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

Figure 9.

Figure 9.

Rapid increase in the colocalization of GluR1 and synaptic NR2B induced by circuit reactivation in TTX-treated neurons. A, Immunofluorescent images of NR2B, synaptophysin, and GluR1 puncta. Triple staining with antibodies to NR2B (a-c), synaptophysin (d-f; Synp), and GluR1 (g-i) are displayed with the same gain and encoded in merged images (NR2B in green, synaptophysin in blue, and GluR1 in red). Scale bar, 5 μm. Ctrl, Control neurons; TTX, TTX-treated neurons. B, Quantification of synaptic NR2B puncta. NR2B puncta colocalizing with synaptophysin were counted and normalized by total synapse number. TTX treatment significantly increased the number of silent synapse (control, 23.9 ± 3.0% of total synapse number, n = 10; TTX, 36.6 ± 3.8, n = 11; *p < 0.05; total synapse number: control, 96.6 ± 11.9 × 103/mm2; TTX, 111.3 ± 7.5 × 103/mm2). Circuit reactivation, achieved by washing out TTX and incubating neurons in extracellular solution with PTX (100 μm) for 30 min, did not increase the number of silent synapse (TTX + activation, 18.4 ± 1.7% of total synapse; n = 10). C, Quantitative analysis of NR2B-positive functional synapse. GluR1 puncta colocalizing with synaptic NR2B puncta were counted and normalized by total synapse number. TTX treatment did not significantly increase the extent of colocalization of GluR1 and synaptic NR2B puncta (control, 15.7 ± 2.4%, n = 10; TTX, 19.3 ± 2.8%, n = 11). Circuit reactivation clearly increased the number of GluR1 puncta colocalizing with synaptic NR2B puncta (TTX + activation, 28.5 ± 3.3; n = 10; *p < 0.05). Total synapse number was 129 ± 15.9/mm2 (n = 10), 148 ± 9.9/mm2 (n = 11), and 119.3 ± 10.0/mm2 (n = 10) for control, TTX, and TTX + activation, respectively. D, Quantification of GluR1 recruitment on synaptic NR2B puncta. Fluorescent intensities of GluR1 on synaptic NR2B puncta were quantified and plotted (left, same experiment in B; right, pooled data from three independent experiments). Circuit reactivation also increased the intensity of GluR1 signals on synaptic NR2B puncta (left; TTX, 176.1 ± 4.8, n = 11; TTX + activation, 198.4 ± 8.5, n = 10) and confirmed (right; pooled data, TTX, 100 ± 2%, n = 23 from 3 experiments; TTX + activation, 116 ± 2%, n = 31 from 3 experiments; ***p < 0.001).