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. 2021 Jan 11;13(2):e12632. doi: 10.15252/emmm.202012632

Figure 5. Knockdown of SLC6A20A expression by antisense oligonucleotide increases whole‐brain glycine levels and NMDAR function and rescues repetitive climbing in PtenΔC / ΔC mice.

Figure 5

  1. Schematic representation for the intracerebroventricular (ICV) injection of Slc6a20a antisense oligonucleotide (ASO) into the lateral ventricle (LV) for the reduction of Slc6a20a expression in the whole mouse brain.
  2. Slc6a20a‐ASO decreases the levels of Slc6a20a mRNAs in the brain of WT and PtenΔC / ΔC mice (P20–32), as shown by qRT–PCR analysis performed 1–2 weeks after injection. (n = 5 mice for WT‐Sal/saline, four for WT‐ASO, three for ΔC‐Sal, and three for ΔC‐ASO, *P < 0.05, **P < 0.01, ***P < 0.001, Student's t‐test for each comparison). The error bars represent SEM.
  3. Slc6a20a‐ASO decreases the levels of SLC6A20 proteins in the brain of WT mice (2–4 months), as shown by immunoblot analysis of whole‐brain lysates 6 days after injection. (n = 4 mice, **P < 0.01, Student's t‐test). The error bars represent SEM.
  4. PtenΔC / ΔC mice (2–4 months) treated with Slc6a20a‐ASO display partially normalized levels of whole‐brain glycine, as shown by the lack of difference between saline‐treated WT and ASO‐treated PtenΔC / ΔC mice. Note that the glycine levels observed here are ~ 4 times lower than those measured in Fig 3A, which could be attributable to different mouse ages (P21 in Fig 3A vs. 2–4 months in Fig 5D), absence and presence of ASO injection, or lot‐to‐lot variation of the ELISA kits. (n = 8 mice for WT‐saline, five for WT‐ASO, seven for ΔC‐saline, and eight for ΔC‐ASO, **P < 0.01, ns, not significant, two‐way ANOVA with Tukey's test). The error bars represent SEM.
  5. PtenΔC / ΔC mice (P20–37) treated with Slc6a20a‐ASO display an NMDA/AMPA ratio at hippocampal SC‐CA1 synapses that is comparable to that in ASO‐treated WT mice. (n = 11 neurons from five mice for WT‐ASO, 10 (4) for ΔC‐ASO, ns, not significant, Mann–Whitney U‐test). The error bars represent SEM.
  6. PtenΔC / ΔC mice (P19–37) treated with Slc6a20a‐ASO display unaltered mEPSC frequency and amplitude in CA1 pyramidal neurons, as compared with ASO‐untreated PtenΔC / ΔC mice. Note that mEPSCs in WT mice are not affected by Slc6a20a‐ASO treatment. (n = 13 neurons from three mice for WT‐saline, 14 (2) for WT‐ASO, 14 (3) for ΔC‐saline, and 12 (3) for ΔC‐ASO, ***P < 0.001, ns, not significant, two‐way ANOVA with Bonferroni's test). The error bars represent SEM.
  7. PtenΔC / ΔC mice (2–4 months) treated with Slc6a20a‐ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT‐saline, seven for WT‐ASO, seven for ΔC‐saline, and eight for ΔC‐ASO, *P < 0.05, **P < 0.01, ns, not significant, two‐way ANOVA with Bonferroni's test. The error bars represent SEM.
  8. Slc6a20a‐ASO treatment has no effect on the self‐grooming of PtenΔC / ΔC mice (2–4 months) in the Laboras test. (n = 6 mice for WT‐saline, seven for WT‐ASO, seven for ΔC‐saline, and eight for ΔC‐ASO, ns, not significant, two‐way ANOVA with Bonferroni's test). The error bars represent SEM.

Source data are available online for this figure.