Stereotaxic frame

Mice were anesthetized with 2.5% isoflurane and 80% O₂ and head-fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) equipped with digital manipulator arms (Stoelting Co, Wood Dale, IL). A nose cone was used to deliver isoflurane to maintain anesthesia. Mice were given a subcutaneous injection of meloxicam (2 mg/kg) and 0.1 mL of 0.25% Marcaine around the incision site. After exposing the skill, craniotomies were made with an electric drill (Stoelting CO, Wood Dale, IL) with a ball bur attached. A Neuros 7000 series 1 µL Hamilton syringe with a 33-gauge needle (Reno, NV) connected to a remote automated microinfusion pump (KD Scientific, Holliston, MA) was used for delivery of tracers or viral constructs at a rate of 100 or 50 nL/min, respectively. Details of dye, viruses, and stereotaxic coordinates are provided in Supplementary Table 4. Following infusion, the needle was left in place for 5 min and then slowly manually retracted. Mice were placed on a heat pad for at least 30 min post-surgery before being returned to their home cage. Mice were monitored for three days post-surgery to ensure recovery. Brains were collected and imaged (see below) to confirm viral expression and injection placements and no misplacements were found.

AAV constructs were obtained from Vector Biolabs (Malvern, PA). All the constructs carried the enhanced GFP reporter under the cytomegalovirus promoter and the shRNA sequence (against 5αR1, 5αR2, or scrambled) under the U6 promoter. Constructs were packaged into the AAV5 capsid, with a titer of 1.5 × 10¹² GC/ml for 5αR1, 2.1 × 10¹² GC/ml for 5αR2, and 1.2 × 10¹² GC/ml for scrambled.
A mix of three different sequences was used to efficiently down-regulated the expression of the target of interest, specifically 5αR1: 5′-ACCGCTA-TGTACAGAGCAGATACTCTCGAGAGTATCTGCTCTGTACATA-GCTTTTT-3′, 5′-ACCGCACCATCAGTGGTACCATGACTCGAGTCA-TGGTACCACTGATGGTGCTTTTT-3′, and 5′-ACCGGGAAACTGGA-TACAAGATACCTCGAGGTATCTTGTATCCAGTTTCCC-TTTTT-3′; and 5αR2: 5′-ACCGTACTTCCACAGGACATTTATTCTC- GAGAATAAATGTCCTGTGGAAGTATTTTT-3′, 5′-ACCGGTACACAG-ATGTGCGGTTTACTCGAGTAAACCGCACATCTGTGTACCTTTT-T-3′, and 5′-ACCGCAGGAGTTGCCTTCCTTTGTCTCGAGACAA-AGGAAGGCAACTCCTGCTTTTT-3′.
Long-Evans rats were anesthetized with xylazine/ketamine (20/80 mg/kg, ip) and then placed onto a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Blunt ear bars were used to avoid damage to the tympanic membrane. Under aseptic conditions, rats were shaved, and their scalp was retracted. Bilateral craniotomies were made above the target site and a 5-μl Hamilton Neuros syringe (Reno, NV) was positioned above bregma. The target locations for brain infusions were as follows: mPFC: anteroposterior (AP) + 3.0 mm, mediolateral (ML) ± 0.5 mm, dorsoventral (DV) = −3.0 mm from the dura mater; NAc: AP + 1.7 mm, ML ± 0.8 mm, DV = −7.8 mm from the dura mater. Coordinates were taken from bregma, according to the stereotaxic brain atlas (38 ). The needle was slowly lowered into the injection site, and 1 μl of AAV was infused into one hemisphere. The syringe was left in place for at least 5 min after completion of the infusion to eliminate backflow and then slowly withdrawn. This process was repeated on the other side to produce bilateral viral injections. After completing injections into both hemispheres, rats were placed back on a warming pad until the body temperature returned to normal and the recovery of normal movement. Rats were given antibiotic therapy for 2 days (enrofloxacin, Bayer HealthCare, Shawnee Mission, KS) and were allowed to recover in their home cages (single-housed) with food and water available. Behavioral testing started 14 days after surgery.

Animals were placed in a stereotaxic frame (David Kopf Instruments; Tujunga, CA, USA) under ketamine/xylazine anesthesia. The ARC and the VMH were targeted bilaterally using a 25-gauge needle (Hamilton; Reno, NV, USA). The injections were directed to the following stereotaxic coordinates: a) for the VMH of rats: 2.8 mm and 3.2 mm posterior to the bregma, ± 0.6 mm lateral to midline and 10.1 mm deep; b) for the VMH of mice: 1.7 mm posterior to the bregma, ± 0.5 mm lateral to midline and 5.5 mm deep c) for the ARC of rats 2.8 mm posterior, ± 0.3 mm lateral to bregma and 10.2 mm deep. For acute treatments, T3 (Sigma-Aldrich, St Louis, MO, USA; 16 ng in 1 μL of 1:50 saline:DMSO; during 12 hr) or vehicle (100 nL of 1:50 saline:DMSO) were given. For chronic nuclei-specific treatments, T3 was given at 4 ng/day (in saline + 1 mM NaOH) and vehicle (saline + 1 mM NaOH) used as control. The selection of these doses was based on previous reports (López et al., 2010 (link), Varela et al., 2012 (link), Martínez-Sánchez et al., 2017 (link)). Nuclei-specific injections were delivered via a permanent 28-gauge stainless steel cannula (Plastics One, Roanoke, VA, USA) inserted bilaterally either in the VMH or ARC. A catheter tube was connected from each infusion cannula to an osmotic minipump flow moderator (Model 1007D; Alzet Osmotic Pumps, Cupertino, CA, USA). These pumps had a flow rate of 0.5 μL/hour during 7 or 28 days of treatment. The osmotic minipumps were inserted in a subcutaneous pocket on the dorsal surface created using blunt dissection and the treatment was given for 7 or 28 days (Imbernon et al., 2013 (link), Contreras et al., 2014 (link)).
Adenoviral (GFP, AMPKα-DN, AMPKα-CA, GRP78-DN, TR-DN and SPTLC1-2; Viraquest; North Liberty, IA, USA and SignaGen; Rockville, MD, USA) or adeno-associated (Cre; SignaGen; Rockville, MD, USA) vectors were delivered in the VMH of rats or mice using the aforementioned coordinates at a rate of 200 nl/min for 5 min for rat and 10 min for mouse (1 μl/injection site) as previously reported (López et al., 2010 (link), Varela et al., 2012 (link), Martínez de Morentin et al., 2014 (link), Contreras et al., 2014 (link)). Animals were treated for 5-7 days (adenovirus) or 30 days (Cre).