Fig 2: (A): Glial connexins and pannexin1 mRNAs quantification measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in the cortex, hypothalamus and DVC of control mice (n = 6, 23 and 24 respectively). ** p < 0.01 and *** p < 0.001 compared to cortex. (B–G): Confocal images of Connexin-43 (Cx43) (red) and Glial Fibrillary Acidic Protein (GFAP) (E–G) or vimentin (F) double labeling immunohistochemistry performed within the hypothalamus and Dorsal Vagal Complex (DVC). Scale bar: 200 μm. Inset in F shows Cx43 and vimentin co-localization in α-tanycytes. (H,I): Confocal images Cx43 (red) and GFAP (green) double immunofluorescence within the DVC. Cx43 is strongly associated with astrocytes GFAP+ (green) processes. (J,K): Confocal images of Cx43 (red) and Ionized Ca2+ Binding Adapter Molecule 1 (IBA1) (green) double immunofluorescence within the DVC. Note that Cx43 labelling is sometimes found associated with thin processes of IBA+ microglia. Scale bars: 20 μm for (H, J) and 2 μm for (I) and (K). AP: area postrema, NTS: nucleus tractus solitarius, cc: central canal; ARC: arcuate nucleus, 3V: third ventricle, ME: median eminence.

Fig 3: (A,B): Connexin-43 (Cx43) and Glial Fibrillary Acidic Protein (GFAP) mRNA expression (normalized with Glyceraldehyde-3-phosphate dehydrogenase gene, GAPDH) was assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) from the hypothalamus (A) and dorsal vagal complex (DVC) (B) of control ad libitum (n = 14), 24 h fasted (n = 13), 24 h fasted plus 2 h refeed (n = 8) or plus 4 h refeed (n = 8) mice. (C): Representative Western blot analysis of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of control ad libitum (n = 5) and 24 h fasted (n = 5) mice. (D,E): Cx43 and GFAP mRNA expressions (normalized with GAPDH) were assessed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) from the hypothalamus (D) and dorsal vagal complex (DVC) (D) of Normal Chow (NC) (n = 9) and High Fat Diet (HFD) fed (n = 9) mice. (F): Representative Western blot analysis and quantification of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of NC (n = 9) and HFD-fed (n = 9) mice. A two-way analysis of variance test was performed between different experimental groups * p < 0.05 and *** p < 0.001 significantly different from control ad libitum. #p < 0.05 and ##p < 0.001 significantly different from 24 h fasted mice. ns: no significant difference.

Fig 4: (A): Confocal images of connexin-43 (Cx43) (red) and bassoon (green) double labeling immunohistochemistry performed within the nucleus tractus solitarius (NTS). Scale bar: 5 µm. Inset: Arrows show Cx43 labelling in close vicinity of synapses labelled by presynaptic bassoon protein. Scale bar 1 µm. (B): An example of a voxel (4 × 4 µm) used for the quantification of Cx43/bassoon appositions. Scale bar: 0.5 µm. (C): Twelve z-stack images defining the voxel illustrated in B and allowing the identification of Cx43/bassoon appositions. (D): Stereological estimation of Cx43/bassoon appositions in the NTS (85.13%) and arcuate nucleus of the hypothalamus (ARC) (80.46%) nuclei. (E): Table gathering the different stereological values acquired or estimated for each studied animal.

Fig 5: (A): Cumulative food intake (g), measured over a 24 h period, of mice having received an i.c.v. injection of either vehicle (Sodium Chloride (NaCl) 0.9%) or TAT-GAP19 (12.5 or 25 µg). The TAT-GAP19-I14A, which includes a point mutation making it ineffective on connexin 43 hemichannels (Cx43 HCs) activity was tested on food intake at the dose of 25 µg. (B): Graph showing meal size over 24 h after i.c.v. injection of vehicle or TAT-GAP19 25 µg. Note that each bar represents a meal and its width the meal duration. The dark period is represented by the shaded box. (C–F): Meal microstructure analysis performed during the dark period for mice that received either vehicle or TAT-GAP19 25 µg allowed quantification of meals number (C), meals size (in mg of food, (D)), meal duration (in min, (E)) and interprandial period (in min, average intervals between meals, (F–G)): Measure of energy expenditure (in Kcal/day-1/kg0.75) within 12 h after injection of 0.9% NaCl vehicle (n = 7) or 50 µg TAT-GAP19 (n = 7). (H,I): Measure of total spontaneous activity (in Arbitrary Unit, UA; (H) and cumulative spontaneous activity (I) within 12 h for the 2 groups. ns: no significant difference; * p < 0.05, ** p < 0.01 and *** p < 0.001, significantly different from NaCl-treated mice.