Cortical interneuron development is affected in 4H leukodystrophy 49 2 To establish whether increased network activity in 4H neuronal networks is due to altered synaptic signalling, modulators for glutamate or GABA signalling were added to the cultures at 14 weeks post plating. Network activity was measured for 10 min before and after addition of modulators. Ionotropic AMPA receptor antagonist DNQX (10 uM) or NMDA antagonist APV (50 µM) were used to measure glutamatergic signalling. The inhibition of either one of these glutamate receptors resulted in a significant decrease of spike count for both 4H and control networks (Supplementary Fig. 1B-C, E-F). In line with the spike counts, burst and network bursts mostly disappeared (data not shown), showing that coordinated network activity depended on glutamatergic signalling and showed no obvious differences between 4H and control lines. To study GABAergic signalling, GABAA receptor antagonists Bicuculline (30 µM) combined with Gabazine (20 µM) or the neurotransmitter GABA (10 µM) were administered. After addition, GABA antagonists evoked network bursts in controls that were mostly absent before (burst before 2.85 ± 2.00, after 13.5 ± 5.00, Z = -2.201, P = 0.028; network burst before 0 ± 0, after 10.5 ± 6.99, Z = -2.023, P = 0.043, Fig. 5G-J). In contrast, the number of bursts and network bursts did not significantly change in 4H patients (burst before 14.13 ± 12.57, after 18.06 ± 11.60, Z = -1.886, P = 0.059; network burst before 11.7 ± 10.91, after 15.00 ± 12.45, Z = -1.527, P = 0.127, Fig. 5G-J). Administration of GABA decreased neuronal activity in both control and 4H cells (control before 215.13 ± 83.50, after 1.68 ± 0.25, t(5) = 5.005, P = 0.004; 4H before 374.73 ± 212.51, after 32.18 ± 50.59, Z = -2.803, P = 0.005, Fig. 5K,L), showing normal post-synaptic GABAergic response. This data shows that the decreased generation of GABAergic synapses correlates to an increase in network activity, and there is a decreased response of 4H neurons to treatment with GABA antagonists.
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