Fig. 5: Acute degradation of PSD-95:mTurq2:mAID is followed by loss of AMPARs at the same synapses.

A Top panels: A rat cortical neuron in culture co-expressing PSD-95:mTurq2:mAID, SEpH:GluA2 and OsTIR1-P2A-mCherry. Bottom panels: Region in yellow rectangle at greater detail, before, and after addition of 5-Ph-IAA. Note that the near complete loss of PSD-95:mTurq2:mAID fluorescence is associated with a noticeable reduction in SEpH:GluA2 fluorescence. Scale bars: 10 µm. B PSD-95:mTurq2:mAID fluorescence measured at 16–41 synapses of each neuron (455 in total) tracked throughout the experiments. Each thin gray line is the average fluorescence measured for the synapses of one neuron (18 neurons from 3 experiments). Fluorescence was normalized to fluorescence measured at time point just before 5-Ph-IAA addition. Thick magenta line is the population average. A subset of neurons was imaged only once every 12 (instead of 3) h to minimize potential confounds related to photobleaching (open diamonds; 10 neurons from the same 3 experiments). C Changes in SEpH:GluA2 fluorescence at the same synapses and neurons of (B). Thick brown line is the population average. D SEpH:GluA2 fluorescence measured at 14–30 synapses of neurons positive for SEpH:GluA2 and OsTIR1-P2A-mCherry but negative for PSD-95:mTurq2:mAID (222 in total). Each thin gray line is the average fluorescence measured for the synapses of one neuron (10 neurons from 3 experiments). Thick gray line is the population average. Open diamonds represent measurements made in a subset of cells imaged only once every 12 h (5 neurons from the same experiments). E Pooled data. All error bars are standard deviations, not SEM. Test for difference between PSD-95:mTurq2:mAID positive and negative cells – unpaired t-test, without assuming equal variances; applied to data obtained at last time point.