulus (In vivo spinal cord electrophysiology and In vivo spinal cord electrophysiology) was applied only once.