Gad65

Three-µm tissue sections were incubated with primary antibodies including 1∶50 to 1∶100 dilution of GABA_A α1, β2/3 and π subunits, GABA_B receptor R1 subtype (Cell-Signaling), R2 subtype (Cell Signaling), GAD67 (Santa Cruz, GAD65 (MILLIPORE) GAT2 (MILLIPORE) or ABAT. Immuno Shot reagents (COSMO BIO) were used to enhance the immune reaction in some assays. Chromogenic immunostaining was performed with an avidin–biotin immunoperoxidase kit (Vectastain Elite kit or ABC/Peroxidase kit, Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (Sigma Fast DAB Tablets, Sigma-Aldrich). The kidneys were lightly counterstained with hematoxylin. Fluorescent immunostaining was performed using secondary antibody of Alexa Flour 568 conjugated goat anti-rabbit antibody and Alexa Flour 488 conjugated goat anti-mouse antibody (Life Technologies). GABA_A π subunit antibody (Abcam: ab26055) was used for immunofluorescence studies. Normal rabbit IgG (Santa Cruz: sc-2027) and mouse monoclonal IgG1 (Abcam: ab81032) were used as negative control. Nuclei were stained by DAPI (Life Technologies). FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) was used for examination and image acquisition.

Brain tissue preparation and immunohistochemistry were performed as described (Bormuth et al.^{28 (link)}). Primary antibodies were directed against Ank3 (1:50, mouse IgG, Santa Cruz Biotechnology), Calretinin (Calb2; 1:1000, polyclonal rabbit; Millipore), HCN1 (1:200, polyclonal rabbit; Biomol), GABA Aα1 receptor (1:500, polyclonal rabbit, Millipore), GABA Aα6 receptor (1:500, polyclonal rabbit, Millipore), GAD67 (1:1000, mouse IgG, Millipore), GAD65 (1:1000, polyclonal rabbit, Millipore), GFAP (1:200 mouse IgG, Chemicon), MAP2 (1:800, mouse IgG, Millipore), NFH (1:200, polyclonal rabbit, Sigma), Parvalbumin (1:1000, mouse IgG, Sigma), Parvalbumin (1:000, polyclonal rabbit, Swant), Pax2 (1:250, polyclonal rabbit, Zymed), PCNA (1:300, polyclonal rabbit, Abcam), PSD95 (1:400, mouse IgG, Affinity Bioreagents), S100beta (1:200 monoclonal rabbit, Abcam), VGLUT1 (1:5000, polyclonal guinea pig, Millipore). Detection was performed with secondary antibodies conjugated to Alexa Fluor 488, 555 and 633 (1:1000, Thermo Fisher Scientific) and biotinylated secondary antibodies followed by diaminobenzidine (DAB; LSAB2 Kit, Dako; Vectastain Kit, Vector Laboratories).

Brain tissue was homogenized in 10 volumes of cold sucrose buffer (0.32 M sucrose, 25 mM HEPES, pH7.4). After a brief centrifugation at 3000×g for 5 min at 4 °C, the supernatant was collected and centrifuged at 10,000×g for 12 min at 4 °C. The outer three-fourths of the pellet was collected and re-suspended using the same sucrose buffer by gentle pipetting, while the dark center containing mitochondria was avoided. After a second centrifugation at 10,000×g for 12 min at 4 °C, the pellet without a dark center was collected in cold HBS (25 mM HEPES, pH 7.4, 150 mM NaCl) as the synaptosome-enriched brain lysate and used for western blot experiments^{51 (link)}. Primary antibodies for immunoblotting included: VGLUT1 (1:1000, guinea pig, SYSY), VGLUT2 (1:1000, rabbit, SYSY), GAD65 (1:1000, rabbit, Millipore), synaptophysin (1:1000, mouse, Millipore), MEF2C (1:500, rabbit, Proteintech), α-tubulin (1:20,000, mouse, Sigma), and β-actin (1:10,000, mouse, Sigma) and followed by appropriate secondary antibodies^{52 (link)}. Note that GAD65 was used instead of VGAT for immunoblotting because the former antibody proved superior forwestern blots. The immunosignals were captured on Kodak x-ray film and quantified using ImageJ version 1.45s (http://rsb.info.nih.gov/ij/). All uncropped western blots can be found in Supplementary Fig. 12.