Research ArticleNeuroscience

The acid-sensing ion channel ASIC1a mediates striatal synapse remodeling and procedural motor learning

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Sci. Signal.  07 Aug 2018:
Vol. 11, Issue 542, eaar4481
DOI: 10.1126/scisignal.aar4481
  • Fig. 1 ASIC1a is enriched in the PSD fraction of the mouse striatum and is involved in regulating motor coordination and learning.

    (A) Quantitative polymerase chain reaction (PCR) assessment of Asic1a mRNA amounts in the mouse cortex, hippocampus (Hipp), and striatum. n = 3 for each group. *P < 0.05 and ***P < 0.001, unpaired Student’s t test. (B and C) Representative immunoblots (B; −/−, striatum from an Asic1a KO mouse) and quantification (C) of ASIC1a protein abundance in the mouse cortex, hippocampus, and striatum. n = 3 for each group. *P < 0.05 and ***P < 0.001, unpaired Student’s t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Enrichment of ASIC1a in the PSD fraction of the striatum shown by immunoblotting. GluN2B was used as a positive control. (E and F) Representative patch-clamp traces (E) and statistical analysis (F) of evoked EPSCs in dorsal striatal MSNs from WT (+/+) and Asic1a KO (−/−) mice before (WT, light gray; KO, pale red) and after (WT, black; KO, red) the application of CNQX (20 μM), d-APV (50 μM), and PTX (100 μM), without or with amiloride (500 μM) (WT, orange; KO, yellow). Arrows (E: left, WT, black; right, KO, red) indicate ASIC-dependent synaptic currents. n = 6 cells from three WT mice and 7 cells from three Asic1a KO mice. *P < 0.05, **P < 0.01, and ***P < 0.001, paired Student’s t test, comparison as indicated. ##P < 0.01, WT versus KO, unpaired Student’s t test. (G) Motor-related behaviors of WT and Asic1a null mice in the incremental fixed-speed rotarod learning test. n = 10 for each group. Two-way repeated-measures analysis of variance (ANOVA), main effect of genotype, F1,100 = 6.824, P = 0.011. *P < 0.05, unpaired Student’s t test. (H and I) Assessment of motor coordination, as time to fall (H) and distance traveled (I) in beam walking tests, in WT and Asic1a null mice. n = 7 to 9 for each group. Two-way repeated-measures ANOVA, main effect of genotype, F1,80 = 24.107, P < 0.001. *P < 0.05 and **P < 0.01, unpaired Student’s t test.

  • Fig. 2 Loss of ASIC1a increases dendritic spine density but disrupts spine maturation in dorsal striatum.

    (A and B) Representative micrographs (A) and statistical analysis (B) of Golgi staining of striatal dendritic segments and spines of WT (Asic1a+/+) and Asic1a KO (Asic1a–/–) mice. Data are mean and distribution of 109 dendritic segments from eight WT mice and 66 dendritic segments from six KO mice. ***P < 0.001, unpaired Student’s t test. (C and D) Cumulative plots showing spine head width (C) and length (D) in MSNs from WT and Asic1a–/– mice. n = 2946 (C) and 2971 (D) spines from three WT mice and n = 2952 (C) and 2826 (D) spines from four KO mice. ***P < 0.001, two-sample Kolmogorov-Smirnov test. (E to G) Representative traces (E) and cumulative distribution plots of amplitudes (F) and frequency (G) of mEPSCs obtained by patch recordings from MSNs in dorsal striatal slices of WT (Asic1a+/+) and KO (Asic1a−/−) mice. Inset bar graphs show average for each; n = 26 cells from eight WT mice and 27 cells from six KO mice. (H to L) Quantification of different subtypes of spines in WT and KO MSNs. n = 24 dendritic segments from three mice for each genotype. *P < 0.05 and ***P < 0.001, unpaired Student’s t test.

  • Fig. 3 PSDs in striata of Asic1a null mice exhibit altered morphology and protein compositions.

    (A) Representative electron micrographs of striatal neurons from WT (Asic1a+/+) and Asic1a KO (Asic1a–/–) mice depicting the synaptic contact and PSD. (B) Cumulative plots showing the PSD thickness in WT and Asic1a KO MSNs. n = 719 PSDs from three WT mice and 967 PSDs from four KO mice. ***P < 0.001, two-sample Kolmogorov-Smirnov test. (C) Cumulative plots showing PSD length in WT and Asic1a KO MSNs. n = 753 PSDs from three WT mice and 1100 PSDs from four KO mice. ***P < 0.001, two-sample Kolmogorov-Smirnov test. (D and E) Immunoblots of PSD and total proteins including GluN1, GluN2A, GluN2B, and PSD95, but not GluA1, GluA2, and PICK1, in striata prepared from WT (+/+) and Asic1a null (–/–) mice: (D) representative immunoblots; (E) statistical analysis of the results. n = 4 to 8 each group. *P < 0.05 and **P < 0.01, WT versus KO, unpaired Student’s t test.

  • Fig. 4 Asic1a null mice exhibit impaired NMDAR function.

    (A and B) Representative traces (A) and statistical analysis (B) of NMDAR (top) and AMPAR (bottom) currents recorded with patch clamp from striatal MSNs in dorsal striatal slices prepared from WT (+/+; left) and Asic1a KO (−/−; right) mice. Data are NMDAR/AMPAR ratios for 14 cells from four WT mice and 12 cells from five Asic1a KO mice. Stimulation artifacts were removed for clarity. **P < 0.01, unpaired Student’s t test. (C and D) Locomotor behaviors of WT and Asic1a null mice in the open field test in response to intraperitoneal injection of NMDAR antagonist MK-801 (0.25 mg/kg, intraperitoneally at time 0). n = 16 for each group: (C) distance traveled of WT (Asic1a+/+) and Asic1a–/– mice in each 10-min interval within the 120-min test. Two-way repeated-measures ANOVA, main effects of genotype, F1,384 = 61.213, P < 0.001. *P < 0.05, **P < 0.01, and ***P < 0.001, Asic1a+/+ versus Asic1a–/–, unpaired Student’s t test. (D) Total distance traveled by Asic1a+/+ and Asic1a–/– mice during the entire 120-min test. ***P < 0.001, unpaired Student’s t test.

  • Fig. 5 Striata of Asic1a null mice exhibit impaired activation of CaMKII-ERK signaling.

    (A to D) Representative immunoblots and statistical analysis of phosphorylated (“p-”) and total protein abundance of CaMKII (A and B) and ERK1 and ERK2 (C and D) in striata from WT (+/+) and Asic1a null (−/−) mice. n = 4 to 6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired Student’s t test. (E to H) Effects of treatment with acid (pH 6.0) on the phosphorylation of CaMKII (E and F) and ERK (G and H). (E and G) Representative immunoblots and analysis showing phosphorylated and total protein abundances of CaMKII (E and F) and ERK1 and ERK2 (G and H) in cultured striatal neurons prepared from WT mice and maintained at pH 7.4 only or exposed to a pH 6.0 external solution for 2 min without or with pretreatment (30 min) of PcTX1 (20 nM) or KN93 (10 μM), as indicated. n = 6 to 15 for each group. **P < 0.01 and ***P < 0.001, compared with pH 7.4 only; #P < 0.05, compared with pH 6.0 alone, unpaired Student’s t test. (I to L) Representative immunoblots and statistical analysis of the effects of the same acid treatment on the phosphorylation of CaMKII (I and J) and ERK (K and L) in cultured striatal neurons prepared from Asic1a null (Asic1a–/–) mice. n = 3 for each group.

  • Fig. 6 Reintroduction of ASIC1a in the striatum rescued defects of CaMKII-ERK signaling, glutamate receptor function, motor coordination, and motor learning in Asic1a KO mice.

    (A) Schematics of AAV vectors engineered to express a control construct [green fluorescent protein (GFP)] or ASIC1a. (B) Verification of AAV injection and expression, showing an example of AAV-mediated enhanced yellow fluorescent protein (EYFP) expression in the striatum. Scale bar, 1 mm. (C) Expression of ASIC1a protein (top) in striata of Asic1a null mice after the injection of AAV-ASIC1a, but not control (GFP), as shown by immunoblotting. A GFP antibody was used to detect both GFP from the AAV-GFP virus and EYFP from the AAV-ASIC1a virus (second panel from the top). Note that EYFP generated from the EYFP-2A-mASIC1a fusion protein by cleavage contained 21 extra amino acids of the 2A peptide and therefore migrated slower than just GFP. The fourth and sixth panels show that AAV-ASIC1a injection into the striata of Asic1a null (Asic1a–/–) mice also restored the phosphorylation levels of CaMKII and ERKs, respectively. (D and E) Statistic results for the ratios of phosphorylated/total CaMKII (D) and ERKs (E). n = 3 experiments for each group. *P < 0.05, unpaired Student’s t test. (F and G) Effects of AAV injection into the striata on the NMDAR/AMPAR ratio in MSNs. (F) Representative current traces of NMDAR (top) and AMPAR (bottom) currents, respectively. (G) Statistic results. n = 12 cells from three mice for each group. **P < 0.01 and ###P < 0.001, unpaired Student’s t test. (H and I) Effects of AAV injection into the striata on the performance in beam walking (H) and learning to cross the 5-mm square beam (I). (H) n = 8 to 10 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the Asic1a+/+ + AAV-GFP group. #P < 0.05 and ##P < 0.01, compared with the Asic1a–/– + AAV-GFP group. (I) n = 10 to 11 for each group. KO: two-way repeated-measures ANOVA, main effects of AAV, F1,110 = 26.706, P < 0.001.

  • Fig. 7 Overexpression of CaMKII in the striatum corrects the defects of CaMKII-ERK signaling, NMDAR function, and motor learning in Asic1a KO mice.

    (A) Schematics of AAV vectors engineered to express a control construct (GFP) or CaMKII. (B) Verification of CaMKII overexpression and its effects on CaMKII and ERK phosphorylation in striata of Asic1a null mice: representative immunoblots. (C and D) Statistic results for the total and phosphorylation levels of CaMKII (C) and the ratios of phosphorylated/total ERKs (D). n = 4 to 5 experiments for each group. *P < 0.05 and **P < 0.01, unpaired Student’s t test. (E and F) Effects of AAV injection into the striata of Asic1a null mice on the NMDAR/AMPAR ratio in MSNs. (E) Representative current traces of NMDAR (top) and AMPAR (bottom) currents, respectively. (F) Statistic results. n = 10 cells from three AAV-GFP–injected mice and 7 cells from three AAV-CaMKII–injected Asic1a KO mice. **P < 0.01, unpaired Student’s t test. (G and H) Effects of AAV injection into the striata of Asic1a null mice on the performance on beam walking (G) and learning to cross the 5-mm square beam (H). (G) n = 7 to 8 for each group. *P < 0.05 and **P < 0.01, unpaired Student’s t test. (H) n = 7 to 8 for each group. Two-way repeated-measures ANOVA, main effects of AAV, F1,75 = 17.844, P < 0.001. *P < 0.05 and **P < 0.01, unpaired Student’s t test.

  • Fig. 8 Proposed mechanism for ASIC1a regulation of striatal synaptic remodeling and motor learning.

    Postsynaptic ASIC1a channels are activated by drops in the pH in the synaptic cleft associated with striatal synaptic activities, leading to cation (such as Na+ or Ca2+) influx. The increase in intracellular Ca2+ activates the downstream CaMKII-ERK signaling pathway. Whereas CaMKII presumably contributes to actin dynamics to promote structural remodeling of dendritic spines and regulates postsynaptic distribution and function of NMDAR, ERK signaling presumably participates in the activity-dependent transcriptional regulation of a set of neuronal proteins, which, in turn, drive long-term synaptic plasticity. Together, the ASIC1a-CaMKII-ERK signaling cascade represents a novel molecular mechanism that facilitates synaptic remodeling in the striatum, which is important for procedural motor learning.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/542/eaar4481/DC1

    Fig. S1. Effects of loss of ASIC1a in mice on Asic2a mRNA expression in different brain regions.

    Fig. S2. Effects of Asic1a gene ablation in mice on basal locomotor activity and motor coordination in a series of behavioral paradigms.

    Fig. S3. Demonstration of Golgi staining and analysis.

    Fig. S4. Input-output relationships of AMPAR- and NMDAR-mediated synaptic currents in striatal neurons from WT and Asic1a KO mice.

    Fig. S5. Effects of loss of ASIC1a on the activation of p38 or JNK signaling in striata.

    Fig. S6. Effects of acidosis on the phosphorylation of ERK, p38, or JNK in HEK-293 cells.

    Fig. S7. Effects of pharmacological inhibition of ASIC1a in the striatum on motor learning in the beam walking test.

    Fig. S8. Representative images showing expression patterns of viral-encoded proteins (represented by EYFP) after AAV-ASIC1a injection into striata of Asic1a KO mice.

    Fig. S9. Effects of reexpression of ASIC1a in striata of Asic1a null mice on spine density and maturation.

    Fig. S10. Effects of reexpression of ASIC1a in striata of Asic1a null mice on the performance in the inclined beam walking test.

  • This PDF file includes:

    • Fig. S1. Effects of loss of ASIC1a in mice on Asic2a mRNA expression in different brain regions.
    • Fig. S2. Effects of Asic1a gene ablation in mice on basal locomotor activity and motor coordination in a series of behavioral paradigms.
    • Fig. S3. Demonstration of Golgi staining and analysis.
    • Fig. S4. Input-output relationships of AMPAR- and NMDAR-mediated synaptic currents in striatal neurons from WT and Asic1a KO mice.
    • Fig. S5. Effects of loss of ASIC1a on the activation of p38 or JNK signaling in striata.
    • Fig. S6. Effects of acidosis on the phosphorylation of ERK, p38, or JNK in HEK-293 cells.
    • Fig. S7. Effects of pharmacological inhibition of ASIC1a in the striatum on motor learning in the beam walking test.
    • Fig. S8. Representative images showing expression patterns of viral-encoded proteins (represented by EYFP) after AAV-ASIC1a injection into striata of Asic1a KO mice.
    • Fig. S9. Effects of reexpression of ASIC1a in striata of Asic1a null mice on spine density and maturation.
    • Fig. S10. Effects of reexpression of ASIC1a in striata of Asic1a null mice on the performance in the inclined beam walking test.

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