Rification of Cannula PositionAfter termination of mice, brains were taken out and fixed by submerging in 4 paraformaldehyde for 48 hours (Sigma-Aldrich, Zwijndrecht, the Netherlands) followed by 30 sucrose (SigmaAldrich, Zwijndrecht, the Netherlands) in PBS for at least 24 hours, until the brain has sank to the bottom of the container. Cannula position was verified in 30 mm thick brain cryosections mounted on microscopic slides. The sections were fixated and defatted in CARNOY solution (100 ethanol, chloroform and acetic acid in a 6:3:1 ratio), hydrated by descending ethanol concentrations (10096-70 ) in 1531364 MilliQ (MQ) water, and a Nissl staining was performed using cresyl violet (Sigma-Aldrich, Zwijndrecht, the Netherlands): 0.9 g cresyl violet, 300 mL MQ, 2.25 mL 10 acetic acid, pH 4.5. The sections were then dehydrated in ascending ethanol concentrations (70-96-100-100 ) followed by 2 times isopropanol and 2 times Histo-Clear (National diagnostics, Atlanta, USA). Cover slips were mounted using xylene, and the cannula position was verified by locating the cannula track in the tissue. When the cannula track ended within the respective ventricle, the cannula was considered to be positioned correctly. The average success rates of LV and 3V cannulation were ,85 and ,60 respectively.Food Intake MeasurementAfter a recovery period of at least 1 week, the mice received a pre-weighed amount of food after which basal food intake was measured for two hours, starting from 09:00 a.m. One day later, mice received an i.c.v. injection of NPY (0.2 mg/kg in 1 mL of artificial cerebrospinal fluid, aCSF) under light isoflurane anesthesia (1.5 in air). Food was weighed before and one and two hours after waking up from the anesthesia to determine NPYinduced food intake.Hepatic VLDL-TG and VLDL-apoB ProductionIn experiments performed under complete anesthesia, 4 h fasted mice were anesthetized with 6.25 mg/kg Acepromazine (Alfasan, Woerden, The Netherlands), 6.25 mg/kg Midazolam (Roche, Mijdrecht, The Netherlands), and 0.31 mg/kg Fentanyl (Janssen-Cilag, Tilburg, The Netherlands). In other experiments, mice were awake throughout the whole experiment, except for the lateral ventricle (LV) or third ventricle (3V) injections, which were performed under light isoflurane sedation (1.5 in air). A basal blood sample was taken from the tail tip into a chilled heparin-coated capillary (Vitrex Medical, Herlev, Denmark), and mice received an intravenous injection of 100 ml PBS containing 100 mCi Tran35S label (MP Biomedicals, Eindhoven, the Netherlands) via the tail vein, resulting in incorporation of 35S into newly produced VLDL-apolipoprotein B. After 30 min, the animals received an intravenous injection of tyloxapol (500 mg/kg body P7C3 site weight; Triton MedChemExpress Sermorelin WR-1339, 24786787 Sigma), as a 10 (w/w) solution in sterile saline, to prevent systemic lipolysis of newly secreted hepatic VLDL-TG [35]. Immediately after the tyloxapol injection, mice received an injection of either NPY (0.2 mg/kg BW, Bachem, St. Helens, UK in 1 mL aCSF) or vehicle (aCSF, 1 mL) into the lateral ventricle (LV) or third ventricle (3V). In the dose-finding study, mice received an LV injection of NPY (0.0002, 0.002, 0.02, 0.2 or 2.0 mg/kg BW in 1 mL aCSF) or vehicle. All dosages were tested once, in the number of mice indicated. In the antagonist study, mice received either an LV injection of Y1 antagonist GR231118 (0.5 mg/kg in 1 mL aCSF) or vehicle (aCSF, 1 mL) or an i.v. injection of PYY3?6 (0.5 mg/kg in 10.Rification of Cannula PositionAfter termination of mice, brains were taken out and fixed by submerging in 4 paraformaldehyde for 48 hours (Sigma-Aldrich, Zwijndrecht, the Netherlands) followed by 30 sucrose (SigmaAldrich, Zwijndrecht, the Netherlands) in PBS for at least 24 hours, until the brain has sank to the bottom of the container. Cannula position was verified in 30 mm thick brain cryosections mounted on microscopic slides. The sections were fixated and defatted in CARNOY solution (100 ethanol, chloroform and acetic acid in a 6:3:1 ratio), hydrated by descending ethanol concentrations (10096-70 ) in 1531364 MilliQ (MQ) water, and a Nissl staining was performed using cresyl violet (Sigma-Aldrich, Zwijndrecht, the Netherlands): 0.9 g cresyl violet, 300 mL MQ, 2.25 mL 10 acetic acid, pH 4.5. The sections were then dehydrated in ascending ethanol concentrations (70-96-100-100 ) followed by 2 times isopropanol and 2 times Histo-Clear (National diagnostics, Atlanta, USA). Cover slips were mounted using xylene, and the cannula position was verified by locating the cannula track in the tissue. When the cannula track ended within the respective ventricle, the cannula was considered to be positioned correctly. The average success rates of LV and 3V cannulation were ,85 and ,60 respectively.Food Intake MeasurementAfter a recovery period of at least 1 week, the mice received a pre-weighed amount of food after which basal food intake was measured for two hours, starting from 09:00 a.m. One day later, mice received an i.c.v. injection of NPY (0.2 mg/kg in 1 mL of artificial cerebrospinal fluid, aCSF) under light isoflurane anesthesia (1.5 in air). Food was weighed before and one and two hours after waking up from the anesthesia to determine NPYinduced food intake.Hepatic VLDL-TG and VLDL-apoB ProductionIn experiments performed under complete anesthesia, 4 h fasted mice were anesthetized with 6.25 mg/kg Acepromazine (Alfasan, Woerden, The Netherlands), 6.25 mg/kg Midazolam (Roche, Mijdrecht, The Netherlands), and 0.31 mg/kg Fentanyl (Janssen-Cilag, Tilburg, The Netherlands). In other experiments, mice were awake throughout the whole experiment, except for the lateral ventricle (LV) or third ventricle (3V) injections, which were performed under light isoflurane sedation (1.5 in air). A basal blood sample was taken from the tail tip into a chilled heparin-coated capillary (Vitrex Medical, Herlev, Denmark), and mice received an intravenous injection of 100 ml PBS containing 100 mCi Tran35S label (MP Biomedicals, Eindhoven, the Netherlands) via the tail vein, resulting in incorporation of 35S into newly produced VLDL-apolipoprotein B. After 30 min, the animals received an intravenous injection of tyloxapol (500 mg/kg body weight; Triton WR-1339, 24786787 Sigma), as a 10 (w/w) solution in sterile saline, to prevent systemic lipolysis of newly secreted hepatic VLDL-TG [35]. Immediately after the tyloxapol injection, mice received an injection of either NPY (0.2 mg/kg BW, Bachem, St. Helens, UK in 1 mL aCSF) or vehicle (aCSF, 1 mL) into the lateral ventricle (LV) or third ventricle (3V). In the dose-finding study, mice received an LV injection of NPY (0.0002, 0.002, 0.02, 0.2 or 2.0 mg/kg BW in 1 mL aCSF) or vehicle. All dosages were tested once, in the number of mice indicated. In the antagonist study, mice received either an LV injection of Y1 antagonist GR231118 (0.5 mg/kg in 1 mL aCSF) or vehicle (aCSF, 1 mL) or an i.v. injection of PYY3?6 (0.5 mg/kg in 10.
