Article Figures & Data
Figures
- Figure 1.
LFU-induced spine shrinkage is independent of ion flux through the NMDAR but requires glutamate binding and p38 MAPK activity. A, Representative images of dendrites from a GFP-transfected hippocampal CA1 neuron at DIV17 showing that LFU at the target spine (yellow crosses) in the presence of the d-serine/glycine binding site NMDAR antagonist 7CK (100 μm) resulted in spine shrinkage (yellow arrowheads). B, LFU-induced spine shrinkage in the presence of 7CK (red filled circles; 15 spines/15 cells) occurred to the same extent as without NMDAR inhibition (black filled circles; 13 spines/13 cells). The volume of the unstimulated neighbors (open red circles) was unaffected. C, Representative images of dendrites from a GFP-transfected CA1 neuron at DIV14 showing that the glutamate binding site NMDAR antagonist CPP (10 μm) and inhibition of p38 MAPK with SB203580 (2 μm) blocked LFU-induced spine shrinkage. D, Inhibition of p38 MAPK in the presence of 7CK (blue filled bar; 12 spines/12 cells) and block of glutamate binding to the NMDAR by CPP (gray filled bar; 10 spines/10 cells) prevented LFU-induced, long-lasting shrinkage of target spines observed at 30 min after LFU in 7CK-only conditions (red filled bar; 13 spines/13 cells). Unstimulated neighboring spines were unaffected (open bars). *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 2.
7CK fully inhibits NMDAR-mediated currents and calcium influx but does not interfere with LFS-induced NMDAR-dependent LTD. A, Representative traces of uncaging-evoked postsynaptic NMDAR currents (uEPSCs) before (black; stim) and after (red; stim+7CK) 7CK. Whole-cell currents were recorded at −65 mV in ACSF containing the following (in mm): 0.1 Mg2+, 3 Ca2+, and 0.01 NBQX. B, NMDAR uEPSCs (black filled bar) were blocked completely by 7CK (red filled bar) to levels indistinguishable from baseline (open bars; 44 spines/10 cells). C, Red fluorescence image of a dendritic segment (left) from a cell transfected with DsRedExpress (red) and GCaMP6 (green) and overlays of red and green fluorescence line-scan images in the absence (middle; veh) or presence (right; 7CK) of 7CK including the target spine (sp) and neighboring dendrite (dend) from the region indicated by the white dashed line. The time of glutamate uncaging is indicated by the yellow arrowhead. Ca2+ transients corresponding to the images shown are displayed below. D, Ca2+ transients (black bar; 20 spines/7 cells) were completely blocked by 7CK (red bar). E, LTD expression, induced by a 1 Hz, 15 min stimulus in acute hippocampal slices from P18–P24 mice, was normal in the presence of 7CK (7 cells) but blocked by the glutamate binding site NMDAR antagonist AP-5 (7CK+AP5; 5 cells). Inset, Representative traces of responses obtained before and after LFS. Scale bars: 10 pA, 100 ms. F, 7CK completely blocks synaptic NMDAR currents (4 cells). NMDAR EPSCs were recorded at 40 mV in the presence of 10 μm NBQX. Inset, Representative traces of baseline and post-7CK responses. Scale bars: 50 pA, 200 ms. ***p < 0.001.
- Figure 3.
Inhibition of ion flux through the NMDAR converts HFU-induced spine enlargement into shrinkage. A, Representative images of dendrites from a GFP-transfected CA1 neuron at DIV14 showing that HFU-induced LTP stimulus (yellow crosses) in the presence of 7CK or MK-801 did not cause continuing spine enlargement but resulted in long-lasting shrinkage of the target spine (yellow arrowheads). B, HFU of glutamate during vehicle conditions led to a stable increase in spine size (blue filled bar; 7 spines/5 cells) 30 min after HFU. However, in the presence of 7CK (red filled bar; 18 spines/12 cells) or MK-801 (black filled bar; 7 spines/7 cells), the same LTP-inducing stimulus caused a significant long-lasting decrease in the volume of the stimulated target spines (red and black filled bars, respectively) at 30 min after HFU. The size of the unstimulated neighboring spines did not change (open bars). C, Representative traces of uncaging-evoked postsynaptic NMDAR currents (uEPSCs) before (black; stim) and after (red; stim+MK-801) MK-801. Whole-cell currents were recorded at −65 mV in ACSF containing the following (in mm): 0.1 Mg2+, 3 Ca2+, and 0.01 NBQX. D, Application of MK-801 (red filled bar) completely blocked NMDAR uEPSCs (black filled bar) and did not differ from baseline (open bars; 28 spines/6 cells). **p < 0.01, ***p < 0.001.
- Figure 4.
Spine shrinkage induced by HFU in the presence of 7CK is independent of group I mGluR activation. A, Representative images of dendrites from a GFP-transfected CA1 neuron at DIV14 showing that the presence of the group I mGluR inhibitors MPEP and CPCCOEt did not block spine shrinkage (yellow arrowheads) induced by HFU (yellow crosses) in the presence of 7CK. B, MPEP/CPCCOEt (black bar; 15 spines/15 cells) did not block spine shrinkage induced by HFU in the presence of 7CK (red bar; 8 spines/8 cells), although HFU stimulation in the absence of drugs resulted in a stable increase in spine size at 30 min (blue bars; 9 spines/9 cells). Spine volume of the respective unstimulated neighbors (open bars) was not changed. *p < 0.05; **p < 0.01.
