Fig. 1: Preferential glycinergic innervation of motor neurons innervating fast-twitch fatigable muscle fibers and loss in a SOD1^G93A mouse model.

Microphotographs depicting glycinergic inputs onto MMP9⁺ fast motor neurons (a–c) and ErrBeta⁺ slow motor neurons (e–g) in a GlyT2^GFP mouse. Motor neuron somata were selected and synapses were reconstructed for quantifications as shown in the masks in (d) and (h). Quantifications (q–r) expressed as synaptic particles and corrected for motor neuron area, show higher synaptic density in MMP9⁺ motor neurons for both synaptophysin (SYN) (MMP9 = 566 ± 60.75 and ErrBeta  = 417.19 ± 17.5; two-tailed t test, P = 0.0317, t = 2.354, df = 16; N = 9 independent mice) and GlyT2^GFP (MMP9 = 274.4 ± 29.25 and ErrBeta = 142.75 ± 39.77; two-tailed t test, P = 0.0008, t = 4.100, df = 16; N = 9 independent mice) markers. Innervation of fast (i–k) and slow (m–o) motor neurons in GlyT2^GFP mice crossed with SOD1^G93A mice at postnatal day (P) 63 and their respective synaptic densities (l, p). Quantifications of synaptic density were performed at P45, P63, and P84 for SYN (s) and GlyT2^GFP (t) markers. MMP9⁺ motor neurons showed progressive reduction of SYN⁺ (one-way ANOVA and Dunnett’s post hoc, F(3,14) = 7.004, P45 P = 0.6992, P63 P = 0.0382, P84 P = 0.0027; N = 3 independent mice per timepoint) and GlyT2^GFP+ inputs (one-way ANOVA and Dunnett’s post hoc, F(3, 14) = 16.742, P45 P = 0.0121, P63 P = 0.0015, P84  P= 0.00007; N = 3 independent mice per timepoint) when compared with ErrBeta⁺ motor neurons (SYN ⁺ one-way ANOVA and Dunnett’s post hoc, F(3, 14) = 2.224, P45 P = 0.7919, P63 P = 0.1285, P84 P = 0.9495; N = 3 independent mice per timepoint) (GlyT2^GFP+ one-way ANOVA and Dunnett’s post hoc, F(3, 14) = 2.732, P45 P = 0.057, P63 P = 0.9999, P84 P = 0.9694; N = 3 independent mice per timepoint). MMP9 (a, i) or ErrBeta (e, m) in blue, SYN (c, g, k, o) in red and GlyT2^GFP (b, f, j, n) in green. Scale bar in (o) =50 μm representative for all images. A minimum of 200 motor neurons was quantified per condition. In all graphs, data are presented as mean values ± SEM. Source data are provided as a Source Data file.