2) Unlike Cdc42 and RhoA, RacGTP is detected almost exclusively in synaptosomes in the brain

2) Unlike Cdc42 and RhoA, RacGTP is detected almost exclusively in synaptosomes in the brain. littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75?/? cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels. for 10 min to collect the pellet, which was subsequently resuspended in the homogenization buffer and overlaid on top Chelerythrine Chloride of a sucrose step gradient (0.8, 1, and 1.2 m). The gradient was centrifuged at 82,500 for 2 h. The resulting fraction that was overlaid onto the interface between 1 and 1.2 m sucrose was collected and overlaid onto 0.8 m sucrose solution and centrifuged at 230,000 for 15 min. The pellet contained both presynaptic and postsynaptic membranes, or synaptosomes. Chelerythrine Chloride RacGTP Assay The synaptosome pellet was resuspended in a lysis buffer containing 25 mm HEPES (pH 7.5), 150 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm sodium orthovanadate, 25 mm NaF, 10 g/ml leupeptin, 10 g/ml aprotinin, and 2 mm PMSF. Chelerythrine Chloride The lysates were subjected to RacGTP assays using pull-down methods as described (3). Immunohistochemistry Brains were sectioned at 30 m in a sagittal or coronal plane using a cryostat and used for immunohistochemistry as described (4). For p-PAK staining, tissues were subjected to antigen retrieval at 50 C for 50 min in 10 mm Tris-HCl (pH 9.0). The images were obtained using a Leica confocal microscope (model TCS SL) at the identical setting for wild type and p75 knock-out sections. Organotypic Cerebellar Slices Freshly dissected brains were cut at 200 m on the sagittal plane at 4 C using a vibratome (Leica), and cerebellar slices that contained all 10 lobules were placed inside a Millicell on the sagittal orientation as described (24). BDNF was added to the underlying media at 50 ng/ml, incubated for 10 min CCL2 at 37 C, and processed for protein extraction. Preparation of Cerebellar Slices for Electrophysiological Recordings Mice (postnatal days 15C25) were anesthetized with halothane and sacrificed by decapitation. Sagittal slices of 300-m thickness were prepared from the vermis of the cerebellum with a vibratome (World Precision Instruments) in ice-cold, oxygenated artificial cerebrospinal fluid (aCSF): 125 mm NaCl, 26 mm NaHCO3, 1.25 mm NaH2PO4, 2.5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, and 10 mm glucose bubbled with Chelerythrine Chloride 5% CO2 and 95% O2 (pH 7.4). Slices were recovered at 35 C for 1 h and then maintained at room temperature (22C24 C) in the aCSF until use. Extracellular Recording Littermate pairs of the wild type and p75?/? mice were used. The recordings from each pair of littermates were performed either on the same day or on two consecutive days under the same experimental conditions. From each animal, 20C40 Purkinje cells from the apex of the lobule VI of the cerebellum were randomly recorded. Prior to recording, the cerebellar slice was mounted in a chamber on the stage of a Nikon E600-FN upright microscope and continuously perfused (2 ml/min) with a modified aCSF (aCSF supplemented with 5 mm kynurenic acid (a broad range ionotropic glutamate receptor antagonist) and 100 m picrotoxin (a GABAA receptor blocker)). The answer was warmed to 33C35 C with an SC-20 in-line option heater (Harvard Equipment). The Rac1 inhibitor, NSC23766 (Calbiochem), was diluted in the customized aCSF to 100 m and used through whole-chamber perfusion. Extracellular field potentials had been recorded using cup electrodes using a suggestion size of 0.5C1 m filled up with aCSF with no synaptic blockers. The pipette suggestion was positioned close to the preliminary axon segment from the Purkinje cell soma. Actions potentials made an appearance as fast harmful deflections of 100C1000 V. Just obviously isolated one cell signals were chosen for subsequent and recording analysis. Signals had been preamplified and filtered (at 3 kHz) by NPI EXT-10C and LPBF-01GX amplifier/filtration system modules before getting digitized and sampled (at 10 kHz) with the built-in Advertisement/DA converter of the EPC10 amplifier (HEKA Consumer electronics Inc.). Data had been examined using the Spike2 plan (Cambridge Electronic Style). Whole-cell Current Clamp Documenting Recording pipettes had been taken from micropipette cup (A-M Systems, Inc., Carlsborg, WA) to 3C5 M and filled up with an intracellular option formulated with 122 mm potassium gluconate, Chelerythrine Chloride 9 mm NaCl, 1.8 mm MgCl2, 0.9 mm EGTA, 9.