Article Figures & Data
Figures
- Figure 1.
HDAC2 suppresses excitatory transmission. A, Western blots of lysates from HEK293 cells expressing HDAC2 probed with anti-FLAG to assess HDAC2 expression or anti-β-actin as a loading control. Examples of KD with the four different HDAC2 shRNA or HDAC1 shRNA sequences are shown, as well as samples lacking HDAC2 (GFP) or with scrambled shRNA as a control. B, Quantification of KD of HDAC2 protein by the four HDAC2 shRNAs (n = 5). C, Western blot analysis of HDAC1 KD with examples of KD using four different HDAC1 shRNA or HDAC2 shRNA sequences and samples lacking HDAC1 (GFP) or with scrambled shRNA as a control. D, Quantification of KD of HDAC1 protein by the four HDAC1 shRNAs (n = 5). E, HDAC2 KD increased mEPSC amplitude compared with neighboring untransfected CA1 neurons, seen as a rightward shift of the cumulative amplitude distribution and a significantly increased mean mEPSC amplitude (p < 0.05, n = 9 and 12). F, HDAC2 KD did not significantly alter mEPSC frequency (n = 9 and 12). G, HDAC1 KD did not significantly alter mEPSC amplitude (n = 10 and 12). H, HDAC1 KD did not significantly alter mEPSC frequency (n = 10 and 12). I, HDAC2 OE reduced mEPSC amplitude compared with neighboring untransfected CA1 neurons (p < 0.05, n = 14 and 14). J, HDAC2 OE did not significantly alter mEPSC frequency (n = 14 and 14). K, GFP expression alone did not significantly alter mEPSC amplitude (n = 12 and 11). L, GFP expression alone did not significantly alter mEPSC frequency (n = 12 and 11). All data are plotted as mean ± SEM.
- Figure 2.
HDAC2 KD lowers the threshold for synaptic potentiation without affecting synaptic depression. A, Using a subthreshold induction protocol, no LTP was observed in neurons transfected with HDAC1 shRNA (104 ± 2%, n = 8), whereas neurons transfected with HDAC2 shRNA exhibited robust LTP (155 ± 2%, n = 12). Example EPSC traces before and after induction are shown inset. B, Neurons transfected with HDAC1 shRNA or HDAC2 shRNA were equally capable of expressing robust LTD (HDAC1, 63 ± 1%, n = 6; HDAC2, 64 ± 2%, n = 10). Calibration: 50 pA, 20 ms. All data are plotted as mean ± SEM.
- Figure 3.
HDAC2 enhances inhibitory synaptic transmission. A, HDAC2 KD reduced mIPSC amplitude compared with neighboring untransfected CA1 neurons (*p < 0.05, n = 12 and 15). B, HDAC2 KD significantly decreased mean mIPSC frequency (*p < 0.05, n = 12 and 15). C, HDAC1 KD did not significantly alter mIPSC amplitude (n = 8 and 7). D, HDAC1 KD did not significantly alter mIPSC frequency (n = 8 and 7). E, HDAC2 OE enhanced mIPSC amplitude compared with neighboring untransfected CA1 neurons (*p < 0.05, n = 12 and 12). F, HDAC2 OE did not significantly alter mean mIPSC frequency (p < 0.05, n = 12 and 12). G, GFP expression alone did not significantly alter mIPSC amplitude (n = 15 and 12). H, GFP expression alone did not significantly alter mIPSC frequency (n = 15 and 12). I, Transfection of neuronal cultures using virus expressing HDAC2 shRNA resulted in a significant reduction in HDAC2 (**p < 0.01, n = 3) but not HDAC1 (p > 0.05, n = 3) protein levels. J, Viral HDAC2 KD reduced mIPSC amplitude compared with cultures transfected with the control virus (*p < 0.05, n = 10 and 7). K, Viral HDAC2 KD did not significantly alter mIPSC frequency (n = 7 and 10). L, HDAC2 KD did not significantly alter tonic GABAAR current (n = 6 and 7). Calibration: 50 pA, 10 s. All data are plotted as mean ± SEM. BIC, Bicuculline.
- Figure 4.
HDAC2 regulates synaptic abundance of GABAARs. A, Viral-mediated HDAC2 shRNA expression in primary neuronal cultures resulted in a strong reduction of HDAC2 but not HDAC1 mRNA levels (p < 0.001, n = 3). HDAC2 KD also resulted in a significant decrease in the GABAAR γ2 subunit mRNA levels (p < 0.01, n = 3) but not the GABAAR α1, α2, β2, or δ subunits, GABAAR AP (adaptor protein), BIG2 (brefeldin A-inhibited GDP/GTP exchange factor 2), or Gephyrin. B, HDAC2 KD resulted in reduced total and surface expression of the γ2 subunit (p < 0.001 and 0.01, respectively) and reduced surface expression of the α2 subunit (p < 0.05) but not the total α2 subunit expression or total or surface expression of the α1 subunit (n = 3–4). C, Broad HDAC2 KD using viral transfection reduced the abundance of synaptic GABAAR α2 subunit labeling (green, GABAAR α2; red, vGAT). The total fluorescence intensity of α2 subunit puncta that were colocalized with vGAT staining was significantly reduced (p < 0.05, n = 3), whereas the density of α2 puncta and the intensity and density of vGAT staining were not significantly altered. D, Sparse HDAC2 KD significantly reduced the intensity of synaptic α2 subunit staining in transfected neurons identified by GFP expression (p < 0.05, n = 3), whereas the intensity of vGAT staining was not significantly altered. The density of α2 subunit and vGAT puncta were significantly reduced at 4 d but not 2 d after transfection (p < 0.01, n = 3). E, Sparse HDAC2 OE resulted in significantly increased intensity of synaptic α2 subunit but not vGAT staining (p < 0.05 n = 3). The density of α2 subunit but not vGAT puncta was significantly increased (p < 0.01 n = 3). Scale bars, 10 μm. All data are plotted as mean ± SEM. Con, Control. *p < 0.05, **p < 0.01, ***p < 0.001.
