Stereotactic frame

The production of AAV-BDNF-shRNA-EGFP, AAV-TrkB-shRNA-EGFP and AAV-Control-shRNA-EGFP has already been described in a previous report (Jiang et al., 2019 (link)). In this study, each mouse anesthetized with 0.5% pentobarbital sodium was fixed in a stereotactic frame (Stoelting, Wood Dale, United States). After cutting the scalp, the skull of each mouse was exposed using 75% ethanol and 1% H₂O₂. Two small drill holes were bilaterally made on the skull, and then, a 10 μl Hamilton syringe was placed at the hippocampus coordinates: AP =−2.3 mm, ML = ± 1.6 mm, DV = + 1.8 mm. AAV-BDNF-shRNA, AAV-TrkB-shRNA or AAV-Control-shRNA was bilaterally infused into the hippocampus region of each mouse using the syringe at a rate of 0.5 μl/min (1.5 μl/each side). Afterwards, 5 min of waiting was needed to prevent AAV reflux. The wound of each mouse was cleaned and sutured. A period of 2 weeks was required for the expression of AAV to be stable in the hippocampus. Furthermore, these animals were subjected to 8 weeks of chronic stress (CUMS or CRS) and another 2 weeks of oroxylin A/vehicle administration, followed by the FST, TST and SPT.
All AAVs were adjusted to 5 × 10¹² TU/ml before use. The nucleotide sequences for BDNF-shRNA, TrkB-shRNA and Control-shRNA were 5′-TGAGCGTGTGTGAC AGTATTA-3′, 5′-GCA​ACC​TGC​GGC​ACA​TAA​A-3′ and 5′-TTCTCCGAACGTGTC ACGT-3′, respectively (Jiang et al., 2019 (link)).

At 17 days post-HI surgery, HI mice were anesthetized (isoflurane 5–10 min; 5% induction and 3–4% maintenance with flow O₂: air 1:1), skin of the head was opened up and a hole was made in the skull manually with a 20 gauge needle, followed by an intracranial injection with 1 μg/2μL CXCL10 protein (300 − 12, Peprotech, London, UK) or 2μL vehicle (D-PBS, Merck KGaA, Darmstadt, Germany) into the ipsilateral hemisphere at 2 mm caudal to bregma, 2 mm right from midline and 2 mm below dural surface (Supplementary Fig. 1) using a stereotactic frame (Stoelting, Illinois, USA). Afterwards, skin was sutured and Xylocaine and Bupivacaine were applied subcutaneously for pre- and post-operative analgesia. Carprofelican (Dechra, ‘s-Hertogenbosch, The Netherlands) was administered as systemic analgesia subcutaneously immediately after and on the two consecutive days after the surgery.

For viral microinjection surgeries into adult mouse PFC (AAV8^{hSyn1- Nsun2GFP}, AAV8^hSyn1-GFP, AAV8^hSyn1-CreGFP), 10–12 week-old C57BL/6J mice were anesthetized with isoflurane and 1 μl of virus per hemisphere (bilateral injection) was injected at a rate of 0.25 μl/min using a Hamilton syringe (Reno, NV), a micropump (Stoelting) and a stereotactic frame (Stoelting). Stereotactic coordinates for injection were as follows: 1.5 mm anterior/ posterior, ±0.5 mm medial/lateral, and 1.5 mm dorsal/ventral. Control animals received 1 μl per hemisphere of AAV8^hSyn1-GFP using the same conditions.