Figure 1.
Triple immunofluorescence labeling of hippocampal cultures using live surface labeling of NR2A or NR2B (555; red), followed by labeling for the presynaptic marker VGLUT (647; blue), and a third protein (488; green) including PSD-95/93, SAP102 (Neuromab), pan-cadherin (P-cad), N-cadherin (N-cad), or catenin. A. NR2A/VGLUT/PSD-95/93; B. NR2B/VGLUT/N-cadherin; C. NR2B/VGLUT/pancadherin. A and B are examples of thick dendrites near the cell soma (shown in upper right) and C shows a thinner dendrite that is more distal to the soma. A and C are fused stacks of confocal sections. B is a single confocal section; note how the cadherin-labeled glial processes seem to enwrap the dendrites directly (even evident in a single section). D1-3. Portion (based on averages of data from neurons) of extrasynaptic NR2s that colocalized with other proteins; NR2A is shown in blue and NR2B in red. There were no significant differences between NR2A and NR2B for any protein. There were no significant differences between proximal (thick) and distal (thin) dendrites except for NR2B with PSD-95/93 (D3; asterisks; p=0.004). For statistical analyses in D1 and D3, N=15 neurons per bar; for D2, N=15 neurons per bar for PSD-95 and SAP102 and 3 neurons per bar for pan-cadherin and N-cadherin. E. Ratio of synaptic to extrasynaptic puncta intensity for PSD-95/93 and SAP102 using Volocity (intensity based on 4095 gray level scale for 12 bit images). Note that extrasynaptic puncta of PSD-95/93 are much less intense than synaptic puncta, as compared to SAP102 (p=6.74×10−6). F. Comparison of synaptic (Sy) and extrasynaptic (Ex) puncta size (μm3) in transfected 2 WIV and 3 WIV NMDARs and in native 3 WIV NMDARs (colabeled with antibodies to PSD-95/93 (95) or SAP102 (102) as indicated; data combined for NR2A and NR2B-see methods). Note that synaptic puncta are larger than extrasynaptic puncta (p<0.01). Scale bars are 5 micrometers.