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. 2012 Jul 18;7(7):e41185. doi: 10.1371/journal.pone.0041185

Figure 2. Rem2 redistribution is dependent on calcium influx via the activation of NMDA receptors.

Figure 2

(A) Neuronal cultures were stimulated for 60 seconds with either 100 µM glutamate/10 µM glycine (glu/gly and No Ca conditions) or 100 µM NMDA/10 µM glycine. In the No Ca condition, neurons were incubated in EBS without 3 mM CaCl2, in the presence of 30 nM TTX to reduce spontaneous activity, then stimulated as above. Ratio after/before of Rem2 stimulated with glutamate/glycine, 5.88±0.47, N = 30; with NMDA/glycine, 3.41±0.49, N = 29, one-way ANOVA followed by Bonferroni post-hoc test p<0.005 compared to glutamate/glycine; no calcium + TTX, 1.58±0.28, N = 26, p<0.001 compared to glutamate/glycine. Numbers within the bars denote N, and error bars represent SEM. (B) Redistribution of Rem2 is blocked by MK-801. Neuronal cultures were treated with the NMDAR open-pore blocker MK-801 (62.5 µM) for 20 minutes, and then stimulated for 60 seconds with glutamate/glycine. Controls experienced the same stimulation protocol in the absence of MK-801. This treatment abolished redistribution (ratio after/before of Rem2 alone, 3.17±0.31, N = 50; Rem2+ MK-801, 1.16±0.10, N = 41; t-test p<0.001). (C) Rem2 redistribution is reversed by removing calcium. (Upper) Serial images of individual cells were taken before and after stimulation for 60 seconds with 100 µM glutamate/10 µM glycine. 5 minutes after stimulation, calcium was removed by replacing the bath solution with chelation solution lacking calcium and containing 1 mM EGTA. Images were taken about 5 minutes and 15 minutes after incubating the neurons in chelation solution. (Lower) Quantitation of Rem2 redistribution reversal. Pixel value variance is significantly different after stimulation (882±177 after vs. 164±21 before, one-way repeated measures ANOVA followed by Dunnett’s post-hoc test: N = 27 for each, p<0.001), but goes back to baseline level following calcium chelation (5 min after chelation, 430±130, N = 27; 12 min after chelation, 237±17, N = 9; difference from before stimulation is NS).