3 mM Na-GTP, pH 7.35 with KOH). Liquid junction potential correction was performed off-line. Paired-pulse ratio (PPR) experiments were carried out to estimate release probability. The peak amplitude of excitatory postsynaptic currents (EPSCs) evoked by two identical electrical stimuli separated by 100 ms was measured. PPR was calculated as the

ratio of the peak amplitude of EPSC2/EPSC1. Stimulus artifacts from the paired-pulse traces have been deleted. To measure evoked EPSCs, we performed whole-cell voltage clamp recording (at −60 mV) in presence of 100 μM picrotoxin (added to aCSF). A stimulating electrode was placed near the VMH (300–500 μm from the recording electrode) as mentioned above. The internal recording solution contained (in mM): CsCH3SO3 125; CsCl 10; NaCl 5; MgCl2 2; EGTA 1; HEPES 10; (Mg)ATP 5; (Na)GTP 0.3; 10 lidocaine N-ethyl bromide (QX-314) (pH 7.35 with NaOH). Cre-dependent selleck adeno-associated viral vector AAV-DIO-mCherry was constructed by modifying the AAV-DIO-ChR2(H134R)-mCherry vector kindly provided by Dr. Karl Deisseroth (http://www.stanford.edu/group/dlab/optogenetics/sequence_info.html).

Briefly, Asc1 and Nhe1 restriction sites were used to replace ChR2-mCherry fusion with mCherry alone (detailed methods in A.S. and B.L.S., unpublished data). The AAV-DIO-mCherry vector was then packaged into serotype 8 through the University of North Carolina Vector Core. AAV-mCherry virus (100 nl) at 1.5 × 1012 viral mol/ml was then stereotaxically injected into the arcuate of AgRP-ires-Cre or POMC-Cre related animals at 4 weeks of age (as described above). 3 weeks later, animals were perfused

and the brains were MS 275 coronally sectioned at 30 μm thickness. Rolziracetam Immunostaining against mCherry with rabbit anti-DsRed primary antibody (Clonetech; 1:2,500) and with Alex-594-anti-rabbit secondary antibody (Invitrogen) was then performed as previously described (Kong et al., 2010). Serial images of proximal dendritic structure labeled with mCherry immunoreactivity were taken under Zeiss confocal microscope (oil objective, 63×). In the coronal sections, primary dendrites or major secondary dendrites within a distance of ∼150 μm from the soma that they originated from were imaged. These images were stacked using ImageJ software (1.44i version) for further analysis. The length of dendrites and diameter of spines were calculated according to the scale bars. Spine density was calculated by dividing total spine number to the length of the targeted dendrites. Each day, for 12 days prior to sacrifice, the mice were acclimated to handling. For immunohistochemical detection of c-Fos and GFP in Npy-GFP mice and in Agrp-ires-Cre, Grin1lox/lox, Npy-GFP mice, animals were sacrificed at 10 AM in either the ad lib fed state or after 24 hr of fasting (food removed at 10 AM on the previous day). The mice were perfused and brains were sectioned as described above. Assessment of c-Fos induction was performed using a previously developed method ( Fuller et al.