To promote axon outgrowth, neurons undergo expansion of the plasma membrane

d at 1 cm from the olfactory epithelium. The puff duration was regulated by a Pneumatic Picopump PV 820 triggered by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19663632 a S48 square pulse electrical stimulator Saracatinib chemical information controlled by software using a custom made protocol developed in pCLAMP 10. Odorant responses were elicited by paired-pulses of 100 ms separated by ISIs of 1, 2, 4, 6, 8, 10 and 15 s. We performed 3 trials per condition separated by 1 min intervals. Drugs All drugs were purchased from Sigma Aldrich. We use a variety of cell-permeable inhibitors to disrupt the catalytic activity of enzymes expressed in the cilia of OSNs as follows: the selective and potent inhibitor of PKA N–5isoquinolinesulfonamide ; the non-specific inhibitor of PDEs dihydrochloride hydrate ; the non-competitive inhibitor of dynamin GTPase activity dynasore hydrate; okadaic acid, inhibitor of protein phosphatases 1, 2A and 2B; and monensin sodium salt, a carboxylic ionophore that interrupts GPCR recycling. We screened the olfactory epithelium with concentrations ranging from two- to ten-fold the half maximal inhibitory concentration to disrupt the activity of each enzyme. The concentrations used were: H-89 at 20 mM, IBMX at 190 mM, dynasore at 25 mM, okadaic acid at 10 mM, and monensin at 10 mM. Aliquots were prepared in DMSO and maintained at 2 20uC. Aliquots were diluted to the proper concentrations in normal ringer solution in the day of the experiment and perfused on the top of the olfactory epithelium at 500 mL/min during 20 min using a peristaltic pump Minipuls. EOG recordings started 60 min after perfusion. The control recordings were performed 60 min after 20 min of perfusion of vehicle. We could not conduct recordings during the perfusion because the EOG signal is lost in the wet epithelium. Our experimental and modeling results provide evidences that the observed effects generated by IBMX, okadaic acid and dynasore result from increased levels of cAMP in the olfactory epithelium. In this way, we used cAMP to mimic the general effects of these pharmacological treatments, and washed out the drug to reverse the effects of increasing levels of cAMP. The tissue was perfused during 20 min with a solution of normal ringer, 0.02% DMSO, and 500 mM of N6,29-O-Dibutyryladenosine 39,59-cyclic monophosphate sodium salt, a cellpermeable cAMP analog. Then, the olfactory epithelium was washed during 20 min with a solution of normal ringer and 0.02% DMSO. All recordings started 60 min after the perfusion. We did not observe significant differences between the L, RT and DT of the EOG signal of the treated and the washed groups. Materials and Methods All procedures were approved by the Institutional Animal Care and Use Committee of the Fundacao para a Ciencia e a ~ Tecnologia. All efforts were taken to comply with the 3Rs. Reduction in the number of laboratory animals was performed by using both the ipsilateral and contralateral olfactory epithelium. The other parts of the brain were used in other experiments conducted in the lab as another effort to reduce the use of laboratory animals. We have developed a publicly available computational model that can be used as a replacement for experiments using animals. Refinement to minimize potential pain, suffering and distress were taken by using appropriate anesthetics. Animal surgery The animals were handled according to European Community guidelines and Portuguese law concerning animal care. The experiments were performed in 4 to 7 weeks old male Wistar rats. Rats wer