Fig. 4: iβARK decreases astrocytic Ca²⁺ signals, synaptic transmission, extracellular concentration of glutamate in ACC, and alleviates mechanical allodynia in neuropathic pain mice.

a Representative images displaying all AQuA-detected events from a 1-minute ex vivo astrocytic GCaMP7b Ca²⁺ imaging experiment from different groups. Upper: representative image in CCI mice; middle: representative image in iβARK-expression mice; below: representative imaging in mCherry-expression mice; Colors indicate detected events. Scale bar: 10 μm. b Graph depicting the frequency of astrocytic Ca²⁺ signals in the ACC of CCI, iβARK and mCherry mice (n = 6 cells from 3 CCI mice; n = 9 cells from 4 iβARK mice; n = 6 cells from 3 mCherry mice; CCI (red) = 1.169 ± 0.1352 min/100 μm², iβARK (blue) = 0.7121 ± 0.08085 min/100 μm², mCherry (pink) = 1.109 ± 0.1086 min/100 μm², p = 0.0088, F = 6.221, one-way ANOVA followed by Dunnett’s test). c Graph showing the amplitude of astrocytic Ca²⁺ signals in the ACC of CCI, iβARK and mCherry mice (CCI (red) = 1.367 ± 0.2643 ▵F/F, iβARK (blue) = 0.7574 ± 0.04621 ▵F/F, mCherry (pink) = 1.150 ± 0.09837 ▵F/F, p = 0.0179, F = 5.068, one-way ANOVA followed by Dunnett’s test). d Representative traces of miniature excitatory postsynaptic currents (mEPSCs) recorded from layer II-III neurons of ACC in CCI (red), iβARK (blue) and mCherry (pink) mice. Summary of mEPSCs frequency (e, n = 8 cells from 3 CCI mice; n = 10 cells from 4 iβARK mice; n = 5 cells from 3 mCherry mice; CCI (red) = 2.317 ± 0.2897 Hz, iβAKR (blue) = 0.9633 ± 0.06506 Hz, mCherry (pink) = 2.067 ± 0.2584 Hz, p = 0.0001, F = 14.22, one-way ANOVA followed by Dunnett’s test) and amplitude (g, CCI (red) = 16.75 ± 1.102 pA, iβARK (blue) = 13.00 ± 0.5591 pA, mCherry (pink) = 17.80 ± 1.188 pA, p = 0.0001, F = 14.22, one-way ANOVA followed by Dunnett’s test) in CCI, iβARK and mCherry mice. Cumulative probability plots of mEPSCs inter-event intervals (IEI) (f, CCI (red) vs. iβARK (blue), p < 0.0001, Kolmogorov-Smirnov test; CCI (red) vs. mCherry (pink), p = 0.3480, Kolmogorov-Smirnov Test) and mEPSCs amplitude (h, CCI (red) vs. iβARK (blue), p < 0.0001, Kolmogorov-Smirnov test; CCI (red) vs. mCherry (pink), p = 0.1779, Kolmogorov-Smirnov Test) in CCI, iβARK and mCherry mice. i Representative eEPSC traces evoked by pair pulse protocol (PPP) with an interval of 50 ms recorded in the ACC in CCI (red), iβARK (blue), and mCherry (pink) mice. j Summary bar graph of paired-pulse ratio (PPR) index (n = 8 cells from 4 CCI mice; n = 7 cells from 3 iβARK mice; n = 7 cells from 3 mCherry mice; CCI (red) = 1.040 ± 0.04728, iβARK (blue) = 1.402 ± 0.1040, mCherry (pink) = 0.9640 ± 0.04784, p = 0.0007, F = 10.91, one-way ANOVA followed by Dunnett’s test) of eEPSCs in CCI, iβARK and mCherry mice. k Extracellular glutamate concentrations in the dialysate in the ACC of CCI, iβARK and mCherry mice (n = 5 CCI mice; n = 6 iβARK mice; n = 6 mCherry mice; CCI (red) = 8.656 ± 0.4981 μM, iβARK (blue) = 5.665 ± 0.3399 μM, mCherry (pink) = 8.117 ± 0.6241 μM, p = 0.0019, F = 10.10, one-way ANOVA followed by Dunnett’s test). l iβARK significantly increased hind paw withdrawal threshold in neuropathic pain mice (n = 6 CCI mice; n = 8 iβARK mice; n = 6 mCherry mice; Day0_CCI (red circle) = 1.567 ± 0.1585 g, Day0_iβARK (blue circle) = 1.367 ± 0.1579 g, Day0_mCherry (pink circle) = 1.494 ± 0.2304 g, Day14_CCI (red circle) = 0.6078 ± 0.1093 g, Day14_iβARK (blue circle) = 1.533 ± 0.2397 g, Day14_mCherry (pink circle) = 0.5956 ± 0.09532 g; Day14_CCI vs. Day14 iβARK, p = 0.0018, Day14_CCI vs. Day14 mCherry, p = 0.9985, two-way ANOVA (F (2, 17) = 14.14, p = 0.0002), Dunnett’s post hoc test). Error bars represent the mean ± SEM.