Figure 1.

ChR2 circuit-assisted mapping of LH/PF^Vgat → Orx input. A, Schematic of the experimental design used to map connectivity between local GABAergic neurons and orexin neurons in brain slices (LH/PF^Vgat → Orx). We injected an AAV-DIO-ChR2-YFP and an AAV-h-orexin-tdTomato into the LH/PF region of Vgat-IRES-Cre mice to produce expression of ChR2-YFP in GABAergic neurons and tdTomato in orexin neurons. Six weeks after the AAV injections, we recorded in brain slices. We photostimulated cell bodies and terminals of local GABAergic neurons while recording from orexin neurons that express tdTomato. B, An example of a Vgat-IRES-Cre mouse injected with AAV-DIO-ChR2-YFP and AAV-h-orexin-tdTomato. Confocal photomicrograph showing, in the perifornical region, local GABAergic neurons expressing ChR2-YFP (green represents immunolabeled against YFP; indicated by arrows) and native tdTomato fluorescence in orexin neurons (red). Scale bars, 40 μm. C, Heatmap of AAV-DIO-ChR2-YFP injection sites of 29 recorded Vgat-IRES-Cre mice showing overlapping regions of ChR2-YFP-expressing neurons. D, Confocal images of ChR2-YFP-expressing neurons (in green) and in situ hybridization for Vgat RNA (red). Scale bar, 20 μm. White arrows indicate double-labeled neurons. Blue arrows indicate individual neurons expressing Vgat mRNA but not ChR2-YFP. E, Confocal images of 3 recorded tdTomato-expressing neurons filled with biocytin and post hoc labeled with streptavidin-conjugated Alexa-405 (in blue) that are double-labeled for orexin immunoreactivity (magenta). Scale bar, 40 μm. F, Map of the distribution of n = 77 recorded neurons that were found after post hoc labeling for biocytin. Red dots indicate 72 recorded neurons (+) for orexin immunoreactivity. Blue dots indicate 5 recorded neurons (−) for orexin immunoreactivity. Atlas levels are per Paxinos and Franklin (2001). opt, Optic tract; cp, cerebral peduncle; mt, mammillothalamic tract; f, fornix.