After incubation with the first antibody, sections were washed with 1× PBS three times for 20 min each, followed by incubation with Alexa 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) for 2–4 hr at room temperature and then washed with 1× PBS. see more Sections were transferred onto slides, mounted with 0.1% paraphenylinediamine in 90% glycerol/PBS, and imaged with a microscope (BX61, Olympus). Acute

slices were prepared according to published procedures (Peça et al., 2011). Briefly, mice were anesthetized with Avertin solution (20 mg/ml, 0.5 mg/g body weight) and perfused through the heart with 20 ml of ice-cold oxygenated (95% O2, 5% CO2) cutting solution containing 105 mM NMDG, 105 mM HCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 26 mM NaHCO3, 25 mM glucose, 10 mM MgSO4, 0.5 mM CaCl2, 5 mM L-ascorbic acid, 3 mM sodium tyruvate, and 2 mM thiourea (pH was 7.4, with osmolarity of 295–305 mOsm). The brains were rapidly removed and placed in ice-cold oxygenated cutting solution. Coronal or transverse hippocampal slices (300 μm) were prepared using a slicer (Vibratome 1000 Plus, Leica Microsystems) and then transferred to an incubation chamber (BSK4, Scientific System Design) at 32°C with carbogenated cutting solution, which Cabozantinib was gradually replaced with artificial cerebral spinal fluid (ACSF) in 30 min through a peristaltic

pump (Dynamax Model RP-1; Rainin Instruments), allowing a precise regulation of fluid flow rates. The slices were then kept in Dipeptidyl peptidase the ACSF that contained 119 mM NaCl, 2.3 mM KCl, 1.0 mM NaH2PO4, 26 mM NaHCO3, 11 mM glucose, 1.3 mM MgSO4, and 2.5 mM CaCl2 (pH was adjusted to 7.4 with HCl, with osmolarity of 295–305 mOsm) at room temperature for at least 30 min. Recordings were performed in oxygenated ACSF. Intracellular solution consisted of 130 mM KMeSO3, 10 mM HEPES, 4 mM MgCl2, 4 mM Na2ATP, 0.4 mM NaGTP, 10 mM Na-phosphocreatine,

and 3 mM Na-L-ascorbate; pH was adjusted to 7.3 with KOH. Recordings were performed at room temperature in ACSF. To evoke APs, we held cells in the current-clamp configuration, and we injected 3–5 nA of current for 2 ms through the recording electrode. Cells were selected if their GCaMP fluorescence was homogeneously distributed in the cytoplasm. Fluorescent signals were imaged by a confocal microscope (Fluoview FV 1000; Olympus) with a 30 mW multiline argon laser, at 5%–10% laser power. The laser with a wavelength of 488 nm was used for excitation, and fluorescence was recorded through a band-pass filter (505–525 nm). The images were acquired using 40× water-immersion objectives (NA = 0.8) with 5 Hz scanning speed. XYT image galleries were collected and average fluorescence intensity in the soma was measured for the quantification by Fluoview data processing software.