Figure 4.
Functional NMDARs are present at axonal growth cones. DIV3–DIV6 primary hippocampal cultures were whole-cell voltage clamped at the cell body, and NMDA (200 μm) was applied to the growth cone or cell body by pressure application (100 ms, at 4–6 psi). A, A composite differential interference contrast (DIC) image of a representative cell from which the currents were recorded in response to NMDA application. The position of the application pipette is shown. Scale bar, 20 μm. Electrophysiological recordings were performed in the presence of TTX (1 μm). To test the specificity of the NMDA-mediated response at growth cones (GC), 100 μm dl-APV was added to the recording solution and NMDA was applied to the growth cone (B, bottom trace). Currents observed after NMDA application were eliminated after application of dl-APV. C, Average peak amplitudes for NMDA currents recorded after application of NMDA at the cell body and growth cones were 265 ± 40.9 and 31 ± 3.4 pA, respectively (n = 10). D, Average charge (in femtocoulombs) of currents elicited by applying NMDA to growth cones, before and after APV application (14.4 ± 5.1 and 0.038 ± 0.5 fC, respectively) (n = 10). Values shown are mean ± SEM and analyzed by unpaired Student's t test, *p < 0.05. E, To examine the area of the pressure application cloud, a recording electrode was loaded with Alexa Fluor 488 and applied into the solution. Time-lapse frames are in 200 ms intervals. Fluorescent signals of the application were pseudocolored based on pixel intensity. The fluorescent images were digitally overlaid onto a DIC image of the experimental system for visualization purposes. Scale bar, 50 μm.