Article Figures & Data
Figures
- Figure 1.
sEPSCs in the GABAergic neurons and tonic inhibition in the excitatory neurons in the contralateral M1 were recorded by in vitro slice electrophysiological recording. A, Sample traces showing sEPSCs (clamped at −60 mV) recorded in the GABAergic neurons fromGAD67-GFP transgenic mice in the Sham, Stroke, and Stroke + EA groups. Scale bars: 2 s, 5 pA. B, C, Quantifications of amplitude (Amp) and frequency (Freq) in the contralateral M1 from the Sham, Stroke, and Stroke + EA groups. The amplitude of sEPSCs was not affected by stroke or EA treatment (B). While there is a significant decrease in the frequency of sEPSCs in the Stroke group compared with that in the Sham group, and this impairment could be improved by EA treatment (C). N (cells) = 8–9 per group. N (mice) = 7, 5, and 5 for the Sham, Stroke, and Stroke + EA groups, respectively. Compared with Sham group, *p < 0.05; compared with Stroke group, #p <0.05. D, Sample traces showing sIPSCs (clamped at +10 mV) recorded in the GABAergic neurons from GAD67-GFP transgenic mice in the Sham, Stroke, and Stroke + EA groups. Scale bars: 1 s, 20 pA. E, F, Quantifications of amplitude (Amp) and frequency (Freq) in the contralateral M1 from the Sham, Stroke, and Stroke + EA groups. The amplitude and frequency of sIPSCs was not affected by stroke or EA treatment. N (cells) = 13–17 per group. N (mice) = 7, 5, and 5 for the Sham, Stroke, and Stroke + EA groups, respectively. G, Sample traces showing the recorded tonic inhibition by changes of holding current induced by picrotoxin (PTX; 100 μm) in the excitatory neurons clamped at +10 mV. Scale bars: 4 s,10 pA. H, The tonic inhibition, calculated as the change of holding current after PTX application divided by the capacitance, showed no significant difference among three groups. N (cells) = 6 per group. N (mice) = 3 per group. Data are shown as mean ± SEM. One-way ANOVA.
- Figure 2.
Expression of PNNs was significantly decreased at 1 and 3 d postinjection of ChABC. A, Representative confocal images of PNNs in the Sham, Stroke, and Stroke + EA group. Scale bars: 100 μm. B, Quantification of the density of PNNs in the different groups. The expression of WFA-positive PNNs was significantly increased in the Stroke group, and reduced in the Stroke + EA group. N (slices) = 6–7 per group. N (mice) = 3 per group. Compared with the Sham group, *p < 0.05; Compared with the Stroke group, ^p < 0.05. C, Left, The atlas of the mouse brain to show the tip points of injection needles. Right, Representative confocal images showed the injection site of the ChABC or P-nase in the M1. The dashed line was drawn at the site where the drug was injected and the ablation of the PNNs was shown in the ChABC-injected mice. Scale bars: 200 μm. D, Representative confocal images of PNNs in the Sham and ChABC-injected (300 nl, 40 U/ml) group. Scale bars: 100 μm. E, Quantification of the density of PNNs in the Sham and the ChABC-injected group. A significant decrease in the expression of WFA-positive PNNs was significantly decreased in the first day postinjected ChABC group (1 d) compared with the Sham group and the third day postinjected ChABC group (3 d), while the expression of PNNs was still less than that in the Sham group. N (slices) = 6 per group. N (mice) = 3 per group, respectively. Compared with the Sham group, **p < 0.01, ##p < 0.01; compared with the 1 d group, ^^p < 0.01. F, Representative confocal images of PNNs in the Sham and P-nase (as a control for ChABC)- injected (300 nl, 40 U/ml) group. Scale bars: 100 μm. G, Quantification of the density of PNNs in the Sham and the P-nase-injected group. The density of PNNs was affected in the P-nase-injected mice at neither first nor third postinjection. N (slices) = 6–10 per group. N (mice) = 4, 3, and 3 for the Sham, 1 d, and 3 d groups, respectively. Data are shown as mean ± SEM. One-way ANOVA.
- Figure 3.
Removal of PNNs in the contralateral M1 affected the expression of c-Fos. A, Schematics of experimental design. B, Representative images showing the expression of c-Fos after P-nase injection or ChABC injection into the contralateral M1 of mice in the Sham, Stroke, and Stroke + EA treatment. Scale bars: 50 μm. C, Quantification of the density of c-Fos expression among three groups after P-nase or ChABC injection into the contralateral M1. The results showed that stroke induction and EA stimulation can induce an enhancement of c-Fos expression in the P-nase-injected group. However, the expression of c-Fos showed no change in the ChABC-injected group. The expression of c-Fos showed a significant decrease in the ChABC-injected mice with stroke induction compared with that in the P-nase-injected mice with stroke induction, and this is the case between the ChABC-injected and P-nase-injected mice with stroke induction and EA treatment. N (slices) = 8–10 per group. N (mice) = 3 per group. Compared with the Sham group, *p < 0.05; Compared with the Stroke group, ##p < 0.01, ^^^p < 0.001; compared with the Stroke + EA group, &&&&p < 0.0001. Data are shown as mean ± SEM. Two-way ANOVA.
- Figure 4.
Effects of removal of PNNs to neuronal activity in the contralateral M1 in vivo. A1, Example raster and spectrum for spike firing displaying activity at three different conditions in the P-nase-injected mice. The total time of represented raster and spectrum is 300 s. A2, Example raster and spectrum for spike firing displaying activity at three different conditions in the ChABC-injected mice. The total time of represented raster and spectrum is 300 s. B, The spike frequency was impaired in the stroke condition but was rescued by EA treatment in the P-nase-injected mice. There was no significant decrease in spike frequency among the groups after PNNs ablation by ChABC. The spike frequency showed a significant increase in the ChABC-injected mice with stroke induction compared with that in the P-nase-injected mice with stroke induction, but there is no effect between the ChABC-injected and P-nase-injected mice with stroke induction and EA treatment. P-nase-injected group: N (units) = 23, 12, and 10 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (units) = 17, 26, and 26 for the Sham, Stroke, and Stroke + EA groups, respectively. N (mice) = 4, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. Compared with the Sham group, *p < 0.05; compared with the Stroke group, #p < 0.05, ^p < 0.05. C, The representative action potential waveforms of the putative excitatory and inhibitory neurons. D, The frequency of putative inhibitory neurons was impaired in the stroke condition in the P-nase-injected mice, and there was no significant decrease in the frequency of putative inhibitory neurons among the groups after PNNs ablation by ChABC. The frequency of putative inhibitory neurons showed a significant increase in the ChABC-injected mice with stroke induction and EA treatment compared with that in the P-nase-injected mice with stroke induction and EA treatment. P-nase-injected group: N (units) = 14, 10, and 11 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (units) = 8, 9, and 12 for the Sham, Stroke, and Stroke + EA groups, respectively. N (mice) = 4, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. Compared with the Sham group, **p < 0.01; compared with the Stroke + EA group, &p < 0.05. E, The frequency of putative excitatory neurons was impaired in the stroke condition but was rescued by EA treatment in the contralateral M1 in the P-nase-injected mice. However, there was no significant decrease of frequency in putative excitatory neurons among the groups after PNNs ablation by ChABC. The frequency showed a significant increase in the ChABC-injected mice with stroke induction compared with that in the P-nase-injected mice with stroke induction, but there was a similar effect between the ChABC-injected and P-nase-injected mice with stroke induction and EA treatment. P-nase-injected group: N (units) = 17, 15, and 13 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (units) = 9, 16, and 12 for the Sham, Stroke, and Stroke + EA groups, respectively. N (mice) = 4, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. Compared with the Sham group, ****p < 0.0001; Compared with the Stroke group, #p < 0.05, ^^p < 0.01, ###p < 0.001; compared with the Stroke + EA group, &p < 0.05. Data are shown as mean ± SEM. Two-way ANOVA.
- Figure 5.
Effects of removal of PNNs to sEPSCs in the excitatory neurons in vitro. A1, Sample traces showing sEPSCs recorded in the excitatory neurons clamped at −60 mV in the P-nase-injected mice. Scale bars: 1 s, 20 pA. A2, Sample traces showing sEPSCs recorded in the excitatory neurons clamped at −60 mV in the ChABC-injected mice. Scale bars: 1 s, 20 pA. B, Quantifications of amplitude (Amp) in the contralateral M1 from Sham, Stroke, and Stroke + EA groups in the P-nase or ChABC-injected mice. The results showed that the amplitude of sEPSCs were no altered in the P-nase-treated or ChABC-treated mice. N (cells) = 10–14 per group. P-nase-injected group: N (mice) = 6, 4, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (mice) = 6, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. C, Quantifications of frequency (Freq) in the contralateral M1 from Sham, Stroke, and Stroke + EA groups in the P-nase-injected or ChABC-injected mice. The results showed that stroke affected the frequency of sEPSCs, and this impairment could be improved by EA treatment in the P-nase-injected mice, while the frequency of sEPSCs in the excitatory neurons showed no difference in the ChABC-injected mice. N (cells) = 10–14 per group. P-nase-injected group: N (mice) = 6, 4, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (mice) = 6, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. Compared with the Sham group, *p < 0.05; compared with the Stroke group, #p < 0.05. D1, Sample traces showing sIPSCs recorded in the excitatory neurons clamped at +10 mV in the P-nase-injected mice. Scale bars: 1 s, 20 pA. D2, Sample traces showing sIPSCs recorded in the excitatory neurons clamped at +10 mV in the ChABC-injected mice. Scale bars: 1 s, 20 pA. E, Quantifications of amplitude (Amp) in the contralateral M1 from Sham, and Stroke, and Stroke + EA groups in the P-nase or ChABC-injected mice. The results showed that the amplitude of sIPSCs were no longer influenced in the P-nase-treated or ChABC-treated mice. N (cells) = 8–15 per group. P-nase-injected group: N (mice) = 6, 4, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (mice) = 6, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. F, Quantifications of frequency (Freq) in the contralateral M1 from Sham, Stroke, and Stroke + EA groups in the P-nase or ChABC-injected mice. The results showed that the frequency of sIPSCs were no longer influenced in the P-nase-treated or ChABC-treated mice. N (cells) = 8–15 per group. P-nase-injected group: N (mice) = 6, 4, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. ChABC-injected group: N (mice) = 6, 3, and 3 for the Sham, Stroke, and Stroke + EA groups, respectively. Data are shown as mean ± SEM. Two-way ANOVA.
- Figure 6.
The dysfunction of swallowing function in stroke or the effect of EA disappeared after the removal of PNNs. A1, The representative traces of EMG at the mylohyoid in the P-nase-injected mice in the Sham, Stroke, and Stroke + EA groups, respectively. Scale bars: 2 s, 0.1 mV. A2, The representative traces of EMG at the mylohyoid in the ChABC-injected mice in the Sham, Stroke, and Stroke + EA groups, respectively. Scale bars: 2 s, 0.1 mV. B, Quantification of the area under the curve of EMG from every group. N (mice) = 5–6 per group. The results showed that EA could rescue the impaired swallowing function induced by stroke, and the phenomenon disappeared when PNNs were removed by ChABC, while a significant increase in the ChABC-injected mice with stroke induction compared with that in the P-nase-injected mice with stroke induction. Compared with the Sham group, *p < 0.05; compared with the Stroke group, #p < 0.05, ^p < 0.05. Data are shown as mean ± SEM. Two-way ANOVA.
Tables
Figures Purpose Experiments Number of mice Figure 1A–F To assess the effect of stroke and EA treatment on the synaptic activity Electrophysiological recording in slices GAD67-GFP mice: Sham group (N = 7), Stroke group (N = 5), and Stroke + EA group (N = 5) Figure 1G,H To validated whether the tonic inhibition was changed Electrophysiological recording in slices 9/3* Figure 2A–G To explore the expressions of PNNs in different conditions and show the injection sites of PNNs degradation and test the efficacy of injection of P-nase or ChABC (1) Injection of P-nase or ChABC
(2) ImmunofluorescenceP-nase-injected group (N = 2)
ChABC-injected group (N = 2)
27/3*Figure 3A–C To explore the role of PNNs in the regulation of neuronal activity in the stroke or EA treatment (1) Injection of P-nase or ChABC
(2) Immunofluorescence18/3* Figure 4A–E To explore the role of PNNs in the regulation of neuronal activity in the stroke or EA treatment (1) Injection of P-nase or ChABC
(2) In vivo electrophysiological recording24/4* Figure 5A–F To record the sEPSCs and sIPSCs (1) Injection of P-nase
(2) Electrophysiological recording in slicesSham group (N = 6), Stroke group (N = 4), and Stroke + EA group (N = 3) Figure 5A–F To record the sEPSCs and sIPSCs (1) Injection of ChABC
(2) Electrophysiological recording in slicesSham group (N = 6), Stroke group (N = 3) and Stroke + EA group (N = 3) Figure 6A,B To validate the function of PNNs in the EA-mediated improvement of swallowing function (1) Injection of P-nase or ChABC
(2) Electromyography recording in the mylohyoid in vivo36/6* Total = 160 mice, GAD67-GFP = 17 mice, C57 = 143 mice Full name Abbreviation Analysis of variance ANOVA ChondroitinaseABC ChABC Central nervous system CNS Chondroitin sulfate proteoglycans CSPGs Lianquan CV23 Electroacupuncture EA Extracellular matrix ECM Electromyography EMG Interspike interval ISI Primary motor cortex M1 Neuronal activity-regulated pentraxin Narp Nogo receptor NgR Optimal cutting temperature OCT Orthodenticle homeobox protein 2 OTX2 Phosphate buffer PB Phosphate buffered saline PBS Paraformaldehyde PFA Penicillinase P-nase Perineuronal nets PNNs Poststroke dysphagia PSD Parvalbumin neurons PV+ Semaphorin3A Sema3A Excitatory postsynaptic currents sEPSCs Inhibitory postsynaptic currents sIPSCs 2,3,5-Triphenyltetrazolium chloride TTC Wisteria floribunda agglutinin WFA
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