13 were shown to be immunoreactive to CART. This incidence was significantly higher than the incidence of the CART- immunoreactive neurons in the total NG neurons, indicating that insulin preferentially targets CART neurons. Insulin Induces Depolarization and Voltage-gated Ca2+ Influx in NG Neurons The resting membrane potential of single NG neurons was 52.761.9 mV under the perforated-patch configuration. As sown in Fig. 2A and B, administration of 1027 M insulin depolarized membrane potential in 5 of 55 NG 9400011 neurons in a reversible manner. In i measurements, repeated administration of 1027 M insulin twice with a washing period of 20,30 min increased i repetitively in NG 
neurons. The amplitude of i response to the first stimulation was somewhat larger than that to the second stimulation . The effects of verapamil and v-conotoxin GIVA, the voltage-gated L- and N-type Ca2+ channel blockers, respectively, were examined for the i responses to S1. The insulin -induced increase in i was markedly suppressed in the presence of verapamil and v-conotoxin GIVA compared to the i responses to insulin observed after washing out the agents. v-Conotoxin GIVA showed a greater inhibition than verapamil. In a Ca2+free condition made with no added Ca2+ and 0.1 mM EGTA, insulin administration failed to increase i in all the neurons examined, and after bringing Ca2+ back to HKRB it elicited i increases. Preincubation for 1 hr with 50 mM LY294002, a PI3K inhibitor, significantly attenuated the amplitude of i responses to insulin in NG neurons compared to control, whereas preincubation with 10 mM U0126, a MAPK inhibitor, 22619121 for 30 min had no effect. Additionally, pretreatment with LY293002 and U0126 slightly decreased the amplitude of i responses to CCK-8, but not that to CAP. These results indicate that IRS2 and PI3K are involved in the BAY-41-2272 chemical information insulin-induced i increases. Insulin Concentration is High in the Pancreas, NG Neurons Innervate the Pancreas, and Insulin Activates the Pancreas-innervating NG Neurons with Greater Incidence A much greater population of NG neurons were recruited to the i response to insulin when its concentration was elevated to 1027 M, a concentration 100,1,000-fold higher than that in circulation. We thought that this result might reflect a situation in which insulin released from pancreatic islets at high concentrations could be immediately sensed by the NG neurons that innervate the pancreas. To verify this, we examined 1) whether insulin concentration in/around the pancreas is actually high, 2) whether neuronal fibers innervating the pancreatic islet express IR, and 3) whether the NG neurons innervating the pancreas could sense insulin particularly at higher concentrations. First, to assess local insulin concentrations in the pancreas, we measured the insulin concentration in the pancreatic vein. Furthermore, to estimate the difference between the insulin concentration in the pancreas and that in circulation, we compared insulin concentration in the pancreatic vein, that in the pancreatic artery and that in the portal vein. Under the condition fasted overnight, insulin concentration in the pancreatic vein was 15-fold higher than that in the pancreatic artery and 5-fold higher than that in the portal vein . In ad lib fed condition, insulin concentration in the pancreatic vein was 64-fold higher than that in the pancreatic artery and 17fold higher than that in the portal vein . Moreover, insulin concentration in the pancreatic vein under ad li
