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1) Product Images from "Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake"
Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake
Figure Legend Snippet: ( A ): Representative images depicting ethidium bromide (EtBr) uptake via hemichannels in hypothalamic (ARC) slices treated with either artificial cerebrospinal fluid (aCSF), 200 μM carbenoxolone (Cbx) or 100 µM the connexin-43 (Cx43) hemichannel blocker TAT-GAP19 for 15 min, then exposed to EtBr for 10 min. After fixation, slices were submitted to Glial Fibrillary Acidic Protein (GFAP) immunostaining and Hoechst labelling. Scale bar: 15 µm. ( B ): Quantification of EtBr fluorescence measured in GFAP+ astrocytes. n = 5, with each n representing the average of three replicates. ** p
Techniques Used: Immunostaining, Fluorescence
Figure Legend Snippet: ( 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
Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay, Western Blot
Figure Legend Snippet: ( 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.
Techniques Used: Labeling, Immunohistochemistry
Figure Legend Snippet: ( 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/kg 0.75 ) 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
Techniques Used: Mouse Assay, Injection, Mutagenesis, Activity Assay