Following the measurement, cells were lysed for protein expression analysis. For coaggregation experiments, COS7 cells were transiently transfected (Fugene, Roche)

and proteins were expressed for 48 hr. Cells were fixed with 4% PFA, 4% sucrose in 100 mM sodium phosphate buffer (pH 7.4) for 15 min at room temperature. Immunostaining was done using standard Selleckchem Palbociclib procedures. Dissociated cultures of mouse cerebellar granule cells were prepared from P5–P7 pups as described previously (Dean et al., 2003). Knockdown of msyd-1a was performed on day 1 (replenished at day 4) with 0.75 μM Accell SMART pool siRNA against msyd-1a or a nontarget control pool (Dharmacon). At day 7, cells were fixed with 4% paraformaldehyde, containing 4% sucrose in 100 mM phosphate buffer (pH 7.4). After antibody staining the coverslips were mounted with ProLong (Invitrogen). For lentiviral delivery of hSYD1A, a lentiviral vector with a dual human synapsin promoter was used to express GFP and hSYD1A (Gascón et al., 2008). All procedures related to animal experimentation were reviewed and approved by the Kantonales Veterinäramt Basel-Stadt. Whole-cell patch-clamp recordings were performed on DIV 8–11 cerebellar granule cell cultures. For rescue, the lentivirus was added at DIV

3. The extracellular solution (pH 7.3) contained see more the following: 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM Glucose, 25 mM Sucrose, and 5 mM HEPES. For all experiments, 300 nM TTX, 0.1 mM Picrotoxin, and 0.1 mM AP5 were used in the solution. The internal solution contained the following: 130 mM CsCl, 10 mM HEPES, 10 mM EGTA, 10 mM Phosphocreatine, 2 mM MgATP, 5 mM NaCl (pH 7.25), 298 mOsm. For acute slice recordings, P11–P16 mice were anesthetized with isoflurane and rapidly decapitated. Three hundred micrometer thick sagittal sections were cut in sucrose substituted artificial cerebrospinal fluid (ACSF) that consisted of 83 mM NaCl, 2.5 mM KCl, GBA3 1 mM NaH2PO4, 26.2 mM NaHCO3, 22 mM glucose, 72 mM sucrose, 0.5 mM

CaCl2, 3.3 mM MgCl2. Slices were allowed to recover at 32°C for 1 hr and then maintained at room temperature in the same sucrose ACSF. For whole-cell recordings, slices were perfused with: 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2, 1.3 mM MgSO4, 11 mM glucose, 0.1 mM Picrotoxin. For all experiments, whole-cell recordings were digitized at 10 kHz and filtered at 2 kHz. Whole-cell patch-clamp recordings of CA1 pyramidal cells were done using 3–6M Ω pipettes and filled with an internal solution that contained: 130 mM Cs-methanesulfonate, 5 mM NaCl, 10 mM EGTA, 10 mM HEPES, 10 mM phosphocreatine, and 2 mM Mg-ATP (pH 7.3) with CsOH, 290–300 mOsm. The cells were held at a holding potential of −70 mV. For mini recordings, slices were also perfused with 500 nM TTX. The mEPSCs were detected using Axograph X software and the mEPSCs were detected using a template based detection algorithm package.