Figure 1

Co-expression of RCaMP and eGFP-gephyrin variants. (A) An illustration of different gephyrin mutations used in the study, along with the labels of signaling pathways that are affected by the mutated residue. The gephyrin-K148R (SUMO1 conjugation site mutant) facilitates scaffolding. The gephyrin-S303A and S305A (PKA and CaMKIIα phospho-null mutant) hamper NMDA receptor activity-induced scaling at GABAergic postsynaptic sites. The gephyrin-DN (lacks part of E domain) disrupts endogenous gephyrin scaffolds in neurons. (B) Representative images of neuron co-expressing RCaMP and eGFP-gephyrin variants after injection of AAVs in L2/3 barrel cortex in vivo. The following combination of viruses were injected into the barrel cortex (same combination used in 2P Ca²⁺ imaging): AAV6-hSyn1-flex-gephyrin variants, AAV6-CaMKIIα-CreER^T2, AAV6-CaMKIIα-RCaMP1.07. All brain sections were stained for CaMKIIα and eGFP. Scale bar: 20 µm. (C) The averaged percentages of L2/3 pyramidal neurons from the field of views that were RCaMP-positive and RCaMP-positive + gephyrin variant-positive. Quantification of average expression of RCaMP-expressing neurons, gephyrin variant-expressing neurons, and neurons co-expressing RCaMP and eGFP-gephyrin mutants after normalization to total CaMKIIα-positive neurons.