KCC2 measurements and analysis of subcellular distribution

KCC2 measurements and analysis of subcellular distribution

The analysis method presented in this study is based on an already published algorithm used to quantify receptor membrane internalization (also see, Dedek et al, 2019, Brain).

This technique was designed to reduce conscious or unconscious biases that can arise from user interventions. Prior to the analyses, the intensity of the immunostaining background noise was defined from white matter of the spinal dorsal horn, region where KCC2 is known to be absent. For each image, the background average intensity was subtracted to the whole image to exclusively calculate the KCC2(+) immunostaining contribution. All neurons, identified by a NeuN(+) labelling, present in the immunocytochemistry confocal image were considered. The distance of each neuron from the grey/white matter border was first estimated. This provides for each neuron a measure of anatomical position in the dorsal horn and will be used for pooling KCC2 intensity profiles. To measure the subcellular KCC2 intensity profiles, the membrane regions of neuronal cells were delineated. For each pixel in the region of interest, the distance to the closest membrane segment was calculated. Using this distance map, the mean pixel intensity and standard deviation of KCC2 fluorescence signal were quantified as a function of the distance to the neuronal membrane (defined as zero). Positive axis values correspond to neuronal intracellular space. KCC2 subcellular intensity profiles were averaged as a function of the distance from grey/white matter border. 

The average pixel intensity profiles for each analyzed neuron were pooled together (Distance bins from grey/white matter border of 20 µm, from 20 µm to 120 µm) to generate a graph of the KCC2 expression as a function of the anatomical position in the dorsal horn. A ratio paired t-test was used to compare KCC2 membrane immunostaining intensities between vehicle and CLP290 at the different distance ranges from the grey matter surface. 

A total of 542 neurons (n = 6 mice) for the vehicle condition and 534 neurons (n = 8 mice) for the CLP290 condition were considered. The KCC2 membrane intensity profile for the bin 100-120 µm (68 neurons from 6 vehicle mice and 68 neurons from 8 CLP290 mice) was shown to illustrate the KCC2 membrane intensity profile. A ratio paired t-test was used to compare KCC2 membrane immunostaining intensities between vehicle and CLP290 at 0, 1, 2 and 3 µm from the membrane.