Protocols for slice preparation and recordings of olfactory bulb, hippocampal and cortical neurons.
Mice were anaesthetized with an intra-peritoneal injection of ketamine (50 mg/ml) and decapitated. The head was quickly immersed in ice-cold (2-4°C) artificial cerebrospinal fluid (CutACSF) of the following composition: 125 mM NaCl, 4 mM KCl, 25 mM NaHCO3, 0.5 mM CaCl2, 1.25 mM NaH2PO4, 7 mM MgCl2 and 5.5 mM glucose (pH = 7.4 oxygenated with 95 % O2/5 % CO2). The osmolarity was adjusted to 320 mOsm with sucrose. Horizontal olfactory bulb (OB) slices (400 µm thick), cortical coronal slices (400µm thick) and hippocampus coronal slices (350 µm thick) were prepared with a vibratome (Leica). Slices were then incubated in a recovery chamber at 30 ± 1°C using an ACSF solution with a composition similar to the Cut ACSF, except for changes to CaCl2 and MgCl2 concentrations (2 mM and 1 mM, respectively). Slices were transferred to a recording chamber mounted on an upright microscope and continuously perfused with oxygenated ACSF (4 ml/min) at 30 ± 1°C. Neurons were visualized using a microscope (Zeiss axioscope) with a 40X objective (Zeiss Plan-APOCHROMAT). Data were acquired with the amplifier RK 400 BioLogic at full sampling frequency of 25 kHz using a 12-bit A/D-D/A converter (Digidata 1440A, Axon Instruments) and PClamp software (PClamp10, Axon Instruments). Patch-clamp whole-cell recordings were achieved with borosilicate pipettes having a resistance 4-8 MΩ and filled with: 126 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 2 mM ATP-Na2, 0.3 mM GTP-Na3, and 10 mM phosphocreatine (pH = 7.3, 290 mOsm). The membrane potential was corrected for the junction potential (-15 mV). All experiments were performed in the presence of ionotropic receptor antagonists (NBQX 10 µM, APV 40 µM and Gabazine 10µM).
Protocol for slice preparation and recordings of striatal neurons . Mice were deeply anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.) and transcardially perfused with an ice-cold N-methyl D-glucamine (NMDG)-based solution containing (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 20 glucose, 10 MgCl2, 93 HCl, 2 Thiourea, 3 sodium pyruvate, 12 N-acetyl cysteine and 0.5 CaCl2(saturated with 95% O2 and 5% CO2, pH 7.2-7.4). The brain was then removed from the skull and glued to the stage of a vibratome (Leica, VT1000S) where it remained submerged in ice-cold oxygenated NMDG-based solution. Coronal slices (250 µm thick) containing the striatum were collected. Slices were immediately transferred to recover in NMDG-based solution at 35°C for 5 min and then stored for at least 1h at room temperature in normal artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 25 NaHCO3 and 11 glucose, to which 250 µM kynurenic acid and 1 mM sodium pyruvate had been added. For the recordings, slices were transferred one at a time to a submersion-type chamber and perfused continuously with warm ACSF (32-34°C) at a rate of 3 ml/min. Solutions were continuously equilibrated with 95% O2 / 5% CO2. All experiments were performed in the presence of ionotropic receptor antagonists (NBQX 10 µM, APV 40 µM and Gabazine 10µM). MSNs were visualized on an upright microscope (Nikon Eclipse FN1) equipped with DIC optic using an IR 40x water-immersion objective (Nikon). Patch-clamp electrodes (4-6 MΩ) were prepared from filamented borosilicate glass capillaries (PG150T-7.5, Harvard Apparatus) using a micropipette puller (PC-10, Narishige) and were filled with an intracellular solution containing (in mM): 126 KMeSO4, 14 KCl, 3 MgCl2, 0.5 CaCl2, 5 EGTA, 10 HEPES, 2 NaATP and 0.5mM NaGTP, 10 Na-Phosphocreatine, pH adjusted to 7.25 with NaOH and osmolarity adjusted to 270-280 mOsm/L. Recordings were obtained using motorized micromanipulators (MP-85, Sutter Instrument), a Multiclamp 700B amplifier, a Digidata 1550B digitizer, and pClamp 10.7 acquisition software (Molecular Devices, San Jose, CA, USA). Signals were low-pass filtered at 10 kHz online and sampled at 10 kHz. Electrode capacitances were compensated electronically during recording. In current-clamp mode, the bridge was continuously balanced and input resistances were monitored. Cells showing more than 20% input resistance variation were excluded from the analysis.