Cortical interneuron development is affected in 4H leukodystrophy 47 2 To investigate whether 4H cultures present changes in the synapses, we analysed synaptic marker expression by immunocytochemistry (Fig. 4A). In our cortical neuronal cultures cells differentiate into GABAergic and glutamatergic neurons (Nadadhur et al., 2017), allowing us to study differentiation efficiency into these neuronal subtypes. At day 56 of differentiation, we studied the total number of synapses by quantifying the number of synaptophysin1 puncta per MAP2 dendritic length. We observed no significant change between the 4H and control cultures (control 0.078 ± 0.017, 4H 0.061 ± 0.018, Fig. 4D). To study whether the fraction of inhibitory synapses changed in 4H cultures, we measured the number of GABAergic presynaptic terminals by co-localization of pre-synaptic protein synaptophysin1 puncta with vesicular GABA transporter (VGAT) puncta. While 4H neurons showed a normal amount of synapses, the percentage of GABAergic synapses was significantly decreased from 46.4% in control to 26.1% in 4H neurons (t(6) = 2.503, P = 0.046, Fig. 4E). The decreased generation of GABAergic synapses confirms that interneuron development is affected in 4H. 4H neurons show altered network activity To assess whether the decreased percentage of GABAergic synapses had consequences for network activity, we recorded spontaneous activity of developing neuronal networks from three controls and four 4H patients plated on multi-electrode arrays (MEAs) (Fig. 5A). The activity was captured in three parameters namely single spike events, local bursts (spike trains) and network wide bursting events (Fig. 5B). Spontaneous action potential firing in the first weeks mainly consisted of single spike events, while local and network wide bursts occurred as the cultures matured (Fig. 5C-F). Tetrodotoxin administration (TTX, 1 uM) confirmed that baseline activity was depending on sodium ion channels (Supplemental Fig. 1A, D). At 14 weeks post plating, three of four 4H patients showed high levels of local and network bursts, which was mostly absent in control cultures (Fig. 5C, F). This is an indication of hyperactivity in 4H networks, although the network activity was not significantly different from control (spikes control 1.19 ± 0.40, 4H 2.41 ± 1.88; bursts control 1.65 ± 0.13, 4H 5.61 ± 5.21; network bursts control 0.33 ± 0.60, 4H 5.17 ± 5.16). Interestingly, the single 4H line (P3) that did not show an increased number of bursts and network bursts was also the 4H line with the highest percentage of GABAergic synapses. A correlation analysis indeed confirmed significant correlations between the percentage of VGAT+ synapses and the number of bursts (r = -0.774, P = 0.041) and network bursts (r = -0.786, P = 0.036), showing a relation between the amount of GABAergic pre-synapses and network activity.
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