Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Acid (pH 6.5)-induced changes in cytosolic Ca 2+ signal, indicated by GCaMP6 fluorescence, in cultured mouse hypothalamic neurons (n = 77 in Vector group, n = 63 in OE ASIC1a group, n = 48 in shASIC1a group, n = 73 in OE ASIC1a + PcTx1 group, n = 60 in OE ASIC1a + Amiloride group). B Representative western blot assay showing the expression of calcium signaling pathway-related proteins in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 4F. C The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of P-CaMKII (n = 3 per group). ** p < 0.01 (one-way ANOVA). D The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). E The bar graphs show the mRNA expression levels of c-Fos in primary hypothalamic neurons detected by qPCR. Experimental treatments were conducted as in Fig. 4F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). F Representative confocal images (scale bar = 10 μm) of c-Fos and CRH in primary hypothalamic neurons overexpressing GFP/ASIC1-GFP or knockdown of ASIC1a for 48 h, before or 15 min after treatment with HBSS at pH 6.5 with or without 100 nM PcTx1 or 200 nM amiloride. G The bar graph illustrated the relative fluorescence intensity of c-Fos shown in Fig. 5F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). H The bar graph shows the secretion levels of CRH in primary hypothalamic neuron culture supernatants detected by ELISA, after treatment with pH 6.5 HBSS or simultaneous treatment with 80 μM T-5224 for 30 min following 48-h overexpression of ASIC1a (n = 3 per group). * p < 0.05 (one-way ANOVA). I Representative western blot assay showing the protein expression levels of CRH, ASIC1a, and c-Fos in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 5H. J The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). K The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of CRH (n = 3 per group). * p < 0.05 (one-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Fluorescence, Cell Culture, Plasmid Preparation, Western Blot, Expressing, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression