Hamilton syringe

For knockdown of PLCβ1 in the mPFC, we injected the adeno-associated virus (AAV)-shPLCβ1-mCherry virus in WT mice. The AAV-shPLCβ1 virus was created as described previously (Kim et al., 2015 (link)). The AAV-shPLCβ1 virus expresses a small hairpin RNA (shRNA) targeting PLC-β1 messenger RNA (mRNA) (target sequence 5′-CCTCCAGTGAGGAGA-TAGAAA-3′). The scrambled shRNA (shSCR) sequence, 5′-AATCG-CATAGCGTATGCCGTT-3′, was used to construct a non-targeting control virus. Both the viruses were created at the KIST Virus Facility. After anesthetizing WT mice with 2% avertin (tribromoethyl alcohol/tertiary amyl alcohol; Aldrich, MA, United States), 0.5 μl of a high-titer AAV preparation (10¹³ GC/ml) was bilaterally (or unilaterally) injected into the mPFC (+1.6 anterior-posterior, ±0.3 mediolateral, and -1.80 dorsoventral) at a rate of 0.05 μl/min by using a Hamilton syringe connected to a microinjection pump (KD Scientific, MA, United States) (Kim et al., 2015 (link)). The methods of histology for PLCβ1 expression in the mPFC and quantitative real-time PCR are included in Supplementary Material.

Knockdown mTMC6 gene of WT mice and re-express TMC6 of TMC6-KO mice in the L4 DRG were achieved by affecting the DRG with AAV9 virus, and were produced from Genechem Co., Ltd (Shanghai, China). The mTMC6 DNA sequence referred to NCBI (GenBank: NM_145439). Vectors construction was GV466 (hSyn promoter-MCS-EGFP-3FLAG-SV40 PolyA). AAV9-mTMC6-cDNA (2.29 × 10¹³ v. g./mL), AAV9-control 323 (1.8 × 10¹³ v. g./mL); AAV9-mTMC6-shRNA (2.33 × 10¹³ v. g./mL), target sequence: CCT​GCA​TCA​TTC​TGG​TAT​A, AAV9-control 533 (1.08 × 10¹³ v. g./mL). Diluted virus to 5 × 10¹² v. g./mL before using. Mice were anesthetized with 0.2 mL 1% Pentobarbital dissolved in saline solution. The above-mentioned AAV9 virus were injected into the DRG (right L4). Mice were anaesthetized with 1% pentobarbital sodium and ganglions were exposed surgically. Viral solution was diluted with equal volume PBS and injected into the exposed DRG at a rate of 0.2 mL/min (2 mL per DRG) with a glass micropipette connected to a Hamilton syringe controlled by a microsyringe pump controller (78–8,130, KD Scientific, United States). The glass micropipette was removed after 5 min, then the skin was sutured, the animal was transferred to a recovery cage.

Mice (6–7 weeks old) were anaesthetized with an Avertin^® (2,2,2-tribromethanol in 2-methyl 2-butanol) and placed in a stereotaxic frame. Briefly, the scalp was opened, and two holes were drilled in the skull (−1.8 mm AP from bregma, ± 1.6 mm ML). AAV-EF1α-mCherry and AAV-EF1α-mCherry-IRES-Cre (AAV5) were purchased from UNC Vector Core. AAV-hSyn-BFP and AAV-hSyn-BFP-Cre were packaged with serotype DJ at KIST Virus Facility. These viruses were bilaterally injected (250 nL per side) into the dentate gyrus area (2.1 mm DV from the dura) through a Hamilton Syringe with a syringe pump (KD Scientific, Holliston, MA, USA) that infused the virus at a speed of 0.1 µL/min. At the injected points, Hamilton Syringe was left in place for 10 min. After injection, the syringe stayed in target place for an additional 10 min.

Mice were anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The skull was exposed, and holes were drilled in the skull bilaterally above dHC. A Hamilton syringe with a 32-gauge needle mounted on a nanopump (KD Scientific, Holliston, MA) was stereotactically inserted into dHC (1.7 mm posterior to bregma, 1.5 mm lateral from midline, and 1.55 mm ventral from dura). AAV-DJ-hSyn-Cre-GFP or AAV-DJ-GFP (5.8 × 10¹² genomic copies/mL, 1 µL per side; Gene Vector and Virus Core, Stanford University) was microinjected at a rate of 0.2 µL/min. The needle was left in place for an additional 5 min following microinjection to ensure complete diffusion of the AAV, and then slowly retracted. The scalp was sutured, and meloxicam (3 mg/kg, s.c.) was administered as an analgesic treatment. Mice were returned to their home cage for 2 weeks to recover from the surgery and to allow viral expression.