Extended Data Fig. 15: SCVsx2::Hoxa10 neurons are necessary and sufficient for the recovery of walking after paralysis.
a, Exploded diagram of a new wireless optoelectronic system that integrates on the same implant red-shifted microLEDs40 to deliver deeply-penetrating photostimulation and electrodes to deliver EES. The zoom shows the microLEDs and electrodes for EES. b, Optogenetic silencing of SCVsx2::Hoxa10 neurons in mice that underwent four weeks of EESREHAB. Experiments and data are shown using the same conventions as in Extended Data Figs. 2 and 139 (n > 20 per mouse, n = 4 mice per group). Bars report n = 4 mice per group; statistics indicate Tukey HSD tests following repeated measures ANOVA, P < 10–15, P = 0.003 and P = 0.002, respectively. c, As in b, but during acute silencing of SCVsx2::Hoxa10 neurons with Gi in mice that underwent four weeks of EESREHAB (n = 4 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 7.2 × 10–10, P = 2.1 × 10–5, P = 0.0006 respectively). d, As in c, but during acute activation of SCVsx2::Hoxa10 neurons with Gq in mice tested at four weeks post-SCI (no EESREHAB) with EESOFF and EESON (n = 4 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 3.1 × 10–5, P = 4.4 × 10–7, P = 3.1 × 10–5, P = 0.0069 respectively). e, As in d, but following the chronic silencing of SCVsx2::Hoxa10 neurons (Gi, CNO in drinking water) in mice that underwent four weeks of EESREHAB, chronic activation of SCVsx2::Hoxa10 neurons (Gq, CNO in drinking water) in mice after SCI, and chronic activation of SCVsx2::Hoxa10 neurons (Gq, CNO in drinking water) in mice that underwent four weeks of rehabilitation without EES (n = 5 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 0.0007, P = 1.6 × 10–5, P = 0.004, P = 0.011, respectively). f, Chronic silencing of SCVsx2::Hoxa10 neurons during EESREHAB altered the connectome and projectome of SCVsx2::Hoxa10 neurons compared to mice that underwent EESREHAB. 3D reconstructions show the labeled neurons in the reticular formation 3 days following the infusion of Cre-dependent PRV Ba2017 in the lumbar spinal cord of Vsx2Cre mice. The density of vGluT1 synapses apposing SCVsx2::Hoxa10 neurons was quantified to evaluate the synaptic projection from large-diameter afferent fibers onto SCVsx2::Hoxa10 neurons. The projectome of SCVsx2::Hoxa10 neurons was vizualized using infusions of AAV-flex-tdTomato in the lumbar spinal cord. The bar plots show the average number of vGluT1 synapses apposing SCVsx2::Hoxa10 neurons (n = 6 mice per group; independent samples two-tailed t-test, t = –3.1, P = 0.018), the number of neurons in the reticular formation (n = 4 and 3 mice per group, respectively; independent samples two-tailed t-test, t = –4.3, P = 0.0076), and the density of projections from SCVsx2::Hoxa10 neurons in the ventral horn (n = 4 mice per group; independent samples two-tailed t-test, t = –9.2, P = 0.0003). g, The impact of the chronic ablation of SCVsx2::Hoxa10 neurons on the natural recovery of mice with thoracic lateral hemisection SCI was evaluated as in Extended Data Fig. 13g, and summarized in the timeline of experiments. Photographs show coronal sections of the hemisected spinal cord at the lesion epicenter, while CLARITY-optimized light sheet microscopy illustrates the interruption of the reticulospinal tract on the hemisected side. Locomotor performances were evaluated as detailed in the other panels. Bar plots, n = 5 mice per group; independent samples two-tailed t-test, t = 6.6, P = 0.0002; t = –3.4, P = 0.09; t = –1.9, P = 0.093, respectively). h, Photographs show coronal sections of the spinal cord with Vsx2 RNAScope, confirming the reduction in the number of SCVsx2::Hoxa10 neurons in the lumbar spinal cord of Vsx2Cre mice after diphtheria toxin injections. Bar plots report the mean number of Vsx2 labeled neurons per section in the spinal grey matter (SCI n = 3, SCI with SCVsx2::Hoxa10 ablation n = 4 mice; independent samples two-tailed t-test, t = 9.5, P = 0.0001). Photographs below show examples of projections from reticulospinal neurons in the lumbar spinal cord in both groups of mice. The plot reports the mean density of reticulospinal projections across the dorsoventral extent of the spinal cord for mice with SCI (n = 3) and mice with SCI and ablation of SCVsx2::Hoxa10 neurons (n = 4). Ribbons show the standard deviation (two-tailed Wilcoxon rank-sum test, W = 2008248, P < 10–15). Bar plots show the mean with individual data points overlaid. Error bars show the standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
