Beuthanasia d

Rats (n = 4) received infusions of helper viruses (AAV8-CA-FLEX-RG and AAV5-EF1a-FLEX-TVAmCherry) into ACC and CAV2-Cre either unilaterally into DMS (IT-targeting) or pons (PT-targeting) as described above. After 21 days, the animals received unilateral infusions of EnvA G-deleted Rabies-eGFP into ACC. The rabies virus was allowed 7 days to express before the rats were anesthetized with Beuthanasia-D (Patterson Veterinary) and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Control animals underwent the same procedures, but did not receive the CAV2-Cre injections. Brains were removed, post-fixed overnight, and switched to PBS the next morning. Brains were sectioned in 40 µM slices using a Leica vibrating microtome. Images across the rostro-caudal axis of the brains were taken with confocal microscopy (×20; Zeiss LSM 710). eGFP-positive cells were quantified in ImageJ (v1.52s; NIH) and were assigned to brain structures based on standard anatomical landmarks using the Rat Brain Atlas.

Rats (n = 3) received an infusion of CTB conjugated to AlexaFluor 488 (CTB-488; C-34775, Thermo Fisher) unilaterally into the pons (350 nL, 70 nL/min), and an infusion of CTB conjugated to AlexaFluor 647 (CTB-647, C-34778, Thermo Fisher) unilaterally into the contralateral DMS (350 nL/140 nL/min). Twenty-one days later, rats were anesthetized with Beuthanasia-D (Patterson Veterinary) and transcardially perfused with PBS, followed by 4% PFA. Brains were extracted, fixed overnight in 4% PFA, and post-fixed in 30% sucrose. Brains were sectioned at 40 µm with a vibrating microtome. Z-stacks of the rostro-caudal axis of the ACC were collected with confocal microscopy (×20; Zeiss LSM 710) and localization of CTB-488 and CTB-647 were quantified using ImageJ (v1.52s; NIH).