Medetomidine

The following mouse strains were used: GENSAT ChAT-GFP Tg(Chat-EGFP)GH293Gsat/Mmcd (#000296-UCD) and Mobp-GFP Tg(Mobp-EGFP)103Gsat/Mmcd (#030483-UCD)^{5 (link)} (MMRRC; www.mmrrc.org); GFPM^{20 (link)}; SST-ires-Cre::Ai9^{6 (link)}; and wild type C57BL/6J (Jax mice; # 000664). As anatomical tracers, we used CTB Alexa Fluor-488 (Invitrogen; #C22841; 0.5 % wt/vol in phosphate buffer) and AAV-GFP with synapsin promoter^{21 (link), 22 (link)}. AAV was produced at the Salk vector core (http://vectorcore.salk.edu) as a chimeric 1/2 serotype^{23 (link)}, purified by iodoxinal gradient and concentrated to 5.3 × 10¹¹ genomic copy per ml. Stereotaxic injections of the tracers were done as described^{24 (link)}. Briefly, the mice were anaesthetized by 1% isoflurane inhalation. A small craniotomy (approximately 300 × 300 μm) was opened over the left primary somatosensory cortex and ~50 nl of virus or 50 nl of 0.05 % CTB Alexa FluorR 488 was injected into layer 2/3 barrel cortex at stereotaxic coordinates: caudal 1.6, lateral 3.2, ventral 0.3 mm relative to bregma. The skin incision was then closed with silk sutures, and the mice were allowed to recover with free access to food and water (meloxicam was given at 1 mg/kg, s.c. for analgesia). The brains were prepared for imaging 10–14 days later (see below).
The mouse brains were prepared for STP tomography as follows. The mice were deeply anesthetized by intraperitoneal (i.p.) injection of the mixture of ketamine (60 mg/kg) and medetomidine (0.5 mg/kg) and transcardially perfused with ~15 ml cold saline (0.9 % NaCl) followed by ~30 ml cold neutral buffered formaldehyde (NBF, 4% w/v in phosphate buffer, pH 7.4). The brains were dissected out and post-fixed in 4% NBF overnight at 4 °C. In order to decrease formaldehyde-induced autofluorescence, the brains were incubated in 0.1 M glycine (adjusted to pH 7. 4 with 1M Tris base) at 4 °C for 2–5 days. The brains were then washed in phosphate buffer (PB) and embedded in 3–5% oxidized agarose as described^{25 , 26 (link)}. Briefly, agarose (Sigma, cat.# A6013) was oxidized by stirring in 10 mM sodium periodate (NaIO₄, Sigma cat.# S1878) solution for 2 hrs at RT, washed 3x and re-suspended in PB to bring the final concentration to 3–5 %. The mouse brain was pat-dried and embedded in melted oxidized agarose using a cube-shaped mold. Covalent crosslinking between brain surface and agarose was activated by equilibrating in excess of 0.5–1 % sodium borohydrate (NaBH_4, Sigma cat.# 452882) in 0.05 M sodium borate buffer (pH = 9.0–9.5), gently shaking for 2–4 hrs at RT (or overnight at 4 °C) (note that after rinsing, activated agarose can be stored in PB at 4 °C for up to one week; sodium borohydrate buffer should be prepared fresh). Covalent crosslinking of the agar-brain interface is important to keep the brain firmly embedded during sectioning and to limit shadowing artifacts by insufficiently cut meninges (see Troubleshooting, below).