Figure 1.
Changes in dendritic architecture during hippocampal development in vitro.
A, Cultured hippocampal neurons were transfected with GFP at 0 DIV with Amaxa nucleofection and fixed at 2, 5, 7, 12, 15 and 20 DIV. Images of representative GFP-expressing hippocampal neurons at the different stages are shown. Scale bar is 25 μm. Inset: Magnified views of boxed areas showing examples of dendritic branches. Note that dendritic arborization increases until 7 DIV and then gradually, but profoundly, drops during the next 2 weeks. Also note that whereas immature neurons display filopodia-like structures, mature neurons are predominantly decorated by dendritic spines. B, Quantification of the average number of primary, secondary or tertiary dendritic branches of neurons expressing GFP at the developmental stages indicated in the graph. C, Sholl analysis of neurons at different stages of development documents that cells at 7 DIV display an increased number of intersections close to the soma (25–50 μm). D–E, Averaged length of primary, secondary and tertiary branches (D) and total outgrowth (E) of hippocampal neurons at different developmental stages. At least 20 neurons, obtained from 3 independent experiments, were analyzed for each stage. Figures show Mean ± SEM.
Figure 2.
Over-expression of NR2B in hippocampal neurons promotes dendritic branching only in immature cells.
Cultured hippocampal neurons were transfected with GFP alone (control) or with NR2B (NR2B) and fixed at 7, 12, 15 and 20 DIV. To express GFP and/or NR2B in neurons for 3 days, neurons were transfected with either CaPO4 (<12 DIV) or magnetofection-based methods (>12 DIV). A–B, Images of representative control hippocampal neurons or expressing NR2B at 7 DIV (A) or 20 DIV (B). Scale bar is 25 μm. Note that NR2B expression induces branching only at 7 DIV, while in hippocampal neurons at 20 DIV NR2B expression results in a dendritic architecture that is similar to that of controls. C–E, Averaged length of secondary (C) and tertiary (D) branches, and total outgrowth (E) of hippocampal neurons expressing GFP alone or GFP plus NR2B at different developmental stages. F, Sholl analysis of control neurons or NR2B-expressing neurons at 7 DIV and 20 DIV. For each developmental stage and condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. *** p<0.001 (ANOVA).
Figure 3.
Expression of NMDAR subunits NR2A and NR2B, and MAGUKs PSD95 and SAP102, at different stages of development.
A, Total protein extracts were obtained from hippocampal neurons at 2, 5, 7, 12, 15, 18, and 20 DIV and immunoblotted with antibodies against NR2B, NR2A, PSD95, SAP102, or N-cadherin as loading control. B, Results of qRT-PCR run with primers designed to determine mRNA levels for NR2B, NR2A, PSD95, or SAP102 at the same developmental stages; results were normalized against levels of GAPDH mRNA. Note that NR2B expression decreases with development, while NR2A and PSD95 increases. Expression of SAP102 remains relatively stable at these developmental stages. C, Immunodetection of endogenous NR2B, NR2A, PSD95, or SAP102 (green) and co-localization with presynaptic marker Bassoon or Piccolo (red) at 5 and 20 DIV. Insets show magnified views of boxed areas showing examples of synaptic clusters located in close apposition to presynaptic clusters.
Figure 4.
Knockdown of PSD95 promote NR2B clustering in mature hippocampal neurons.
A, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP plus shRNA-PSD95. At 20 DIV, cultures were fixed and stained with anti-PSD95 antibody. Confocal images (one optical section of 1 μm) show transfected cell (green) and PSD95-IR (red). Scale bar: 20 μm. B, Representative images of neuronal branches expressing GFP alone (Control; left images) or GFP plus shRNA-PSD95 (shRNA-PSD95; right images) and immunostained against PSD95 (red). Note that shRNA-PSD95 causes a significant loss of mature spines and an increase in the number of filopodia-like structures. Images were constructed from a stack of optical section (total 1.5 μm). Scale bar: 5 μm. C, Representative images of neuronal branches expressing GFP plus NR2B (NR2B; left images) or GFP plus NR2B and shRNA-PSD95 (NR2B+shRNA-PSD95; right images) and immunostained against NR2B (red). Images were constructed from a stack of optical section (total 1.5 μm). Scale bar: 5 μm. D, Quantification of total NR2B cluster number per 100 μm of neurite length, in NR2B neurons or those expressing both NR2B and shRNA-PSD95. For each condition at least 9 neurons were analyzed. Figures show Mean ± SEM. * p<0.05 (t-test).
Figure 5.
Simultaneous over-expression of NR2B and knockdown of PSD95 induce dendritic branching in mature hippocampal neurons.
A, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP alone (control; left image), GFP plus shRNA-PSD95 (shRNA-PSD95; middle image), or GFP plus NR2B and shRNA-PSD95 (NR2B+shRNA-PSD95; right image). At 20 DIV, cultures were fixed and images taken. Scale bar is 25 μm. Inset: Magnified views of boxed areas showing examples of dendritic branches. Note that neurons that express NR2B+shRNA-PSD95 display a more complex dendritic architecture compared to control or shRNA-PSD95-expressing cells. B–C, Quantification of the average number of secondary (B) and tertiary (C) processes: neurons expressing NR2B+shRNA-PSD95 have more branches relative to control neurons and to cells transfected with shRNA-PSD95. D–E, Total outgrowth (D) and Sholl analysis (E) of hippocampal neurons expressing the different constructs as indicated. For each condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. *** p<0.001 (ANOVA).
Figure 6.
The C-terminal domain of NR2B is required to promote dendritic branching.
A, Schematic representation of a wild-type NR2B subunit (NR2B), the chimera NR2AheadBtail (NR2AhBt), a wild-type NR2A subunit (NR2A), and the chimera NR2BheadAtail (NR2BhAt). B–C, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP and the different wild-type and chimeric NR2 constructs, as indicated. As controls, cells were transfected with GFP alone (control; red continuous lines) or GFP plus shRNA-PSD95 (shPSD95; red dotted lines). For transfection with constructs that contain the NR2B tail (NR2B and NR2AhBt), PSD95 was knocked-down to allow clustering of these NR subunits. At 20 DIV, cultures were fixed, images taken and average number of secondary (B) and tertiary (C) dendritic branches was quantified. Note that in hippocampal neurons, the expression of a NR2 construct containing the C-terminal of the NR2B is a prerequisite for an increase in the number secondary and tertiary dendritic branches relative to control and shPSD95 conditions. For each condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. * p<0.05, ** p<0.01, and *** p<0.001 (ANOVA) for branches relative to control conditions (control or shRNA-PSD95); and ### p<0.001 (t-test) for branches between the indicated conditions. N.S. = not significantly different.