Design and characterization of a FLEX switch for ChR2mCherry. A, FLEX switch recombination sequence for stable inversion proceeds in two steps: (1) inversion followed by (2) excision. loxP and lox2272 are orthogonal recombination sites. B, Construct design for FLEX-for-ChR2mCherry and FLEX-rev-ChR2mCherry. CAG: CMV enhancer/β-globin chimeric promoter, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, ITR: inverted terminal repeat. C, Images showing mCherry fluorescence in HEK 293 cells for FLEX-for-ChR2mCherry and FLEX-rev-ChR2mCherry in the presence and absence of Cre. D, Colocalization of EGFP and mCherry fluorescence (yellow arrowheads) in HEK 293 cells cotransfected with FLEX-rev-ChR2mCherry and Cre-IRES-EGFP.

Cre-dependent ChR2mCherry expression in transgenic mice using rAAV-FLEX-rev-ChR2mCherry. A, Distribution of fluorescent neurons resulting from a large co-injection (150 nl) of rAAV-ChR2-EGFP and rAAV-FLEX-rev-ChR2mCherry into the hypothalamus of wild type mice. Extensive fluorescence from EGFP (left) but no fluorescence from mCherry in brain slices (right) shows the absence of background expression with rAAV-FLEX-rev-ChR2mCherry. Background image of slice obtained from DAPI fluorescence. B, Top: Schematic showing location of the imaged area in caudal arcuate nucleus. Bottom: mCherry fluorescence only in the arcuate nucleus after a large injection of rAAV-FLEX-rev-ChR2mCherry into the hypothalamus of pomc-cre;rosa26-loxSTOPlox-eyfp mice. Compare distribution of fluorescence with A. C, Colocalization of mCherry and EYFP fluorescence in arcuate nucleus. D-E, Similar to B-C in this case agrpcre;rosa26-loxSTOPlox-eyfp mice were used with rAAV-FLEX-rev-ChR2mCherry virus injections. F, Top: Image showing neuron morphology from the arcuate nucleus of labeled POMC neurons. Bottom: Higher magnification image of boxed area. G-H, Axonal projections of AGRP neurons infected with rAAV-FLEX-rev-ChR2mCherry. Strong axonal labeling observed in the paraventricular nucleus of the hypothalamus (G) and paraventricular thalamus (H). 3V: third ventricle; D3V: dorsal third ventricle.

Photostimulation of AGRP and POMC neurons in the hypothalamus. A-B, Whole cell voltage clamp recordings performed from mCherry positive neurons in hypothalamic slices from agrp-cre or pomc-cre mice infected with rAAV-FLEX-rev-ChR2mCherry. Light pulses (500 ms) of varying power elicited ChR2mCherry-mediated inward currents. C-D, The peak current is plotted as a function of laser power for AGRP (C) and POMC (D) neurons. E-F, Perisomatic repetitive stimulation with 1 ms light pulses at 10 Hz in AGRP (E) and POMC (F) neurons. Blue dashes mark timing of light flashes.

Channelrhodopsin assisted circuit mapping for hypothalamic neuronal circuits: AGRP→PVN and POMC→PVN. A, Diagram of a sagittal hypothalamic section depicting anatomy of connections between ARC and PVN. B, Coronal slices containing PVN, but not arcuate nucleus, were used for whole cell voltage clamp recordings from PVN neurons. C, Fluorescence image showing POMC axonal projections to PVN. Blue box outlines region of laser stimulation in E, PVN boundary is outlined in red, location of recorded cell body is marked by a star, and recording pipette is outlined in yellow. D, Overlay of POMC→PVN inhibitory postsynaptic currents resulting from three photostimulation trials at a site perisomatic to a voltage clamped PVN neuron. E, Synaptic input map shows mean current responses over 100 ms time window as a colormap in voltage clamped PVN neuron resulting from LSPS of axons originating from POMC neurons. Position of soma marked with a star. F-H, Similar to C-E but in this case projections arise from AGRP neurons.