Fig 2: Immunocytochemical analysis of synapse formation during cultivation of spinal cord culture. For excitatory synapse analysis, colocalization of VGLUT1 and Homer1 was examined (A,C,D), while colocalization of synaptic markers VGAT and Gephyrin was used for analysis of inhibitory synapses (B,E,F). Formation of new excitatory synapses was the most prominent between day in vitro (DIV) 7 and 15 (C). A similar pattern was observed in colocalization analysis of inhibitory synapses (E). VGLUT1, Homer1, and VGAT colocalization ratios were stable during cultivation (D,F), while an increase of ratio of Gephyrin marked synapses that colocalized with VGAT occurred between DIV7 and DIV15 (F). Scale bars: 25 µm. Data are presented as means + SEM of N = 3 biological replicate cultures; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Artemisinin treatment has a specific postsynaptic effect, decreasing gephyrin cluster volume(A) Description of experimental design; preparation of primary hippocampal cultures and treatment with artemisinins before electrophysiology and immunocytochemistry experiments.(B) Molecular structure of artemisinins (Kasaragod et al., 2019) and diagram of molecular interaction of artemisinins and GABAAR intracellular loops between transmembrane domains 3–4 binding to the gephyrin universal binding pocket on the GephE domain.(C) Following 1-h control (DMSO) and 50 µM artemisinin treatment, neurons were co-immunolabeled for vGAT (presynaptic marker, green), gephyrin (postsynaptic marker, red), and MAP2 (dendrite, blue). The colocalization of vGAT and gephyrin puncta represent inhibitory puncta.(D) Quantitative analyses of inhibitory puncta per 10 µm of dendrite (ordinary one-way ANOVA p = 0.0137, Dunnett’s multiple comparisons test DMSO versus artemether p = 0.4367, DMSO versus artemisinin p = 0.1475, DMSO versus artesunate p = 0.9308. Sample size is equivalent to number of regions imaged: DMSO n = 12, artemether n = 10, artemisinin n = 16, artesunate n = 13).(E) 3D dSTORM reconstructions of colocalized and pre- and postsynaptic clusters with localizations color coded by probe (vGAT, blue; gephyrin, pink).(F) Quantitative analyses of gephyrin cluster volume (Kruskal-Wallis test p = 0.0001, Dunn’s multiple comparisons test DMSO versus artemether p = 0.0011, DMSO versus artemisinin p = 0.0003, DMSO versus artesunate p = 0.4738. Sample size is equivalent to number of regions imaged: DMSO n = 7, artemether n = 7, artemisinin n = 9, artesunate n = 6).(G) vGAT cluster volume (Kruskal-Wallis test p = 0.0872, Dunn’s multiple comparisons test DMSO versus artemether p = 0.9122, DMSO versus artemisinin p > 0.9999, DMSO versus artesunate p = 0.19991. Sample size is equivalent to number of regions imaged: DMSO n = 7, artemether n = 7, artemisinin n = 9, artesunate n = 6).(H) Distance between center of vGAT and gephyrin colocalized clusters (Kruskal-Wallis test p = 0.8732, Dunn’s multiple comparisons DMSO versus artemether p > 0.9999, DMSO versus artemisinin p > 0.9999, DMSO versus artesunate p > 0.9999. Sample size is equivalent to number of regions imaged: DMSO n = 7, artemether n = 7, artemisinin n = 9, artesunate n = 6).(I) Proposed schematic of how pre- and postsynapse centroid distance is unchanged following the loss of gephyrin. Graphs are mean ± SEM. Significance reported as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. NS denotes non-significance.

Fig 4: Image analysis pipelineSingle molecule localizations of the same probe are clustered according to the DBScan algorithm. Molecules not localized to a cluster are eliminated from further analysis. Cluster a shapes are determined and further analysis is conducted on gephyrin volume, vGAT-gephyrin centroid distance, and density. (Representative clusters obtained from data generated in Guzikowski and Kavalali,1 Images have been manipulated to represent potential outcomes of this protocol).