Animals
Wild-type and VGAT-tdTomato mice of C57BL/6 backgrounds [18] were used in this study. All procedures for the care and treatment of animals were performed according to the Japanese Act on the Welfare and Management of Animals and the Guidelines for Proper Conduct of Animal Experiments issued by the Science Council of Japan. The experimental protocol was approved by the Institutional Committee of Gunma University (No. 18–019; 19–028). All efforts were made to minimize suffering and reduce the number of animals that were used.
Viral Vectors
We used BBB-penetrating AAV-PHP.B [19] except for Fig. 1, which used AAV-PHP.eB [20]. The expression plasmids pAAV, comprising a polyadenylation signal sequence and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), were designed to express GFP under the control of the mouse glutamic acid decarboxylase 65 (mGAD65) promoter or the mouse Dlx gene enhancer combined with a minimal promoter (mDlx enhancer, hereafter) [16]. The mGAD65 promoter and mDlx enhancer were inserted into the pAAV plasmid upstream of the GFP gene at restriction enzyme sites for XhoI and AgeI. pAAV-PHP.B and pAAV-PHP.eB were constructed from the pAAV2/9 plasmid [19], which was provided by Dr. James M. Wilson at the University of Pennsylvania. Recombinant single-strand AAV-PHPB/PHP.eB vectors were produced by co-transfection into HEK293T cells (HCL4517; Thermo Fisher Scientific; Waltham, MA, USA) with 3 plasmids: the expression plasmid, pHelper (Stratagene, La Jolla, CA, USA), and a packaging plasmid (pAAV-PHP.B or pAAV-PHP.eB), as described previously [13, 21]. Briefly, HEK293T cells, which were cultured in Dulbecco’s Modified Eagle Medium (D-MEM; D5796-500Ml, Sigma-Aldrich, St Louis, MO, USA) supplemented with 8% fetal bovine serum (Sigma-Aldrich), were transfected with three plasmids: pAAV/[mDlx enhancer or mGAD65]-EGFP-WPRE-SV40 or pAAV/mDlx enhancer-tdTomato-WPRE-SV40, pHelper (Stratagene, La Jolla, CA, USA), and pAAV-PHP.B/PHP.eB using polyethylenimine. Viral particles were harvested from the culture medium 6 days after transfection and concentrated by precipitation with 8% polyethylene glycol 8000 (Sigma-Aldrich) and 500 mM sodium chloride. The precipitated AAV–PHP.B/PHP.eB particles were resuspended in D-PBS and purified with iodixanol (OptiPrep; Axis-Shield Diagnostics, Dundee, Scotland) continuous gradient centrifugation. The viral solution was further concentrated in D-PBS using Vivaspin 20 (100,000 MWCO PES, Sartorius, Gottingen, Germany). The genomic titers of the viral vector were determined by real-time quantitative PCR with the THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) using the 5’- CTGTTGGGCACTGACAATTC-3’ and 5’-GAAGGGACGTAGCAGAAGGA-3’ primers, which targeted the WPRE sequence. The expression plasmid was used as the standard.
Intravenous injection
Eight to fourteen week-old mice were used in this study. After inducing deep anesthesia via the intraperitoneal injection of ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW), 100 µL of AAV-PHP.B (5.0 × 1013 vg/ml) or AAV-PHP.eB (3.0 × 1013 vg/ml) was injected into the orbital sinus using a 0.5 ml syringe with a 30-gauge needle (08277; Nipro, Osaka, Japan) for 30 to 40 seconds. For Fig. 6, equal titers (5.0 × 1012 vg) of AAV-PHP.B-expressing tdTomato under the control of the mDlx enhancer and AAV-PHP.B expressing GFP under the control of the mGAD65 promoter were mixed in advance and injected as described above.
Direct cerebellar injection
The AAV-PHP.B vector was directly injected into the cerebellar tissue. After inducing deep anesthesia, the mice were placed in a stereotactic frame. The skin covering the occipital bone was cut, and a burr hole was made 7 mm caudal from the bregma. The tip of a Hamilton syringe (33 gauge) with an attached micropump (UltraMicroPump II; World Precision Instrument (WPI) Sarasota, FL, USA) was inserted 1.8 mm below the pia mater of the cerebellar vermis. Ten microliters of viral solution (1 × 1013 vg/ml) was injected at a rate of 400 nl/min using a microprocessor-based controller (Micro4; WPI).
Immunohistochemistry
Depending on the antibodies used, we employed different protocols for immunohistochemistry (see Supplementary Table for details). Three weeks after injection with AAV-PHP.B vectors, the mice were deeply anesthetized and perfused intracardially with 4% paraformaldehyde phosphate buffer (pH 7.4). Their brains were removed and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer at 4 °C. Floating (50 µm thick) and cryostat (20 µm thick) brain sections were prepared using a vibratome (VT1000S, Leica, Wetzlar, Germany) and cryostat (CMS3050S, Leica), respectively. The slices were permeabilized, blocked with an appropriate blocking solution, and treated in blocking solution containing the following antibodies: mouse monoclonal anti-CaMKII (1:100; 05-532; Merck, Germany), rat monoclonal anti-GFP (1:1000; 04404-84; Nacalai, Kyoto, Japan), goat polyclonal anti-PV (1:200; PV-Go-Af460; Frontier Institute, Hokkaido, Japan), rat monoclonal anti-SST (1:100; MAB354; Merck, Germany), goat polyclonal anti-Ankyrin G (P-20) (1:50, SC-31778, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-calbindin D-28 k (1:500; Swant, Bellinzona, Switzerland), or mouse monoclonal anti-mGluR2 (1:1000; ab15672; Abcam, Cambridge, UK). After rinsing several times with PBS or PBS containing Triton X-100 at room temperature (24–26 °C), the slices were incubated with relevant secondary antibodies (Thermo Fisher Scientific) (see Supplementary Table). They were mounted on glass slides after rinsing several times with PBS or PBS containing Triton X-100 at room temperature (24–26 °C). As for mounting medium, we used Prolong Gold/Diamond Antifade Reagent (Thermo Fisher Scientific) and CC/Mountant antifade reagent (Diagnostic BioSystems, Pleasanton, CA, USA) for floating and cryostat sections, respectively.
Confocal microscopy
Most of the fluorescent images of the brain sections were acquired using a laser-scanning confocal microscope (LSM 800, Carl Zeiss, Oberkochen, Germany) with 20 × or 40 × objectives, and z-stack images of different focal planes were generated. The acquired images were combined using the ImageJ software with the MosaicJ plugin [22].
Relative GFP intensity in the whole brain
Bright-field and native GFP fluorescence images of the whole brain were acquired using a fluorescence stereoscopic microscope (VB-700; Keyence, Osaka, Japan). To measure the GFP fluorescence intensity of the forebrain and hindbrain, the margin of the corresponding part of the brain was traced on the bright-field image except for the olfactory bulb, superior and inferior colliculi, and brainstem. GFP fluorescence intensity within the enclosed area was measured using ImageJ (Fiji). Autofluorescence of the brain was measured in 6 non-injected VGAT-tdTomato mice, and the averaged value was subtracted from the GFP fluorescence values of all the samples. Finally, data were normalized to the mean value (100) from the forebrains of the VGAT-tdTomato mice that received AAV-PHP.B expressing GFP under the control of the mDlx enhancer.
Specificity and efficiency of promoters
To determine the specificity and efficiency of the mGAD65 promoter and mDlx enhancer, GFP and tdTomato fluorescent images were obtained from sagittal brain slices (50 µm thickness) using a fluorescent microscope (BZ-X700; Keyence) with a 20 × objective. The number of GFP (+) and/or tdTomato (+) cells were counted manually.
Proportion of interneuron subtypes in cortical GABAergic interneurons
To measure the ratio of the PV (+) or SST (+) cells to transduced GABAergic interneurons in the cerebral cortex, the wild-type mice were intravenously infused with AAV-PHP.B expressing GFP under the control the mGAD65 promoter. Three weeks after the viral injection, sagittal brain slices (50 µm thickness) were immunolabeled for PV and SST. Fluorescent images were obtained using confocal microscopy with a 40 × objective. Then, the ratio of PV (+) cells or SST (+) cells to total GFP (+) cells was calculated.
Statistical analysis
We used GraphPad PRISM version 7 (GraphPad Software, San Diego, CA, USA) for the statistical analysis and production of graphic images.
Statistical methods were shown in the text and/or each figure legend. The data were expressed as the mean ± standard error of the mean.