Extended Data Figure 2. Innervation of histamine neurons in the TMN by GABAergic neurons in the POA and overlap of lentivirus labeling of GABAPOA→TMN neurons with GAD expression and with c-Fos labeling after sleep rebound.
a, In GAD2-Cre mice injected with AAV-DIO-ChR2-eYFP in the POA (left), ChR2-eYFP expressing axons (green) are observed in the TMN area (red, histidine decarboxylase (HDC)). Mouse brain figure adapted with permission from ref. 31. b, Inhibitory postsynaptic current (IPSC, red) recorded in an example TMN histamine neuron, evoked by optogenetic activation of POA GABAergic axons. Light-evoked responses (red) were blocked by bicuculline (black). Blue tick, laser stimulus. c, Amplitudes and latencies of IPSCs recorded from TMN histamine neurons (n=7, from 2 mice). Each symbol represents data from one cell. Error bar, ±s.e.m. d, Single cell reverse-transcription PCR (RT-PCR) identification of HDC-expressing histamine neurons. e, Schematic of RV-mediated transsynaptic retrograde tracing from TMN histamine neurons. Fluorescence image of the TMN shows starter cells (yellow, expressing both GFP and mCherry). Inset, enlarged view of the region in white box. f, Left, fluorescence image showing input neurons in the POA. Right, enlarged view of the region in white box, showing RV-GFP labeling (green) and GAD1/2 expression (red, FISH for mRNA encoding GAD1/2). Arrowheads, RV labeled cells that are GAD1/2+. 79.0±1.4 % of RV-GFP labeled neurons contained mRNA encoding GAD1/2 (n=2 mice). g, Overlap between c-Fos expression induced by sleep rebound and RV-GFP labeling. Left, fluorescence image showing input neurons in the POA. Right, enlarged view of the region in white box, showing RV-GFP labeling and c-Fos expression. 46.9±1.9% of RV-GFP labeled neurons expressed c-Fos (n=6 mice). h, Expression of eYFP or ChR2-eYFP in the POA induced by injecting rEIAV-DIO-TLoop-nls-eYFP or rEIAV-DIO-TLoop-ChR2-eYFP into the TMN of GAD2-Cre mice and their overlap with GAD1/2 expression (FISH for mRNA encoding GAD1/2). Arrowheads indicate cells co-labeled with GAD-FISH probe and eYFP or ChR2-eYFP. 96.2±1.4% of eYFP labeled neurons and 95.4±3.7% of ChR2-eYFP labeled neurons contained mRNA encoding GAD1/2 (n=4,5). i, Expression of ChR2-eYFP in the POA induced by injecting rEIAV-DIO-TLoop-ChR2-eYFP into the TMN of a GAD2-Cre mouse and its overlap with c-Fos expression following sleep rebound (arrowheads).