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. 2019 Jan 24;6(1):ENEURO.0292-18.2018. doi: 10.1523/ENEURO.0292-18.2018

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Copyright © 2019 Chander et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 4. — Knock-out of NSG2 decreases mEPSC frequency. A, Representative confocal images of primary hippocampal neurons at DIV15 showing robust NSG2 (magenta) in MAP2⁺ (blue) neurons transduced with control CRISPR GFP lentivirus (cyan; top panels), whereas neurons transduced with CRISPR KO NSG2 lentivirus (cyan, bottom panels) show the absence of NSG2 (arrow; bottom panels); NSG2 (magenta) is present in an adjacent neuron not transduced with the CRISPR KO NSG2 lentivirus in the same field (arrowhead; bottom panels). B, Representative traces from whole-cell patch clamp recordings from neurons expressing either control CRISPR GFP (upper trace, black) or CRISPR KO NSG2 (bottom trace, green). Averaged mEPSCs from both control (black, n = 10) and NSG2 KO (green, n = 13) are shown to the right. C, Pooled data revealed a significant decrease in mEPSC frequency in neurons expressing NSG2 KO compared to cells expressing control CRISPR GFP (**p = 0.001). The amplitude of mEPSCs was not significantly different between groups (p = 0.34). Data in the KO group were derived only from neurons devoid of NSG2 confirmed by post-recording immunostaining of Lucifer yellow injected neurons (Extended Data Fig. 4-1). NSG2 KO did not alter outward potassium I/V relationship (Extended Data Fig. 4-2). D, Quantification of presynaptic marker Synapsin1⁺ punctae (control, n = 10; NSG2 KO, n = 10; p = 0.46) and postsynaptic marker PSD95⁺ punctae (control, n = 9; NSG2 KO, n = 11; p = 0.18). E, Representative confocal images illustrate PSD95 immunofluorescence (cyan) in neurons expressing CRISPR KO NSG2 (right panels) or controls CRISPR GFP (left panels). GFP expression from both groups (top panels) is presented in grayscale for clarity. Quantification revealed a significant reduction in PSD95 fluorescence intensity in neurons expressing NSG2 KO (n = 11) compared to controls (n = 9; *p = 0.029). F, Pooled data show that the number of surface GluA1⁺ punctae (left; control, n = 10 and NSG2 KO, n = 9; p = 0.86) and surface GluA2⁺ punctae (right; control, n = 10 and NSG2 KO, n = 9; p = 0.18) remained unchanged between groups. Bars represent mean ± SEM. Scale bars = 10 μm (A) and 1 μm (E).