GABA_AR-independent gephyrin declustering during sustained activity induced by NMDA stimulation.

<p><b>A</b>–<b>D</b>: Effect of CysA treatment on NMDA-driven dispersal of GABA_AR and gephyrin clusters. Left: Examples of GABA_AR (<b>A</b>, <b>B</b>) and gephyrin (<b>C</b>, <b>D</b>) immunoreactivity in neurons incubated with NMDA for 30 min, with (<b>B</b>, <b>D</b>) and without CysA (<b>A</b>, <b>C</b>). Scale bars, 5 µm. Right: Normalized fluorescence intensities (averages ± SEM) of synaptic GABA_AR (<b>A</b>, <b>B</b>) and gephyrin (<b>C</b>, <b>D</b>) clusters. *: <i>p</i><0.05, ***: <i>p</i><0.005, Welch's <i>t</i>-test. Con, CysA, CysA+NMDA: n = 30 cells/condition, NMDA: n = 25 cells/condition (3 cultures). CysA suppressed NMDA-induced dispersal of GABA_AR clusters, but not that of gephyrin clusters. <b>E</b>: NMDA-induced gephyrin dispersal under GABA_AR XL. Top: Gephyrin immunoreactive clusters in neurons with (+XL) and without (−XL) surface GABA_AR XL after NMDA stimulation. Bottom: Effects of GABA_AR XL and NMDA treatment on the normalized fluorescence intensity of gephyrin clusters (average ± SEM). ***: <i>p</i><0.005, Welch's <i>t</i>-test, n = 30 cells/condition (3 cultures). <b>F</b>–<b>H</b>: Comparison of the Ca²⁺ influx level induced by 4AP and NMDA. Increase in Ca²⁺ after the addition of 4AP (black) or NMDA (red) (<b>F</b>). Gray horizontal bars in the traces indicate the presence of 4AP or NMDA. Peak amplitudes (<b>G</b>) and areas under the curve (<b>H</b>) for F/F0-time plots during 90 s after the onset of stimulation. Values indicate averages ± SEM. ***: <i>p</i><0.005, Welch's <i>t</i>-test. 4AP: n = 21 cells, NMDA: n = 23 cells (3 cultures).