Figure 6. Effect of patient’s antibodies on the density of GABA_AR clusters in cultures of hippocampal neurons.

Live 14-day-in-vitro cultures of dissociated rat hippocampal neurons were stained with patient’s CSF containing GABA_AR antibodies (green), then fixed and stained with commercial GABA_AR antibodies (red; A). Quantification of colocalisation between patient’s CSF antibodies and the commercial GABA_AR antibody shows that 89% (SE 3%) of receptors labelled by patient’s antibodies were colabelled with the commercial antibody against GABA_AR (B). In a similar assay, neurons were incubated with patient’s CSF for 48 h and subsequently stained for postsynaptic GABA_AR (green) and presynaptic vesicular GABA transporter (vGAT) (red; C). The synaptic GABA_ARs (shown as yellow clusters in control conditions) were greatly reduced after treatment with patient’s CSF (C). The number of GABA_AR clusters along dendrites of neurons treated with patient’s CSF is not different from neurons treated with control CSF (Mann-Whitney test p=0·6; D). The number of GABA_ARs localised in synapses, however, decreased significantly in neurons treated with a patient’s CSF compared with neurons treated with control CSF (40% [3%] compared with control as 100%; p<0·0001; E). Patients’ CSF did not affect the clusters of post-synaptic gephyrin colabelled with presynaptic vGAT when compared with the effects of control CSF (p=0·5; F).