Figure 5

Effect of carbachol on PVN-CRF neurons. (a) YFP signal (left) and regions of interest (ROIs, right) of a representative brain slice. The brain region is indicated by the red box in the upper illustration. Numbers superimposed over the ROIs align with the dorsoventral axis (from dorsal to ventral). D dorsal, V ventral, L lateral, M medial, scale bar: 100 μm. (b–e) Z-scores of the YFP/CFP ratio recorded from each representative brain slice, (b) and (c) are from the brain slice shown in (a). Upper graphs show traces of individual ROIs (light blue: dorsal PVN, light red: ventral PVN) and mean value (black). Black bars indicate the application timing (2 min) of each substance, carbachol (CCh) in (b), (d) and (e), glutamate in (c). Lower heat maps show Z-scores of individual ROIs indicated by the color bars at right. The blue and red color bars at left indicate the dorsal (D) and ventral (V) portions of the PVN. CCh was applied twice in (d) and (e). The nicotinic acetylcholine (ACh) receptor-selective antagonist hexamethonium was applied 3 min before the second application of CCh in (e). (f) Box plots of ratio of the area under the curve of the Z-scores of the dorsal PVN between the first and second application of CCh with and without antagonists: hexamethonium (100 μM), the nicotinic ACh receptor-selective antagonist mecamylamine (1 μM) and the muscarinic ACh receptor selective-antagonist atropine (100 nM). Dots show individual data. ***p < 0.001, Kruskal–Wallis test followed by Dunn’s test.