Fig 2: Effect of knockdown of TrkB or p75NTR in inferior olivary neurons on CF synapse elimination. a Schema of vector constructs for TrkB-KD, TrkB-Res and p75NTR-KD. b, c Bright field (BF) and fluorescent images showing the expression of EGFP in inferior olivary neurons b and CFs in the cerebellum c from a P21 mouse. The box in b is enlarged in the inset. Dotted lines in c indicate the PC layer. Scale bars: b, 200 mm; c, 50 μm; inset in b, 20 μm. d Representative traces of CF-EPSCs recorded from a control PC (left, from a P21 mouse), a PC associated with TrkB-KD CFs (middle, from a P26 mouse) and a PC associated with CFs expressing both TrkB-KD and TrkB-Res (right, from a P21 mouse). Single traces recorded at different stimulus intensities were superimposed. Holding potential was −10 mV. Scale bars, 10 ms and 1 nA. e Frequency distribution histogram for the number of CFs innervating each PC for control PCs (open columns, n = 46 PCs from 11 mice), PCs associated with TrkB-KD CFs (blue columns, n = 64 PCs from 11 mice) and PCs associated with CFs expressing both TrkB-KD and TrkB-Res (light blue columns, n = 23 PCs from 3 mice) during P21–P59. (P = 0.0303, control vs. TrkB-CF-KD; P = 0.026, TrkB-CF-KD vs. TrkB-CF-Res/TrkB-CF-KD; P = 0.509, control vs. TrkB-CF-Res/TrkB-CF-KD, Mann–Whitney U-test). Control PCs and PCs associated with TrkB-KD CFs were recorded in the same mice. *P < 0.05. f, g Similar to d, e but for results from PCs associated with p75NTR-KD CFs and control PCs during P22–P25. CF-EPSCs in f were recorded in cerebellar slices from a control PC (left, from a P21 mouse) and a PC associated with p75NTR-KD CFs (right, from a P21 mouse). Scale bars, 10 ms and 1 nA. Sample sizes for g were 32 control PCs and 34 PCs associated with p75NTR-KD CFs in the same 5 mice (P = 0.832, Mann–Whitney U-test). ns indicates no significant difference between the groups

Fig 3: Characterization of Schwann cell constructs following self-assembly fabrication process using phase-imaging, immunocytochemistry, and confocal microscopy. (A,B) Phase microscopy was utilized to visualize Schwann cells seeded in a 300 µm ID agarose hydrogel micro-column. (A) At 1 day in vitro, Schwann cells were found adhering to the collagen ECM coating the inner lumen of the micro-column. (a) Higher magnification revealed the cells begun to self-assemble into cables and exhibit process-bearing morphology. (B) By 4 days in vitro, the Schwann cells formed dense bundles within the inner lumen of the agarose micro-column. (b) These bundles appeared highly organized comprised of Schwann cells with longitudinally-aligned processes. (C) Morphometric assessment of the dense bundles at 4 days in vitro revealed Hoechst (HST)-positive cells with elevated expression of S100b and NGFR p75, common Schwann cell markers. (c) Under higher magnification, most of the Schwann cells co-localized with NGFR p75R and exhibited bipolar morphologies with elongated processes, a common phenotype during nervous system development and regeneration. (c') Max projection image showing a high density of aligned Schwann cells denoted by arrows. (c'') Single z-plane frame from the same image illustrating NGFR p75 expression on the membrane of highly aligned S100ß positive Schwann cells denoted by arrows. Scale bars: (A,B) 500 µm, (a,b) 50 µm, (C) 100 µm, (c) 50 µm, (c?) 100 µm.

Fig 4: Aberrant HCs in ectopic topology and abnormal pillar cells are formed in the Sox2 CKO.Patches of HCs in the base of the E18.5 Sox2 CKO cochlea are covered by a tectorial membrane with HCs in the topology of inner HCs displaying both large diameter (a,a’) and small diameter (a,a”) stereocilia reminiscent of inner and outer HCs, respectively. (b) Vestibular HCs show normal organization of stereocilia but many display variability in stereocilia diameter in a single HC, normally associated with either type I or type II vestibular HCs. (c-d’) Scattered Myo7a positive HCs are detected in the area corresponding topologically to the organ of Corti (OC); however, forming atypical organ of Corti (“OC”) in the mutant. Immunostaining of Myo7a reveals formation of HCs in the ectopic topologies, medial to OC, in the GER, as well as lateral to OC (in the area of Hensen/Claudius cells) (white arrows) in addition to the area of “OC” in the E18.5 Sox2 CKO. (e-f”) The combination of p75 and Myo7a immunolabeling shows an unusual configuration and distribution of p75 positive cells near the remaining Myo7a positive HCs in E18.5 Sox2 CKO compared to the single row of p75+ inner pillar cells in control littermates (e). Scale bars: 10 µm (a,e-f”), 1 µm (a’,a”,b), 100 µm (c-d’). GER, greater epithelial ridge; H/Cl, Hensen/Claudius cells; OC, organ of Corti; “OC”, atypical organ of Corti in the mutant; SL, spiral limbus.

Fig 5: CF synapse elimination in TrkB-PC-KO and p75NTR-PC-KO mice. a Representative traces of CF-EPSCs in a PC from a control (left, P21) and a TrkB-PC-KO (right, P26) mouse. Holding potential was −10 mV. Single traces recorded at different stimulus intensities were superimposed. b Frequency distribution histogram for the number of CFs innervating each PC in control (open columns, n = 67 PCs from 7 mice) and TrkB-PC-KO (filled columns, n = 71 PCs from 7 mice) mice during P21–P31 (P = 0.612, Mann–Whitney U-test). ns indicates no significant difference between the two groups. c, d Similar to a, b but for results from p75NTR-PC-KO and control mice during P21–P41. CF-EPSCs in c were recorded in cerebellar slices from a control (left, P39) and a p75NTR-PC-KO (right, P38) mouse. Sample sizes for d were 54 PCs from 6 control mice and 69 PCs from 8 p75NTR-PC-KO mice (P = 0.548, Mann–Whitney U-test). ns indicates no significant difference between the two groups. Scale bars: a and c, 10 ms and 1 nA