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. 2021 Jul 15;15:699315. doi: 10.3389/fncel.2021.699315

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Copyright © 2021 Chai, Chen, Xu, Zhang, Li, Li, Zhang, Zhang, Zhang, Mao, Ding, Xu and Zhou.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Reduced firing rate in vitro with CA1 PTEN knock down (KD). (A) Immunofluorescence staining of NeuN and PTEN on lentivirus-infected dorsal CA1 cells labeled with EGFP. In LV-shRNA-PTEN-GFP group, cells labeled with EGFP were not labeled with anti-PTEN antibody. Calibration bar, 150 μm. (B) Merged bright-field with EGFP fluorescence image showed representative fEPSP recording in virus-affected CA1 region of the dorsal hippocampus by stimulating the Schaffer collaterals in brain slices. Calibration bar, 500 μm. (C) Traces for fEPSP recordings of input-output curve. (D) The input-output curve plotted with stimulus intensity against slope was not significant difference among the groups (n = 8 from 3 mice for Vehicle, n = 7 from 3 mice for the other two groups; Two-way ANOVA with Sidak’s post hoc test. Factor of stimulus intensity x treatment, F_(8,76) = 1.956, P = 0.0636; Factor of stimulus, F_(4,76) = 99.92, P < 0.0001; Factor of treatment, F_{(2, 19)} = 1.338, P = 0.2860; P > 0.05). (E) The input-output curve plotted with stimulus intensity against fiber volley was not significant difference among the groups (n = 8 from 3 mice for Vehicle, n = 7 from 3 mice for the other two groups; Two-way ANOVA with Sidak’s post hoc test. Factor of stimulus intensity x treatment, F_{(8, 76)} = 1.994, P = 0.0584; Factor of stimulus, F_{(4, 76)} = 124.0, P < 0.0001; Factor of treatment, F_{(2, 19)} = 1.326, P = 0.2890; P > 0.05). (F) The input-output curve plotted with slope against fiber volley. (G) A form of short-term synaptic plasticity, the paired-pulse facilitation, was not significant different among the groups (n = 8 from 3 mice for Vehicle, n = 7 from 3 mice for the other two groups, P > 0.05). (H) Another form of short-term synaptic plasticity, the short-term augmentation at 20 Hz, was also not significant different among the groups (n = 8 from 3 mice for Vehicle, n = 7 from 3 mice for the other two groups, P > 0.05). (I) Long-term potentiation was not significant different among the groups (n = 6 from 3 mice for Vehicle, n = 7 from 3 mice for the other two groups, P > 0.05). (J) Whole-cell recordings via the fluorescence of CA1 neurons. Calibration bar, 20 μm. (K) Examples of voltage trace elicited by a constant current injection for 1 s at 300 pA. (L) The first three inter-spike intervals (ISIs) were monitored. The shRNA-PTEN group showed higher ISI in contrast to non-infected and shRNA-NC groups (n = 17 for non-infected from 5 mice, n = 18 for shRNA-NC from 4 mice, n = 18 for shRNA-PTEN from 6 mice; 1st, P = 0.0037. χ2 = 11.18. 1st Vehicle vs. 1st shRNA-NC, P = 0.9594; 1st Vehicle vs. 1st shRNA-PTEN,P = 0.0035; 1st shRNA-NC vs. 1st shRNA-PTEN, P = 0.0664; 2nd, P = 0.0061. χ2 = 10.20. 2nd Vehicle vs. 2nd shRNA-NC, P > 0.9999; 2nd Vehicle vs. 2nd shRNA-PTEN,P = 0.0081; 2nd shRNA-NC vs. 2nd shRNA-PTEN, P = 0.0448; 3rd, P = 0.0059. χ2 = 10.26. 3rd Vehicle vs. 3rd shRNA-NC, P > 0.9999; 3rd Vehicle vs. 3rd shRNA-PTEN,P = 0.0096; 3rd shRNA-NC vs. 3rd shRNA-PTEN, P = 0.0326). (M) The firing rate of the evoked action potentials was significantly reduced in shRNA-PTEN neurons compared with NC or non-infected neurons (n = 17 for non-infected from 5 mice, n = 18 for shRNA-NC from 4 mice, n = 18 for shRNA-PTEN from 6 mice; ^∗∗∗P < 0.001). (N) Sample traces of after-hyperpolarization (AHP) of action potentials elicited by a constant current injection for 1 s at 300 pA. (O) Increased AHP at 25 ms after the action potential onset in shRNA-PTEN neurons compared with NC and non-infected neurons (n = 17 for non-infected from 5 mice, n = 18 for shRNA-NC from 4 mice, n = 18 for shRNA-PTEN from 6 mice; P = 0.0002. χ2 = 17.59. non-infected vs. shRNA-NC, P > 0.9999; non-infected vs. shRNA-PTEN,P = 0.0004; shRNA-NC vs. shRNA-PTEN, P = 0.0027). (P) Representative response of voltage steps by injecting hyperpolarization current from resting membrane potential. (Q) No significant difference in resting membrane potential (RMP) among the groups (n = 18 for shRNA-PTEN from 6 mice, n = 17 for non-infected from 5 mice, n = 16 for shRNA-NC from 4 mice, P > 0.05). (R) Input resistance was significantly different among the groups (n = 18 for shRNA-PTEN from 6 mice, n = 17 for non-infected from 5 mice, n = 16 for shRNA-NC from 4 mice; P = 0.0368, χ2 = 6.602. Dunn’s multiple comparisons test, non-infected vs. shRNA-NC, P = 0.4762; non-infected vs. shRNA-PTEN, P = 0.7410; shRNA-NC vs. shRNA-PTEN, P = 0.0307). (S) No sag ratio difference among the groups (n = 18 for shRNA-PTEN from 6 mice, n = 17 for non-infected from 5 mice, n = 16 for shRNA-NC from 4 mice, F_{(2, 48)} = 1.392, P = 0.2585). Data presented as mean ± SEM. Statistical analysis was performed by using Kruskal–Wallis test followed by Dunn’s post hoc analysis and two-way or one-way ANOVA followed by Tukey’s post hoc analysis.