Calretinin

To evaluate synaptic vesicles, retinal expression of synaptophysin was assessed. For fluorescence staining, samples were pre-embedded in 3% agar in deionized water. Vibratome sections (50 μm) were collected and washed several times in PBS. Sections were incubated in 10% normal donkey serum in PBS for 1 h at room temperature to block nonspecific binding activity, then with anti-rabbit synaptophysin (Cell signaling, Danvers, MA, USA) overnight at 4°C. After several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546 (Molecular Probes). For double-labeling studies, sections were incubated with anti-mouse PKCα (Santa Cruz Biotechnology, Santa Cruz, CA), parvalbumin (Sigma, St. Louis, MO), calretinin (Millipore, Temecula, CA, USA), or SMI-32 (Covance, Emeryville, CA, USA) in 0.1 M PBS containing 0.5% Triton X-100 overnight at 4°C, rinsed for 30 min with 0.1 M PBS, and incubated with goat anti-mouse Alexa 488 (Molecular Probes) for 1 h 30 min at room temperature. After further washes in 0.1 M PB for 30 min, the sections were mounted using VectaShield Mounting Medium with DAPI (Vector Laboratories, H-1200). Slides were washed, covered with coverslips, and examined by confocal laser scanning microscopy (Zeiss LSM 510; Carl Zeiss Co. Ltd.).

Protein lysates were prepared from olfactory bulbs and hippocampi of 5 months old WT and KO female mice using RIPA buffer with protease inhibitors followed by sonication by ultrasound and clearing via centrifugation at maximum speed using Eppendorf tabletop centrifuge. Protein concentration was quantified using BCA reagent (Thermo Fisher Scientific, Rockford, IL). Protein extracts (40 μg/well) were subjected to Western immunoblot analysis. We used antibodies against the following proteins: PRDX1 (Sigma), ACC1, pACC1, AMPKα, pAMPKα, NGFR, TrkB, Bcl-xL, Rack1 (all from Cell Signaling Technology), Calretinin (Millipore), LC3 (Abcam). The band intensity was determined using ImageJ software, normalized to loading control (β-actin). The data are presented as a relative intensity of WT bands over KO bands. Phospho-index was determined as a ratio of phosphorylated/total protein intensities.

The midbrain was dissected from the entire brain and stored at − 80 °C until the day of the experiment. Tissue was homogenized in RIPA buffer containing (in mM) 50 Tris-HCl pH 7.5, 150 NaCl, 5 MgCl₂, 1 EDTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, 1 sodium orthovanadate, 5 b-glycerophosphate, 5 NaF and protease inhibitor cocktail, and incubated on ice for 30 min [50 (link), 83 (link)]. The samples were centrifuged at 15,000 g for 20 min and the protein concentration of the supernatant was determined by the Bradford method.
Proteins were applied to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. Blotting analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using ImageJ.
Primary antibodies: Calbindin-D28K (1:200, Sigma Aldrich; #C9849; RRID: AB_476894); Calretinin (1:200, Millipore; #MAB1568; RRID: AB_94259); Actin (1:10000; Sigma Aldrich; #A5060; RRID: AB_476738).
Secondary antibodies: goat anti-mouse IgG (1:3000; Bio-Rad; #1706516; RRID: AB_11125547), goat anti-rabbit IgG (1:3000; Bio-Rad; #1706515; RRID: AB_2617112).
Membranes were stripped using Re-Blot Plus Strong Solution (Millipore) for 15 min at room temperature.
For each age, both genotypes were analyzed simultaneously. The different ages were analyzed in different blots.

Mice were anesthetized with IP ketamine/xylazine (100/10 mg/kg) and perfused with 4% (w/v) PFA freshly prepared in phosphate-buffered saline (PBS, 0.9% NaCl in 0.1 M PB). As described previously^{11 (link)}, vestibular labyrinths were extracted from the temporal bone of three C57BL/6 mice and immunohistochemistry was performed on microdissected vestibular organs with antibodies to ChAT (1:100, Millipore) and calretinin (1:1000; Millipore). Anti-ChAT was used to label vestibular efferent fibres and varicosities while anti-calretinin was used primarily to counterstain hair cells in the otolithic macula and calyx afferents in the crista central zone^{11 (link)}. Whole mount projections were acquired either with a Zeiss Axio Imager motorized upright multifluorescent microscope fitted with an Apotome slider system (Zeiss Imaging Systems, Oberkochen, Germany) or Olympus FV1000 Laser Scanning Confocal microscope. Images were postprocessed using Axiovision, Olympus FV1000, and/or Adobe Photoshop CS6 (Adobe Systems, San Jose, CA) software to adjust contrast and brightness within the linear range.

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).