Новости

5/session; mixed-effects model, t = –6.40, P = 2.9 × 10–8). e, f, The role of SCVsx2::Hoxa10 neurons in the reorganization of muscle responses to EES was evaluated using Cre-dependent expression of Gi, e, or Gq DREADDs in SCVsx2::Hoxa10 neurons, f. The timelines summarize the timing of the various experimental procedures. The photographs illustrate the expression of DREADD receptors in SCVsx2::Hoxa10 neurons. Long-latency muscle responses to single-pulse of EES were quantified before and 30 min after CNO injections that either silenced (Gi) or activated (Gq) SCVsx2::Hoxa10 neurons. Bar plots report the integral of the RMS of long-latency muscle responses for each each experimental condition (n = 5 mice per group (Gi); paired samples two-tailed t-test, t = 2.4, P = 0.046; n = 5 mice per group (Gq); paired samples two-tailed t-test, t = 2.8, P = 0.047). g, Three complementary strategies were used to evaluate the role of SCVsx2::Hoxa10 neurons during basic unskilled walking in uninjured mice: targeted injections of AAV5-flex-hm4di (Gi) or AAV5-flex-Jaws in the lumbar spinal cord of Vsx2Cre mice to evaluate the short-term or immediate impact of silencing SCVsx2::Hoxa10 neurons, and targeted injection of AAV-flex-DTR to evaluate the long-term impact of the complete ablation of SCVsx2::Hoxa10 neurons. Locomotor performance was evaluated during overground walking using the procedures detailed in Extended Data Fig. 2e (n > 20 gait cycles per mouse, n = 5 mice per group). The bar plot reports locomotor performance, quantified as average scores on PC1 (n = 5 mice per group except for DTR with n = 8 mice per group; paired samples two-tailed t-test, Gi: t = 1.4; P = 0.23; LED: t = 1.5, P = 0.21; DTR: t = 1.1, P = 0.33), and the number of SCVsx2::Hoxa10 neurons per tissue section (SCVsx2::Hoxa10 ablation, n = 7 mice; no ablation, n = 4 mice; independent samples two-tailed t-test, t = 12.3, P = 6.2 × 10–7). 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./p>

 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./p>