Article Figures & Data
Figures
- Figure 1.
Exogenous expression or knockdown of NMDAR subunits shifts the kinetics of NMDAR-mediated EPSCs. A, Normalized averaged traces of evoked NMDAR-mediated EPSCs recorded from control tectal neurons and cells exposed to ifenprodil. Retinal ganglion cell axons were stimulated at the optic chiasm and responses from tectal cells were recorded by whole-cell patch clamp. B, Quantification of the decay time constant τ (control, 0.244 ± 0.023 s; Ifenprodil, 0.167 ± 0.02 s; *p < 0.03, n = 9–14 cells in each group). C, Normalized averaged traces of evoked NMDAR-mediated EPSCs from control and exogenous NR2A- and NR2B-expressing tectal neurons. D, Quantification of the decay time constant τ (control, 0.251 ± 0.027; NR2A, 0.142 ± 0.018; *p < 0.02; NR2B, 0.632 ± 0.097, *p < 0.01; n = 5–9 cells in each group). E, Normalized averaged traces of evoked NMDAR-mediated EPSCs from cells expressing control-, NR2A- and NR2B-MOs. F, Quantification of the decay time constant τ (control MO, 0.253 ± 0.046; NR2B MO, 0.155 ± 0.019, *p < 0.05; NR2A MO, 0.4 ± 0.045, *p < 0.05; n = 11–25 cells in each group). Error bars are SEM and all statistical tests are Mann–Whitney U tests.
- Figure 2.
Exogenous expression of NR2A and NR2B decreases local dendritic branch clusters. A, Images and drawings of representative control and exogenous NR2A- and NR2B-expressing cells imaged once daily over 3 d. Axons present in the images are not shown in the drawings. B, Pixel density analysis shows areas of high pixel density with yellow and green colors, and areas of low pixel density with turquoise and blue colors, and highlights the differences in dendritic arbor architecture between control and NR2-expressing neurons. The inset shows the entire reconstructed cell and a red box surrounds the area seen in the pixel density analysis. C, Analysis of the IBTDs (see inset diagram) as neurons develop over 3 d. The distances are measured for every branch tip from one branch tip to every other branch tip on the dendritic tree. D, The median IBTD of each cell was calculated and the average per group is shown. NR2A- and NR2B-expressing cells have longer IBTDs than control cells (control, 37.94 ± 3.80 μm; NR2A, 56.30 ± 6.32 μm, *p < 0.03; NR2B, 56.45 ± 5.23 μm, *p < 0.02). E, Analysis of the interbranch point distance measured from one branch point to every other branch point (see diagram inset) confirms the different branch arrangement. F, Three-dimensional Sholl analysis (see diagram inset) characterizes arbor structure and complexity. G, H, Quantification of the average TDBL (G) and of the total BTN (H). The general growth parameters are similar between the groups on all 3 d: TDBL on days 1, 2, and 3, respectively (in μm), control, 412.97 ± 62.88, 889.40 ± 55.3, and 1156.08 ± 81.26; NR2A, 488.96 ± 62.87, 984.43 ± 80.11, and 1369.29 ± 139.58; NR2B, 406.57 ± 61.58, 896.54 ± 61.03, and 1236.25 ± 103.37; BTN on days 1, 2, and 3, respectively, control, 46.3 ± 6.83, 102.6 ± 6.54, and 128.8 ± 9.07; NR2A, 50.6 ± 4.8, 103.8 ± 9.38, and 125.2 ± 9.8; NR2B, 45.8 ± 4.35, 92.6 ± 6.59, and 122 ± 13.74; n = 10 cells per group. Error bars are SEM and all statistical tests are Mann–Whitney U tests.
- Figure 3.
Knockdown of NR2A and NR2B leads to distinct rearrangements of the dendritic arbor architecture. A, Images and drawings of representative neurons imaged on day 1 and day 3 after single-cell electroporation of control, NR2A and NR2B MOs. Axons present in the images are not shown in the drawings. B, Pixel density analysis of the dendritic arbor highlights areas of high and low branch density. C, IBTD analysis shows that cells expressing NR2B MO have shorter IBTDs, whereas neurons expressing NR2A MO have longer IBTDs. Control cells have IBTDs that are in between the two groups. D, Cumulative frequency plot of the IBTDs (control MO vs NR2A MO, *p < 0.01; control MO vs NR2B MO, *p < 0.05; NR2A MO vs NR2B MO, *p < 0.0001). E, Quantification of the averaged median IBTD for each group (control MO, 45.40 ± 3.31 μm; NR2A MO, 52.19 ± 4.77 μm; NR2B MO, 38.78 ± 3.62 μm; *p = 0.05). F, Three-dimensional Sholl analysis of dendritic arbor structure and complexity. G, TDBL is similar at day 1 and day 3 for all three groups [days 1 and 3, respectively (in μm): control MO, 509.77 ± 55.1, 1280.69 ± 166.77; NR2B MO, 511.06 ± 63.15, 1007.61 ± 144.48; NR2A MO, 462.49 ± 63.29, 1208.19 ± 118.39; n = 9–14 cells per group]. H, BTNs are smaller for cells expressing NR2A MO compared with controls on day 3 [days 1 and 3, respectively: control MO, 47.29 ± 5.71, 131.21 ± 16.97; NR2B MO, 49.6 ± 5.94, 109.9 ± 16.1; NR2A MO, 39.89 ± 6.21, 85.33 ± 6.13; *p < 0.05). I, The change in TDBL and BTN over 3 d normalized to the starting size at day 1 shows no significant difference in growth rate or rate of branch tip additions [TDBL and BTN, respectively: control, 1.94 ± 0.44, 2.64 ± 0.66; NR2B MO, 1.21 ± 0.25, 1.3 ± 0.19; NR2A MO, 2.04 ± 0.46, 1.98 ± 0.77]. Error bars are SEM. The statistical tests are Mann–Whitney U tests, and the Kolmogorov–Smirnov test for the cumulative frequency plots.
- Figure 4.
Exogenous NR2B-expressing cells are more dynamic than control or exogenous NR2A-expressing cells. A, Images and drawings of representative cells imaged every 2 h over 8 h, 1 d after single-cell electroporation. Axons present in the images are not shown in the drawings. B, Quantification of branch dynamics in relation to a reference time point (see diagram inset with time point t4 as an example) categorizes dynamic events in the context of the time points immediately before and after the reference time point [addition, loss, stable, transient, respectively (in percent): control, 26.67 ± 2.64, 22.67 ± 2.71, 36.02 ± 1.47, 42.01 ± 1.37; NR2A, 23.78 ± 2.7, 21.8 ± 2.85, 36.6 ± 1.56, 43.88 ± 1.89; NR2B, 25.56 ± 3.03, 24.93 ± 3.37, 31.82 ± 1.31, 45.77 ± 1.9; *p < 0.05]. C, Analysis of the persistence of branches present at t0 [see diagram inset; t0→t2, →t4, →t6, and →t8, respectively (in percent): control, 47.65 ± 3.65, 32.99 ± 3.11, 27.32 ± 2.41, 22.31 ± 2.55; NR2A, 45.44 ± 2.16, 32.7 ± 2.21, 27.06 ± 2.3, 22.21 ± 2.44; NR2B, 39.52 ± 1.99, 26.55 ± 1.76, 19.98 ± 1.52, 16.7 ± 1.16; *p < 0.05]. D, Analysis of the fraction of all transient branches at any single time point (see diagram inset; control, 54.56 ± 1.67%; NR2A, 53.63 ± 2.87%; NR2B, 57.55 ± 2.0%; *p < 0.05). E, Analysis of the life times of transient branches according to their number of appearances during the imaging protocol [number of appearances 1, 2, 3 (in percent): control, 87.25 ± 0.74, 10.55 ± 0.64, 2.2 ± 0.28; NR2A, 89.85 ± 1.19, 8.85 ± 1.13, 1.3 ± 0.4; NR2B, 88.28 ± 1.61, 10.85 ± 1.76, 0.87 ± 0.31; *p < 0.05]. Error bars are SEM and all statistical tests are Mann–Whitney U tests.
- Figure 5.
Exogenous expression of NR2B blocks an activity-dependent increase in growth rate. A, Images and drawings of representative neurons imaged 1 d after single-cell electroporation. The neurons were imaged before and after a 4 h period in the dark and then after a 4 h period of visual stimulation. Axons present in the images are not shown in the drawings. B, Growth rates in the dark and light of all analyzed cells are shown in gray with the average highlighted in black. C, Average growth rates for the groups of cells imaged in the dark and in the light [dark and light, respectively (in μm): control, 60.99 ± 11.67, 95.27 ± 12.73, *p < 0.05; NR2A, 82.35 ± 17.36, 77.53 ± 14.58; NR2B, 46.98 ± 12.27, 45.92 ± 15.18; n = 15 cells per group]. D, Total branch length additions and retractions in the dark and in the light. The average branch length added by new branches or retracted through the loss of old branches is highlighted in gray. The remaining difference to the total represents the branch length that was added or retracted through extension or shortening of existing branches [dark and light, respectively (in μm): total branch length addition, control, 162.72 ± 10.55, 223.17 ± 21.06; NR2A, 177.59 ± 15.40, 181.56 ± 15.42; NR2B, 151.4 ± 18.61, 174.95 ± 20.63, *p < 0.02; total branch length retraction, control, −172.31 ± 15.4, −204.97 ± 21.92; NR2A, −183.67 ± 17.82, −200.23 ± 18.61; NR2B, −164.04 ± 18.65, −190.47 ± 18.19]. D, Dark, L, light. Error bars are SEM. The Wilcoxon signed-rank test was used to compare growth rates and the Mann–Whitney U test to compare branch length additions and retractions.
- Figure 6.
Exogenous expression and knockdown of NR2A and NR2B decrease AMPAR-mediated synaptic transmission. A, Representative traces of AMPAR-mediated mEPSCs in cells expressing exogenous NR2A or NR2B. B, The average AMPA mEPSC amplitudes of exogenous NR2A- and NR2B-expressing neurons are smaller compared with controls (control, 9.75 ± 0.61 pA; NR2A, 7.67 ± 0.37 pA, *p < 0.02; NR2B 7.31 ± 0.41 pA, *p < 0.01; n = 12–20 per group). C, Cumulative frequency plot of the AMPA mEPSC amplitudes shows the distribution toward smaller amplitudes in exogenous NR2-expressing cells (*p < 0.001 for both NR2A and NR2B vs control). D, The average frequency of AMPA mEPSC events is similar between control and exogenous NR2A- and NR2B-expressing cells (control, 0.96 ± 0.2 Hz; NR2A, 0.96 ± 0.31 Hz; NR2B, 1.08 ± 0.23 Hz). E, Representative traces of AMPA mEPSCs in cells expressing control-, NR2B- and NR2A-morpholinos. F, AMPA mEPSC amplitudes are reduced in NR2B MO and NR2A MO expressing cells (control MO, 9.12 ± 0.49 pA; NR2B MO, 7.80 ± 0.35 pA, *p < 0.05; NR2A MO, 7.08 ± 0.39 pA, *p < 0.01; n = 10–20 per group). G, Cumulative frequency plot of the AMPA mEPSC amplitudes shows the distribution toward smaller values for neurons expressing NR2B MO (*p < 0.001) and NR2A MO (*p < 0.001). H, The average frequency of AMPA mEPSC events is not significantly different between cells expressing control-, NR2A-, or NR2B-MO (control MO, 1.04 ± 0.22 Hz; NR2B MO, 0.68 ± 0.12 Hz; NR2A MO, 1.25 ± 0.79 Hz). Error bars are SEM. The Mann–Whitney U test was used to compare the amplitudes and frequencies, and the Kolmogorov–Smirnov test to compare the cumulative frequency plots.
- Figure 7.
NMDARs as coincidence detectors. Model of the influence of the NR2A and NR2B subunits on the NMDAR as a coincidence detector. NR2A confers a short, NR2B a long integration window for detection of coincident inputs, whereas control cells with signaling through both receptors have an intermediate window. We suggest that the ability of control cells to detect a wider range of correlated inputs that carry useful information for the neuron is critical for the establishment of normal dendritic arbor structure. The detection of inputs is marked by X.
