Figure 3. Excitatory synapse formation... | Rockefeller University Press

Figure 3.

$Figure 3. Excitatory synapse formation and dendritic spine morphogenesis were reduced after presynaptic Fer deficiency. (A) Localization of synaptophysin-GFP (Syn-GFP) and PSD-95 in hippocampal neurons transfected at 10 DIV with synaptophysin-GFP together with control or Fer shRNA. White outlines indicate the localization of synaptophysin-GFP clusters. Arrowheads indicate PSD-95 puncta colocalizing with synaptophysin-GFP puncta. Note the reduced colocalization of PSD-95 immunoreactive puncta at synaptophysin puncta in Fer shRNA–transfected neurons (arrows). (B) The fractional coverage by PSD-95 of synaptophysin-GFP puncta. The proportion of the coverage of major axis was normalized to that of the control vector–transfected neurons. n = 9 (vector) and 10 (Fer shRNA). (C) Representative images of presynaptic control or Fer shRNA vector–transfected neurons visualized with GFP (blue) and postsynaptic mOrange-transfected neurons (black). (D) Spine density of mOrange-positive neurons. Spine numbers along 10-μm dendrites of mOrange-positive dendrites. n = 18 (vector) and 19 (Fer shRNA). (E) The number of GFP-positive axons overlapping mOrange-positive dendrites per mOrange-positive dendrite length. n = 11 (vector) and 12 (Fer shRNA). NS, P = 0.058. (F) Imaging at 14 DIV of hippocampal neurons transfected at 10 DIV with synaptophysin-GFP together with control or Fer shRNA vector. Cells were loaded with FM4-64 (red) at 14 DIV. (G) The percent coverage by FM4-64 of synaptophysin-GFP puncta length. n = 9 (vector) and 8 (Fer shRNA). (H) Imaging of vGlut1-pHluorin in 14 DIV hippocampal neurons transfected at 10 DIV with vGlut1-pHluorin, synaptophysin-mOrange (not depicted), and control or Fer shRNA. The vGlut1-pHluorin signal has been processed to indicate signal intensity. After initial imaging, cells were treated with NH4Cl to elevate the pH of internal vesicles to pH 7.4 using NH4Cl, which revealed the presence of vGlut1-pHluorin puncta in Fer-deficient synaptophysin-mOrange–positive neurons, confirming that the low intensity was not caused by a failure in transfection or protein expression. (I and J) Graph lines indicating mean fluorescence intensities at denoted distances from the centers of the vGlut1-pHluorin puncta (I) and synaptophysin-mOrange (Syn-mOrange) puncta (J). Gray lines indicate control vector–transfected conditions, whereas red lines indicate Fer shRNA–transfected conditions. n = 30 (vector) and 57 (Fer shRNA). *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Error bars represent SEM. Bars, 5 μm.$

Excitatory synapse formation and dendritic spine morphogenesis were reduced after presynaptic Fer deficiency. (A) Localization of synaptophysin-GFP (Syn-GFP) and PSD-95 in hippocampal neurons transfected at 10 DIV with synaptophysin-GFP together with control or Fer shRNA. White outlines indicate the localization of synaptophysin-GFP clusters. Arrowheads indicate PSD-95 puncta colocalizing with synaptophysin-GFP puncta. Note the reduced colocalization of PSD-95 immunoreactive puncta at synaptophysin puncta in Fer shRNA–transfected neurons (arrows). (B) The fractional coverage by PSD-95 of synaptophysin-GFP puncta. The proportion of the coverage of major axis was normalized to that of the control vector–transfected neurons. n = 9 (vector) and 10 (Fer shRNA). (C) Representative images of presynaptic control or Fer shRNA vector–transfected neurons visualized with GFP (blue) and postsynaptic mOrange-transfected neurons (black). (D) Spine density of mOrange-positive neurons. Spine numbers along 10-μm dendrites of mOrange-positive dendrites. n = 18 (vector) and 19 (Fer shRNA). (E) The number of GFP-positive axons overlapping mOrange-positive dendrites per mOrange-positive dendrite length. n = 11 (vector) and 12 (Fer shRNA). NS, P = 0.058. (F) Imaging at 14 DIV of hippocampal neurons transfected at 10 DIV with synaptophysin-GFP together with control or Fer shRNA vector. Cells were loaded with FM4-64 (red) at 14 DIV. (G) The percent coverage by FM4-64 of synaptophysin-GFP puncta length. n = 9 (vector) and 8 (Fer shRNA). (H) Imaging of vGlut1-pHluorin in 14 DIV hippocampal neurons transfected at 10 DIV with vGlut1-pHluorin, synaptophysin-mOrange (not depicted), and control or Fer shRNA. The vGlut1-pHluorin signal has been processed to indicate signal intensity. After initial imaging, cells were treated with NH₄Cl to elevate the pH of internal vesicles to pH 7.4 using NH₄Cl, which revealed the presence of vGlut1-pHluorin puncta in Fer-deficient synaptophysin-mOrange–positive neurons, confirming that the low intensity was not caused by a failure in transfection or protein expression. (I and J) Graph lines indicating mean fluorescence intensities at denoted distances from the centers of the vGlut1-pHluorin puncta (I) and synaptophysin-mOrange (Syn-mOrange) puncta (J). Gray lines indicate control vector–transfected conditions, whereas red lines indicate Fer shRNA–transfected conditions. n = 30 (vector) and 57 (Fer shRNA). *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Error bars represent SEM. Bars, 5 μm.