Acid-sensing ion channel 1a drives AMPA receptor plasticity following ischaemia and acidosis in hippocampal CA1 neurons

Abstract

Key points: The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (AMPAR) plasticity - an anoxic form of long-term potentiation and a delayed increase in Ca(2+) -permeable (CP) AMPARs - contribute to this susceptibility by increasing excitotoxicity. In CA1, the acid-sensing ion channel 1a (ASIC1a) is known to facilitate LTP and contribute to ischaemic acidotoxicity. We have examined the role of ASIC1a in AMPAR ischaemic plasticity in organotypic hippocampal slice cultures exposed to oxygen glucose deprivation (a model of ischaemic stroke), and in hippocampal pyramidal neuron cultures exposed to acidosis. We find that ASIC1a activation promotes both forms of AMPAR plasticity and that neuroprotection, by inhibiting ASIC1a, circumvents any further benefit of blocking CP-AMPARs. Our observations establish a new interaction between acidotoxicity and excitotoxicity, and provide insight into the role of ASIC1a and CP-AMPARs in neurodegeneration. Specifically, we propose that ASIC1a activation drives certain post-ischaemic forms of CP-AMPAR plasticity.

Abstract: The CA1 region of the hippocampus is particularly vulnerable to ischaemic damage. While NMDA receptors play a major role in excitotoxicity, it is thought to be exacerbated in this region by two forms of post-ischaemic AMPA receptor (AMPAR) plasticity - namely, anoxic long-term potentiation (a-LTP), and a delayed increase in the prevalence of Ca(2+) -permeable GluA2-lacking AMPARs (CP-AMPARs). The acid-sensing ion channel 1a (ASIC1a), which is expressed in CA1 pyramidal neurons, is also known to contribute to post-ischaemic neuronal death and to physiologically induced LTP. This raises the question does ASIC1a activation drive the post-ischaemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD), and dissociated cultures of hippocampal pyramidal neurons (HPNs) exposed to low pH (acidosis). We find that both a-LTP and the delayed increase in the prevalence of CP-AMPARs are dependent on ASIC1a activation during ischaemia. Indeed, acidosis alone is sufficient to induce the increase in CP-AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP-AMPARs. By demonstrating that ASIC1a activation contributes to post-ischaemic AMPAR plasticity, our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells, and provide insight into the role of ASIC1a and CP-AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP-AMPAR plasticity, and that inhibiting ASIC1a affords neuroprotection.

Figure 1. Anoxic LTP relies on ASIC1a in OHSCs

A, theta‐burst stimulation (TBS) induced changes in the slope of AMPAR‐mediated fEPSPs measured in the CA1 stratum radiatum of OHSCs from WT (○, n = 4) and ASIC1a KO (●, n = 5) mice. Inset: representative baseline (black trace) and 30 min after TBS (grey trace) recordings; Scale bars: 25 ms and 1 mV. B, experimental protocol for a‐LTP experiments. C, changes of fEPSP slope induced by 10 min OGD in WT (○, n = 3) and ASIC1a KO (●, n = 4) OHSCs. The increase in slope observed in WT corresponds to a‐LTP. Inset: representative traces before (black trace) and 30 min after OGD (grey trace). Scale bars: 25 ms and 1 mV. D, representative Western blots showing changes in GluA2 subunit expression 1 h after OGD. OHSCs from WT or ASIC1a KO animals were exposed either to control (−) or to OGD (+) conditions. Some WT slices were treated with psalmotoxin 1 (PcTx1, 100 nm) during or after OGD; actin was used for normalization. E, relative GluA2 subunit abundance compared with controls expressed as a percentage (n = 6–14). Error bars, SEM; *P < 0.05; n.s., not significant.

Figure 2. OGD induced increase of CP‐AMPARs is ASIC1a dependent in slice cultures

A, experimental protocol. B and C, effect of NASPM (100 μm) on fEPSP slope before (0 h), 6 h after, and 12 h after OGD. Representative recordings (B), before (black traces) and after (grey traces) application of NASPM; scale bar, 25 ms and 0.5 mV. fEPSP slope ratio (C), after vs. before NASPM. D–I, changes in AMPAR‐mediated EPSCs recorded in CA1 neurons in cells derived from WT (D–F, n = 4–6) and ASIC1a KO (G–I, n = 4) mice. D and G, representative EPSC traces at +40 and −70 mV recorded 6 h (left hand side) and 12 h (right) after 10 min OGD treatment; scale bar, 25 ms and 25 pA. E and H, current–voltage plots at 6 h and 12 h after OGD. F and I, EPSC rectification index; RI_+40/−40 = EPSC amplitude at +40 mV/−40 mV. J and K, changes in GluA2 subunit expression 12 h after OGD treatment. Cells from WT or ASIC1a KO animals were exposed either to control (−) or to OGD (+) conditions. Some WT slices were treated with psalmotoxin 1 (PcTx1, 100 nm) during or after OGD. J, representative Western blots, obtained for GluA2 subunits, NeuN and actin (for normalization). NeuN level is relatively stable. K, relative GluA2 abundance, compared with controls expressed as a percentage (n = 8–10). Error bars, SEM; *P < 0.05, **P < 0.005, ***P < 0.0005; n.s., not significant.

Figure 3. GluA1 subunit is downregulated in OHSCs exposed to OGD

A and B, changes in GluA1 subunit expression 12 h after OGD treatment. OHSCs from WT animals were exposed either to control (−) or to OGD (+) conditions. A, Western blots, obtained for GluA1 subunits, GluA2 subunits and actin (for normalization) in three representative experiments (Exp. 1–3). B, relative GluA1 abundance, compared with controls expressed in percentage (n = 8). GluA1 subunit abundance decreases in a similar way to GluA2 in total protein extracts of OHSCs. Error bars, SEM; ***P < 0.0005.

Figure 4. Acidosis induces an ASIC1a‐dependent switch in AMPAR composition in cultured neurons

A, experimental protocol. B, I–V plots of AMPAR currents measured in outside‐out patches from hippocampal pyramidal neurons (HPNs) exposed to pH 7.4 (control, ○, n = 3) or pH 6.0 (acidosis, ●, n = 7). C, AMPAR current rectification index, in control cells and following pH 6.0 treatment; effect of pH 6.0 on RI is blocked by psalmotoxin 1, (PcTx1, 20 nm) (RI_+60/−60, n = 11–26). D, typical current traces at +60 and −60 mV. For ease of comparison, currents at −60 mV are scaled to the same peak amplitude. Upper traces: HPNs exposed to pH 7.4 (left) or pH 7.4 + PcTx1 (right). Lower traces: HPNs exposed to pH 6.0 (left) or pH 6.0 + PcTx1 (right). E, AMPAR single‐channel conductance (n = 7–16). F, bar graph comparing the effects of pH 6.0 treatment in the presence of d‐AP5 (50 μm), cadmium (200 μm) and TTX (1 μm) on AMPAR rectification index. G, Western blots of total and cell surface expression of GluA2 in HPNs 12 h after treatment, characterized by cell surface biotinylation followed by Western blotting (WB) (n = 8). H and I, pooled data from pH 7.4 and pH 6.0 treated hippocampal cultures assayed for total and surface (membrane) GluA2. Error bars, SEM; *P < 0.05, **P < 0.005, ***P < 0.0005; n.s., not significant.

Figure 5. Acidification does not alter GluA1 expression in pyramidal neurons

A, Western blot analysis of total and cell surface GluA1 from cultured hippocampal pyramidal neurons (HPNs) 12 h after exposure to either pH 7.4 or pH 6.0. B and C, pooled data from 5 independent cell surface biotinylation experiments demonstrating no change in cell surface expression of GluA1 following acidification (P > 0.05, n = 5). D, Western blot analysis of the ratio of total protein expression level of GluA1 to the total of GluA2 (GluA1/GluA2) following acidification. This ratio increased significantly (0.70 ± 0.12 to 2.0 ± 0.32; P < 0.05, n = 5).

Figure 6. Combining ASIC1a and CP‐AMPAR inhibitors does not provide further neuroprotection against ischaemia or acidosis

A, evaluation of OGD‐induced degeneration in organotypic cultures with propidium iodide (PI) uptake quantification. Representative pictures of PI uptake in slices at 12 and 24 h after OGD. Scale bar = 250 μm. WT slices exposed to OGD were treated for the first 6 h with either saline (control), PcTx1 (100 nm), NASPM (100 μm) or both inhibitors together. B and C, quantification of the neuroprotection by ion channel inhibitors in the CA1 region at 12 and 24 h after OGD. The percentage of neuroprotection was calculated as detailed in Methods (n = 3 cultures, number of slices/condition = 6–12). D, confocal images of hippocampal pyramidal cells 24 h after a 15 min treatment with pH 7.4, pH 6.0, pH6.0 + PcTx1 (20 nm, during pH 6.0), pH 6.0 + NBQX (10 μm, after pH 6.0), or pH 6.0 + PcTx1 and NBQX together. Top left, double staining for MAP2 (green) and DAPI (blue) confirms the purity of HPN cultures. Other pictures, neuronal injury and death in HPN cultures assessed with caspase‐3 cleaved form staining (red) and the percentage of abnormal nuclei (stained in blue with DAPI) as markers. Scale bar = 25 μm. Typical nuclei are indicated by an arrowhead and represented at high magnification in the right bottom corner. E and F, quantification of the cleaved caspase‐3 signal, and of the percentage of abnormal nuclei. G, representative images of propidium iodide uptake in hippocampal slices from WT and ASIC1a knockout at 2 and 24 h after OGD. H, pooled data showing the protective effect of ASIC1a knockout on OGD mediated cell death (n = 4). Error bars, SEM; *P < 0.05, **P < 0.005, ***P < 0.0005 vs. pH 7.4; n.s., not significant.