We chose to test this possibility in hippocampal neurons because the hippocam pus displays high levels of IL 1B and its receptor, and be cause the physiopathological effects of IL 1B in this brain region are well characterized. Methods Ethics approval All experiments were approved by the Ethics committee selleck products of the Center for Neurosciences and Cell Biology, Faculty of Medicine, University of Coimbra. All animals used in the study were handled in accordance with EU guidelines. Animals Male Wistar rats aged 8 weeks old, were used for total, synaptic and sub synaptic membrane preparations. Rats were maintained in the ani mal facilities and handled only at the time of sacrifice, al ways at the same hour of the day because there is circadian regulation of IL 1B levels in the brain.

Rats were deeply anesthetized with halothane before being killed by decapi tation. Total and synaptic membranes were prepared from the same group of animals and another group of rats was used for preparing sub synaptic membranes. Embryos from 2 to 4 months old female Wistar rats were used for the primary neuronal cultures. Pregnant females Inhibitors,Modulators,Libraries were anaesthetized with halothane on the eight eenth day of pregnancy, and the embryos removed. Preparation of total membranes from the hippocampus The purification of total membranes from the rat hippo campus Inhibitors,Modulators,Libraries was performed essentially as described previously. After removal of the brain, the hippocampi were iso lated and homogenized in a sucrose solution at 4 C. This Inhibitors,Modulators,Libraries homogenate was separated by centrifugation Inhibitors,Modulators,Libraries at 3,000 g for 10 minutes at 4 C.

The supernatant was removed and again separated by cen trifugation at 100,000 g for 30 minutes at 4 C. The obtained pellets contained the total cytoplasmic membranes and were resuspended in 5% SDS with 0. 1 mmol l of PMSF and fi nally, after determination of protein density using the bicinchoninic acid method, diluted in SDS PAGE buffer, 30% glycerol, 10% SDS, 0. 6 mol Inhibitors,Modulators,Libraries l DTT and 0. 012% of bromophenol blue and boiled at 95 C during 5 minutes for western blotting analysis. Preparation of hippocampal synaptosomes The preparation of hippocampal synaptosomes from rats was carried out essentially as selleck chem AZD9291 described previously. After removal of the brain, the dissected hippocampi were homogenized in the same sucrose solution described above, and the homogenates were separated by centrifugation at 3,000 g for 10 minutes at 4 C. The supernatant was removed and again separated by centrifugation at 14,000 g for 12 minutes at 4 C. The resulting pellet was resuspended in 1 ml of a 45% Percoll solution prepared in a Krebs HEPES Ringer solution at 4 C. This hom ogenate was then separated by centrifugation at 12,650 g for 2 minutes at 4 C in an Eppendorf microcentrifuge.