Fig. 4: Synaptic transmission is severely impaired and surface GluA2 is reduced in 11 weeks old IQSEC2-mutant neurons.

a A representative trace of excitatory postsynaptic currents (EPSCs) that were measured in a control neuron at 5 weeks post-differentiation. b A representative trace of EPSCs measured in an IQSEC2-mutant neuron at 5 weeks post-differentiation. c The average amplitude of synaptic events was increased in the IQSEC2-mutant neurons (p = 0.053). d The average rate of synaptic events was not significantly different between control and IQSEC2-mutant neurons. e The cumulative distribution of the amplitude of synaptic events is right-shifted in the IQSEC2-mutant neurons indicating higher amplitudes of synaptic events. f A representative trace of synaptic activity measured in a control neuron at 7 weeks of age. g A representative trace of synaptic activity measured in an IQSEC2-mutant neuron at 7 weeks. h The average amplitude of synaptic events is similar between IQSEC2-mutant and control neurons at 7 weeks of age. i The average rate of synaptic events was not significantly different between control and IQSEC2-mutant neurons at 7 weeks. j The cumulative distribution of the amplitude of synaptic events shows a slight right shift in IQSEC2-mutant neurons. k A representative trace of synaptic activity measured in a control neuron at 11 weeks. l A representative trace of synaptic activity measured in an IQSEC2-mutant neuron at 11 weeks. m The average amplitude of synaptic events is similar between IQSEC2-mutant and control neurons at 11 weeks post-differentiation. n The average rate of synaptic events is significantly decreased in IQSEC2-mutant neurons compared to control neurons at 11 weeks. o The cumulative distribution of the amplitude of synaptic events. p Immunoblots after BS₃-crosslinking of samples to assess surface (crosslinking “+”) and total protein expression of AMPARs (crosslinking “−”). q Quantification of relative total protein levels of AMPA receptor subunits in samples not cross-linked. r Quantification of relative surface protein levels of AMPA receptor subunits in samples treated with BS₃ (crosslinking “+”). Surface expression is the ratio of s-GluA2 to the total GluA2 signal in the lane. Signal intensities normalized to actin signal and wild type as 100%. s Representative images of labeled total (tGluA2) and surface (sGluA2) GluA2 in IQSEC2-mutant and control DG granule neurons. t Quantification of the percentage of sGluA2 to tGluA2 as a measure surface GluA2 expression. u (left). An image of the FACS gating of a control neuronal culture at 7 weeks after the start of differentiation. The live neurons that passed all the gating (GFP positive indicating PROX1 expression and live) were ~55% of the cells in the culture (marked with the red line). u (right). An image of the FACS gating of an IQSEC2-mutant neuronal culture at 7 weeks. The live neurons that passed all the gating were ~41% of the cells in the culture. v (left). An image of the FACS gating of a control neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 36% of the cells in the culture. v (right) An image of the FACS gating of an IQSEC2-mutant neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 14% of the cells in the culture. w (left) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example control neuronal culture. w (right) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example IQSEC2-mutant neuronal culture. x An increased average percentage of neurons stained for caspase3 (out of the total MAP2 positive neurons) in the IQSEC2-mutant neuronal cultures compared to the control neuronal cultures indicate an increased number of apoptotic neurons. In this figure, asterisks indicate statistical significance by the following code: *p value < 0.05, **p < 0.01. Error bars represent the standard error.