3% Triton X-100 (PBST) and 4% bovine serum albumin (BSA, Sigma) for 1 day at 4°C; (2) a biotinylated antirabbit IgG solution (1:1000, Vector Laboratories) in PBST; and (3) an ABC-peroxidase

solution (1:1000, Vector Laboratories) both for 60 min at room temperature. Finally, sections were stained using 3,3′-diaminobenzidine-4 HCl and Nickel solution (DAB-Ni; Vector Laboratories) in order to obtain dark purple staining. Four washes in PBST were performed between each step. The c-Fos-stained sections were incubated in a goat antiserum to orx/hcrt (1:4000; Ku-0059436 price Santa Cruz) in PBST/BSA 4% for 2 days at 4°C. Amplification steps were similar to those described above but the final step was performed in DAB solution without nickel in order to obtain brown staining. Finally, the sections were mounted on slides, dried, and coverslipped with permaslip. Orx/hcrt-positive and orx/hcrt+c-Fos-positive neurons were counted by an investigator blind to experimental conditions, within at least six coronal sections per animal evenly spaced throughout the rostrocaudal extent of the lateral hypothalamus (Bregma −1.06 to −2.14) to avoid rostrocaudal bias. Colocalization of c-Fos and orx/hcrt immunostaining was detected using a high magnification objective.

No significant differences were found in the number of c-Fos-positive orx/hcrt neurons between vehicle injection experiments. This work was funded primarily by the European CHIR 99021 Research Council (ERC FP7 Starting Grant to D.B.). M.M.K. was also supported by Osk. Huttunen Foundation (PhD studentship). L.d.L. was supported by the NIH, and A.A. by the NIH, FRS-FNRS, and NARSAD. L.F. was funded by the

Lundbeck Foundation and the MRC in UK and Denmark. We thank Dr. Methisazone Peter Voshol and Sylvia Osborn of mouse core facilities at Medical Research Council Centre for Obesity and Related Metabolic Diseases (MRC CORD) for assisting with experiments in Figure 2. MMK performed and designed most experiments, analyzed data, and contributed to writing of the paper. J.A.-S. contributed to the experiments shown in Figure 2. A.A. and L.d.L. contributed to the experiments shown in Figure 3. L.F. and L.T.J. created and provided orx/hcrt-eGFP mice used in the project. DB conceived the idea, obtained funding, coordinated the project, and wrote the paper. “
“Mammalian central neurons rely on the dynamic interplay between transmitter receptors and voltage-gated ion channels on their dendrites for signal processing. For example, the A-type voltage-gated K+ channels (IA) on the dendrites of CA1 hippocampal pyramidal neurons regulate neuronal signaling by filtering fast synaptic potentials and regulating action potential back propagation, synaptic integration and long-term potentiation (LTP) (Kim and Hoffman, 2008). This IA derives primarily from Kv4.2 (Birnbaum et al., 2004, Kim et al.