Skip to main content
. 2021 Feb 2;296:100364. doi: 10.1016/j.jbc.2021.100364

Figure 2.

Figure 2

The effect of removing KCC2 from developing and mature networks on maintenance of dendrites, spines, and synapses.A, 1024 × 1024 max projected confocal images of primary cultured neurons from P0 pups infected with AAV-GFP or AAV-Cre at DIV3 and DIV18 and fixed at DIV21 and DIV24, respectively. The cells were immunostained for KCC2 and GFP to amplify the signal, and confocal images were acquired using a 60× objective (Scale bar = 10 μm). B, quantification of the normalized mean fluorescent KCC2 signal within GFP-positive neurons for DIV3-infected and DIV18-infected cells. (DIV3: nKCC2+/+ = 18, nKCC2−/− = 20 neurons, DIV18: nKCC2+/+ = 16, nKCC2−/− = 16 neurons from 4 to 6 individually infected cultures). C, neuronal reconstruction of control and knockout neurons infected with the AAV-GFP or AAV-Cre for Sholl analysis using the Simple Neurite Tracer Fiji plugin. The number of intersections (y-axis) are plotted against distance from the soma (x-axis) and as a cumulative distribution. (DIV3: nKCC2+/+ = 22, nKCC2−/− = 19 neurons, DIV18: nKCC2+/+ = 9, nKCC2−/− = 9 neurons from 4 to 6 individually infected cultures) (Scale bar = 10 μm). D, cropped confocal images of GFP-expressing neuronal processes with dendritic spines (Scale Bar = 10 μm) with spine reconstructions below, made using Neuron Studio (48). Quantification of the number of spines per 100 μm of dendritic process (DIV3: nKCC2+/+ = 24, nKCC2−/− = 22 dendritic segments, DIV18: nKCC2+/+ = 13, nKCC2−/− = 22 dendritic segments from 4 to 6 individually infected cultures). E, immunoblots showing the levels of the excitatory postsynaptic marker PSD95 and the inhibitory postsynaptic marker gephyrin in AAV-GFP and AAV-Cre infected cells at DIV3 and DIV18 (n = 3 replicates).