Article Figures & Data
Figures
- Figure 1.
Tiam2 knockdown reduces baseline AMPAR- and NMDAR-mediated neurotransmission in CA1 pyramidal neurons. A, Tiam2 immunolabeling in whole hippocampal slice, (inset) CA1 cell body layer (scale bar, 200 μm). B, Western blot showing shRNA-mediated reduction of Tiam2 expression in dissociated rat hippocampal neurons. C, Schematic representation of electrophysiological recording setup for CA1 pyramidal neurons. D, E, Scatterplots show eEPSC amplitudes for pairs of untransfected and transfected cells (open circles) with M ± SEM (filled circles). (Insets) Representative current traces from control (black) and transfected (yellow) neurons with stimulation artifacts removed (scale bars: 20 pA for both AMPAR-eEPSCs and NMDAR-eEPSCs, 20 ms for AMPAR, 50 ms for NMDAR). Barplots show average AMPAR and NMDAR-eEPSC amplitudes (±SEM) of CA1 pyramidal neurons expressing Tiam2 shRNA (yellow) normalized to their respective control cell average eEPSC amplitudes (black). Tiam2 shRNA expression decreases AMPAR-eEPSC and NMDAR-eEPSC amplitude in CA1 pyramidal neurons (p = 0.048; n = 10 pairs for AMPAR-eEPSC; p = 0.038; n = 8 pairs for NMDAR-eEPSC; Wilcoxon signed-rank test). F, Paired-pulse facilitation ratio (M ± SEM) for Tiam2 shRNA expressing CA1 pyramidal neurons and paired control neurons with no detectable difference in facilitation (p = 0.88; n = 5 pairs; Student's unpaired t test). Representative scaled current traces from control and transfected (yellow) neurons (scale bars: 20 pA, 20 ms). G, H, CV analysis of AMPAR- and NMDAR-eEPSCs from pairs of control and Tiam2 shRNA expressing CA1 pyramidal neurons. CV−2 values are plotted against corresponding ratios of M amplitudes within each pair (open circles) with M ± SEM (filled circle; for AMPAR-eEPSCs, n = 10 pairs; for NMDAR-eEPSC, n = 8 pairs,). *p < 0.05, n.s., not significant. See Extended Data Figure 1-1 for more details.
- Figure 2.
Tiam2 depletion results in reduced spine density in CA1 pyramidal neurons. A, Schematic of areas of image acquisition from CA1 pyramidal neuron dendrites. B, G, Representative dendritic segments and corresponding reconstructed filaments (scale bars: 4 μm). C, Boxplots show spine density is reduced in CA1 pyramidal neurons expressing Tiam2 shRNA (yellow) compared with GFP-expressing control neurons (gray; p = 0.0000411 for spine density; p = 0.05031 for spine head volume; p = 0.22 for spine length; for GFP n = 9 segments, n = 6 cells; for Tiam2 shRNA n = 9 segments, n = 9 cells; Wilcoxon rank-sum test). D, E, Scatterplots with AMPAR- and NMDAR-eEPSC amplitudes, respectively, plotted against paired control neuron eEPSCs (open circles) with M ± SEM (filled circles). (Insets) Representative current traces from control (black) and transfected (yellow) neurons with stimulation artifacts removed (scale bars: 20 pA for both AMPAR-eEPSCs and NMDAR-eEPSCs, 20 ms for AMPAR, 50 ms for NMDAR). Barplots show average AMPAR and NMDAR-eEPSC amplitudes (±SEM) of transfected CA1 pyramidal neurons normalized to respective control cell average eEPSC amplitudes (black). Tiam2 cDNA expression restores AMPAR- and NMDAR-eEPSC amplitude in CA1 pyramidal neurons coexpressing Tiam2 shRNA (for AMPAR-eEPSCs p = 0.4; n = 9 pairs; for NMDAR-eEPSC p = 0.2; n = 8 pairs; Wilcoxon signed-rank test). F, Schematic of areas of image acquisition from CA1 pyramidal neuron dendrites. G, H, No significant differences were detected in any spine parameters in Tiam2 shRNA and Tiam2 cDNA coexpressing spines in CA1 pyramidal neurons compared with corresponding GFP-expressing control neurons. H, Boxplots show unaltered spine density in CA1 pyramidal neurons expressing Tiam2 cDNA and Tiam2 shRNA (green) compared with GFP-expressing control neurons (gray boxes; p = 0.7413 for spine density; p = 0.7376 for spine head volume; p = 0.5681 for spine length; for GFP n = 12 segments, n = 5 cells; for Tiam2 shRNA n = 16 segments, n = 11 cells; Wilcoxon rank-sum test). ****p < 0.0001. See Extended Data Figure 2-1 for more details.
- Figure 3.
Tiam2 depletion is not rescued by DH1 domain-lacking Tiam2 in CA1 pyramidal neurons and has no effect on neurotransmission in DG granule neurons. A, Illustration of Tiam2's protein domains; full-length Tiam2 (upper) and Tiam1 ΔDH (lower). B, Immunoblot of shRNA-resistant Tiam2 ΔDH and Tiam2 cDNA coexpressed with Tiam2 shRNA in HEK293 cells. C, D, F, G, Scatterplots of AMPAR- and NMDAR-eEPSC amplitudes for CA1 pyramidal neurons expressing Tiam2 shRNA (yellow) and coexpressing Tiam2 shRNA and Tiam2 ΔDH (red), respectively, plotted against paired control neuron eEPSCs (open circles) with M ± SEM (filled circles). (Inset) Representative current traces from control (black) and transfected (yellow or red) neurons stimulation artifacts removed (scale bars: 20 pA for AMPA, 20 ms for AMPA). Barplots show average AMPAR and NMDAR-eEPSC amplitudes (±SEM) of transfected (yellow or red) CA1 pyramidal neurons normalized to respective control cell average eEPSC amplitudes (black). C, D, Tiam2 ΔDH does not restore AMPAR- and NMDAR-eEPSC amplitude in CA1 pyramidal neurons coexpressing Tiam2 shRNA (for AMPAR-eEPSCs p = 0.016, n = 8 pairs; for NMDAR-eEPSC p = 0.016, n = 7 pairs; Wilcoxon signed-rank test.). E, Schematic representation of electrophysiological recording setup for DG granule neurons. F, G, Tiam2 knockdown has no effect on AMPAR- and NMDAR-eEPSC amplitude in DG granule neurons (for AMPAR-eEPSCs p = 0.67, n = 16 pairs; for NMDAR-eEPSC p = 0.08, n = 13 pairs; Wilcoxon signed-rank test) *p < 0.05, n.s., not significant. See Extended Data Figure 3-1 for more details.
- Figure 4.
Tiam1 and Tiam2 are dispensable for functional LTP in CA1 pyramidal neurons. A, Illustration of Tiam1 and Tiam2 protein domains; full-length Tiam1 (upper) and Tiam2 (lower). B, C, Scatterplots with AMPAR- and NMDAR-eEPSC amplitudes for CA1 pyramidal neurons coexpressing Tiam2 shRNA and Tiam1 cDNA (orange), respectively, plotted against paired control neuron eEPSCs (open circles) with corresponding M ± SEM (filled circles). (Inset) Representative current traces from control (black) and transfected (green) neurons stimulation artifacts removed (scale bars: 20 pA for AMPA, 20 ms for AMPA). Barplots show average AMPAR and NMDAR-eEPSC amplitudes (±SEM) of CA1 pyramidal neurons normalized to respective control cell average eEPSC amplitudes (black bar). B, C, Tiam1 cDNA expression restores AMPAR- and NMDAR-eEPSC amplitude in CA1 pyramidal neurons coexpressing Tiam2 shRNA (for AMPAR-eEPSCs p = 0.5, n = 8 pairs; for NMDAR-eEPSC p = 0.8, n = 6 pairs, Wilcoxon signed-rank test; insets). D, E, LTP induction is unaltered by Tiam1 or Tiam2 depletion in CA1 pyramidal neurons. Plots of M ± SEM AMPAR-eEPSC amplitude of control untransfected CA1 pyramidal neurons (black) and CA1 pyramidal neurons electroporated with Tiam1shRNA (green) or Tiam2shRNA (yellow) normalized to the M AMPAR-eEPSC amplitude before an LTP induction protocol (arrow; Tiam1: control, n = 9 neurons; Tiam1shRNA, n = 7 neurons; Tiam2: control, n = 9 neurons; Tiam2 shRNA, n = 7 neurons). Sample AMPAR-eEPSC current traces from control (black) and electroporated (green or yellow) neurons before and after LTP induction are shown to the right of the graphs. (Scale bars: 20 ms, 20 pA.) *p < 0.05, Wilcoxon signed-rank test. See Extended Data Figure 4-1 for more details.
Extended Data
Figure 1-1
Antibody and shRNA validation A Tiam1 immunolabelling (white) in whole hippocampal slice co-localized with DAPI (blue), peptide control shows specificity of Tiam2 antibody (right panel) (scale bar: 500μm). B Immunoblot of DIV 21 rat hippocampal lysate transduced with no virus, GFP- and Tiam2 shRNA- expressing virus probed with Tiam2 antibody and actin (control). Download Figure 1-1, TIF file.
Figure 2-1
Spine imaging and rescue of Tiam2 depletion in CA1 pyramidal neurons A-H No significant differences were detected in other spine parameters in Tiam2 knockdown or Tiam2 rescue in CA1 pyramidal neurons compared with respective GFP-expressing control neurons. Boxplots show spine parameters for CA1 pyramidal neurons transfected with Tiam2 shRNA (yellow) or Tiam2 shRNA and Tiam2 cDNA (green) compared to GFP expressing control neurons (grey) (for Tiam2 knockdown: for GFP n = 9 segments, n = 6 cells, for Tiam2 shRNA n = 9 segments, n = 9 cells, p = 0.06253 for Spine Neck Diameter, p = 0.01876 for Spine Neck Volume, p = 0.06253 for Spine Diameter, p = 0.1359 for Spine Volume; for Tiam2 rescue: GFP n = 12 segments, n = 5 cells, Tiam2 shRNA and Tiam2 cDNA n = 16 segments, n = 11 cells, p = 0.2098 for Spine Neck Diameter, p = 0.1894 for Spine Neck Volume, p = 0.1894 for Spine Diameter, p = 0.3758 for Spine Volume; Wilcoxon rank-sum test). Download Figure 2-1, TIF file.
Figure 3-1
Tiam2 depletion has no effect on spine parameters in DG granule neurons A Schematic of areas of image acquisition from CA1 pyramidal neuron dendrites. E, F Boxplots show spine parameters for DG granule neurons transfected with Tiam2 shRNA (yellow) or GFP expressing control neurons (grey) (for Tiam2 knockdown: for GFP n = 16 segments, n = 8 cells, for Tiam2 shRNA n = 15 segments, n = 10 cells, p = 0.3599 for Spine Density, p = 0.6965 for Spine Head Volume, p = 0.1728 for Spine Length, p = 0.7618 for Spine Neck Diameter, p = 0.6334 for Spine Neck Volume, p = 0.9654 for Spine Diameter, p = 0.4598 for Spine Volume; Wilcoxon rank-sum test). n.s. – not significant. Download Figure 3-1, TIF file.
Figure 4-1
A Tiam1 antibody validation Tiam1 immunolabelling (white) in whole hippocampal slice co-localized with DG granule neuron specific Prox1 (red) and DAPI (blue), (inset) DG granule cell body layer (scale bar: 500μm). B Western blot showing shRNA-mediated reduction of Tiam1 and unaltered Tiam2 expression in Tiam1 shRNA expressing mouse hippocampal neurons, C Western blot showing shRNA-mediated reduction of Tiam2 and unaltered Tiam1 expression in Tiam2 shRNA expressing mouse hippocampal neurons. Barplots show mean ± SEM of experimental replicates (Tiam1 shRNA: Tiam1 n = 5, Tiam2 n = 4, Tiam2 shRNA: Tiam2 n = 4, Tiam1 n = 3, ****p < 0.0001, Mann- Whitney test). Download Figure 4-1, TIF file.
In this issue
