Fig 1: Changes in ASIC2a expression affected the intrinsic excitability of CA1 pyramidal neurons. (a) Confocal images of CA1 pyramidal neurons expressing GFP (green) and labelled with neurobiotin (blue). Scale bar = 10 µm. (b) Representative traces of action potential firing in response to 200 pA current injections in CA1 pyramidal neurons of the negative control, ASIC2a overexpression, and ASIC2a knockdown groups, respectively. (c) Number of action potentials in CA1 pyramidal neurons from the various groups at different current injection steps. Data are presented as means ± standard errors and were analysed using 1- or 2-way ANOVA and Dunnett's multiple comparisons test. *P < 0.05, ASIC2a OE group compared with negative control group; #P < 0.05, ASIC2a KD group compared with negative control group. Abbreviations, ASIC2a: acid-sensing ion channel 2a; GFP: green fluorescent protein; OE: overexpression; KD: knockdown.
Fig 2: Cellular localisation of TFCP2 and ASIC2a in the epileptic rats’ CA1 regions and glucose-deficient cells. (a) Double immunofluorescence labelling for TFCP2 and ASIC2a in the epileptic rats’ CA1 regions during acute and latent post-seizure phases. (b) Cellular localisation of TFCP2 and ASIC2a expression in PC12 cells grown for 24 h in high-glucose media (control), low-glucose media, no-glucose media, and STF-31 (10 µM)-loaded media. Proteins were probed with anti-ASIC2a (green) and anti-TFCP2 (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DAPI: 4′,6-diamidino-2-phenylindole.
Fig 3: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in patients with TLE. (a) Patient 4’s pre-surgical assessment results: magnetic resonance imaging (left) was negative, electroencephalography (middle) showed spike waves in the temporal lobe, and fluorodeoxyglucose positron emission tomography (right) revealed hypometabolic lesions in the right hippocampus. (b) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression in patients with TLE (n = 13) and control patients (n = 10). ß-actin was used as a loading control. (c,d) Normalised densitometry bar graphs of TFCP2 and ASIC2a for the control subjects and patients with TLE. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P < 0.05, **P < 0.01 compared to controls. Uncropped western blot images are shown in Supplementary Fig. 1. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; TLE: temporal lobe epilepsy.
Fig 4: Hippocampal ASIC2a overexpression increased seizure susceptibility. (a) Representative images showing GFP immunoreactivity in the hippocampal CA1 region following adeno-associated virus (AAV) vector infusion. Scale bar = 100 μm. (b) The time interval from pilocarpine injection to Racine IV seizures was significantly shorter in the ASIC2a overexpression group (26.3 ± 1.3 min) than in the negative control AAV group (31.9 ± 1.9) (n = 30 rats/group). (c) The proportion of rats with Racine IV seizures after pilocarpine treatment was significantly higher in the ASIC2a overexpression group (93.3 ± 3.33%) than in the negative control AAV group (66.7 ± 3.33%). Data are presented as means ± standard errors and were analysed using unpaired t-tests or Chi-square tests. *P < 0.05 compared with negative control group. Abbreviations, ASIC2a: acid-sensing ion channel 2a; GFP: green fluorescent protein; DAPI: 4′, 6-diamidino-2-phenylindole; OE: overexpression.
Fig 5: Glucose deficiency influenced TFCP2 and ASIC2a expression in PC12 cells. (a) Representative immunoblot and densitometric analyses showing that cells grown in low-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 12 and 24 h of growth. (b) Representative immunoblot and densitometric analyses showing that cells grown in no-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 6, 12, and 24 h of growth. (c) Representative immunoblot and densitometric analyses showing that STF-31-treated PC12 exhibited significant downregulation of TFCP2 and significant upregulation of ASIC2a relative to DMSO-treated control cells. (d) Representative immunoblot and densitometric analyses showing TFCP2 and ASIC2a expression in 2-deoxy-D-glucose-treated PC12 cells cultured in no-glucose media. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01 compared to controls; #P < 0.05, ##P < 0.01 compared to the DMSO group. Uncropped western blot images are shown in Supplementary Fig. 1. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DMSO: dimethyl sulfoxide; 2-DG: 2-deoxy-D-glucose; Con: control.
Supplier Page from Abcam for Anti-ASIC2 antibody [EPR11029]