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. 2005 Apr 20;25(16):4040–4051. doi: 10.1523/JNEUROSCI.4115-04.2005

Figure 5.

Figure 5.

Reexpression of NR2B-NMDARs and their participation in synaptic function induced by diminished neuronal activity. A, Representative traces of NMDA currents with (dotted line) or without (thick line) ifenprodil in control (Ctrl) and TTX-treated (TTX) neurons. B, Mean ifenprodil sensitivity of NMDA currents in control and TTX-treated neurons (Ctrl, 20.8 ± 6.1%, n = 17; TTX, 52.1 ± 3.7%, n = 17; *p < 0.05). C, Analysis of immunofluorescence of NR2 subunits. NR2A and NR2B subunits were stained with specific antibodies. Immunofluorescent puncta densities for NR2A (left) and NR2B (right) are plotted for each condition (NR2A: control, 13.4 ± 1.1 × 104 puncta/mm2; TTX treated, 11.8 ± 0.7 × 104 puncta/mm2; n = 11; NR2B: control, 6.7 ± 1.6 × 104 puncta/mm2; TTX treated, 17.2 ± 1.7 × 104 puncta/mm2; n = 10; p < 0.01). D, Experimental protocol for assaying the ratio of synaptic and extrasynaptic NMDARs. First, NMDA was applied to the soma and dendritic region of neurons to evaluate surface-expressed NMDARs (NMDA test pulse). Next, the neurons were treated with MK-801, a powerful reagent that blocks activated NMDARs. During treatment, neuronal synapses were allowed to be activated by presynaptically released glutamate during spontaneous neuronal firing (left) or by quantal released glutamate without cell firing (right; recorded in TTX- and Mg2+-free solution). The amount of synaptic NMDARs was estimated as the proportion of NMDAR currents blocked by MK-801, whereas extrasynaptic NMDARs were unblocked currents. NMDAR currents are plotted as a function of treatment time with MK-801. The synaptic NMDARs activated by spontaneous synaptic transmission were blocked with a similar time course in both control (5.56 ± 0.60% of level before MK-801 treatment; n = 8) and TTX-treated (4.74 ± 0.97% of level before MK-801 treatment; n = 8) neurons. Quantal released glutamate also blocked most NMDARs within 10 min of MK-801 treatment (control, 25.6 ± 6.8% of level before treatment, n = 6; TTX-treated, 28.7 ± 4.8% of level before treatment; n = 7).