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Fig. 3 Recovery of excitatory/inhibitory balance during spinal cord regeneration. A UMAP plot showing 30 neuron subclusters identified with 0.6 resolution parameter. B Classification of neuron clusters based on neurotransmitter properties. Clusters enriched for at least one of the glutamatergic and GABA/glycinergic markers are identified as excitatory and inhibitory, respectively. Clusters that express serotonergic, cholinergic or a mix of glutamatergic and glycinergic markers are labeled as ?Other?. One neuron cluster that does not express any of these marker genes is labeled ?Unidentified?. C Pie charts show the proportions of excitatory, inhibitory and ?Other? neuron populations at 0, 1, 3 and 6 wpi. D in silico calculation of E/I ratios during SC regeneration. E, F Immunostaining for RFP, GFP, HuC/D and Hoechst in SC cross sections from Tg(vglut2a:RFP) and Tg(gad1b:GFP) zebrafish at 0, 1, 3 and 6 wpi. White arrows point to RFP+ HuC/D+ neurons in E and GFP+ HuC/D+ neurons in (F). Insets depict double channels for RFP and HuC/D in E and for GFP and HuC/D in (F). Single channel insets of vglut2a:RFP or gad1b:GFP are shown in dorsal and ventral SC domains, respectively. G, H Quantification of glutamatergic and GABAergic neurons after SCI. Percent vglut2a+ HuC/D+ neurons (ANOVA p: 0.0086) and gad1b+ HuC/D+ neurons (ANOVA p: 0.0043) were normalized to the total number of HuC/D+ neurons. I in vivo quantification of E/I ratios during SC regeneration. Ratios of glutamatergic to GABAergic neurons were calculated (ANOVA p: 0.0268). Data points in all bar charts indicate individual animals and sample sizes are indicated in parentheses. SC cross sections 450 µm rostral to the lesion were analyzed. Brown-Forsythe and Welch ANOVA were performed in (G, H) with Dunnett?s T3 multiple comparisons performed across time points with 95% CI. Ordinary one-way ANOVA with Tukey multiple comparisons was performed in (I). All statistical tests are two-sided. In all bar charts, data are presented as mean values +/- SEM. *p ? 0.05, **p ? 0.01; Scale bars, 50 µm. Source data are provided as a Source Data file.