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Vector Biolabs aav5 cag flex mcherry
Aav5 Cag Flex Mcherry, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 7. Optogenetic activation of PVINs improves performance in Scn8a+/-mice while common AED, VPA has no effect. (A) Mice received a bilateral injection with the FLOX-ChR2 virus (scale bar = 500 µm), and then were implanted bilaterally with optical fibers for delivering blue light during the cue (40 Hz, 5ms pulse width). (B) With gamma stimulation, there is a significant effect of light (two-way ANOVA, F (1, 22)=6.973, p=0.0173, n=7 mice) during the intermediate cue (p=0.0044). (C) Timeline of saline and VPA injections (200 mg/kg) during AET testing. (D) While Scn8a+/- mice had reduced performance in comparison to WT, there was no effect of VPA on performance (three-way ANOVA, group effect, F (1, 35)=5.850, p=0.0191, VPA effect, F (1, 35)=0.010, p=0.943; n=3 and 6 for WT and Scn8a+/- mice, respectively). See also Figure 7—figure supplements 1 and 2.

Journal: eLife

Article Title: Prefrontal PV interneurons facilitate attention and are linked to attentional dysfunction in a mouse model of absence epilepsy

doi: 10.7554/elife.78349

Figure Lengend Snippet: Figure 7. Optogenetic activation of PVINs improves performance in Scn8a+/-mice while common AED, VPA has no effect. (A) Mice received a bilateral injection with the FLOX-ChR2 virus (scale bar = 500 µm), and then were implanted bilaterally with optical fibers for delivering blue light during the cue (40 Hz, 5ms pulse width). (B) With gamma stimulation, there is a significant effect of light (two-way ANOVA, F (1, 22)=6.973, p=0.0173, n=7 mice) during the intermediate cue (p=0.0044). (C) Timeline of saline and VPA injections (200 mg/kg) during AET testing. (D) While Scn8a+/- mice had reduced performance in comparison to WT, there was no effect of VPA on performance (three-way ANOVA, group effect, F (1, 35)=5.850, p=0.0191, VPA effect, F (1, 35)=0.010, p=0.943; n=3 and 6 for WT and Scn8a+/- mice, respectively). See also Figure 7—figure supplements 1 and 2.

Article Snippet: resource Designation Source or reference Identifiers Additional information Strain, strain background (musculus males and females) B6;129P2- Pvalbtm1(cre)Arbr/J The Jackson Laboratory Stock No: 008069; RRID: IMSR_JAX:008069 Strain, strain background (musculus males and females) C3Fe.Cg- Scn8amed/J The Jackson Laboratory Stock No: 003798; RRID: IMSR_JAX:003798 Strain, strain background (musculus males and females) B6.Cg- Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J (VGAT- ChR2) The Jackson Laboratory Stock No:014548 RRID:IMSR_JAX:014548 Strain, strain background (musculus males and females) Ai32(RCL- ChR2(H134R)/EYFP) (Ai32) The Jackson Laboratory Stock No: 014548 RRID:IMSR_JAX:012569 Transfected construct (musculus) AAV- EF1a- DIO- EYFP University of North Carolina Vector Core N/A Transfected construct (musculus) AAV5- CAG- FLEX- GCaMP6s Addgene Cat# 100842- AAV5 Transfected construct (musculus) AAV5- FLOX- chR2- mCherry Addgene Cat# 20297- AAV5 Chemical compound, drug DNQX Sigma D0540 Chemical compound, drug CPP Sigma C104 Chemical compound, drug Valproic acid sodium salt Sigma P4543 Software, algorithm Prism GraphPad Software Inc https://www.graphpad.com/ scientific-software/prism/ Software, algorithm MATLAB MathWorks https://www.mathworks. com/ Ferguson et al. eLife 2023;12:e78349.

Techniques: Activation Assay, Injection, Virus, Saline, Comparison

a , Body weight support system enabling overground walking and wireless implantable pulse generator operating in closed loop, connected to a paddle lead targeting the dorsal roots that innervate lumbosacral segments. b , Chronophotography showing the transitioning from sitting to walking in a representative participant. c , 18 FDG-PET projected onto a personalized model of the spinal cord elaborated from high-resolution MRI (participant ID DM002), showing the metabolic activity of the spinal cord—expressed as standardized uptake value (SUV bw )—in response to walking before and after EES REHAB . d , Bar plots reporting the relative change in normalized FDG-PET metabolic activity during walking before and after EES REHAB , the lower limb motor scores, and the distance covered during the 6-min walk test ( n = 9; metabolic activity mixed-effects model: t = −3.2, P = 0.002; lower limb motor scores, paired samples two-tailed t -test: t = 3.7, P = 0.0063; distance covered, paired samples two-tailed t -test: t = 3.5; P = 0.0076). e , Left, body weight support system enabling overground walking in mice, with implantable electrodes to deliver EES. Right, spinal cord visualization of projections from neurons in the motor cortex and glutamatergic (vGluT2 ON ) neurons in the reticular formation, traced with AAV5-CAG-COMET-GFP and AAV5-CAG-DIO-COMET-tdTomato, respectively. Scale bars, 1 mm. f , Chronophotography of representative mice with SCI only (SCI, EES OFF ) or SCI with EES REHAB (EES REHAB , EES OFF ). g , Lumbar spinal cord expression of cFos following walking with EES ON following SCI or SCI with EES REHAB . Scale bars, 500 μm. h , Walking performance of uninjured mice ( n = 3), mice with SCI ( n = 10), and mice with SCI and EES REHAB tested with EES OFF ( n = 10) or EES ON ( n = 10) (one-way ANOVA; Tukey’s honest significant difference for SCI versus EES REHAB →EES OFF : P = 3.3 × 10 –11 ). i , The number of neurons expressing cFos(cFos ON ) (mice with SCI with EES ON , n = 4; mice with EES REHAB and EES ON , n = 4; independent samples two-tailed t -test: t = –5.7; P = 0.001). h , i , Bars show mean ± s.e.m. with individual points overlaid. * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Body weight support system enabling overground walking and wireless implantable pulse generator operating in closed loop, connected to a paddle lead targeting the dorsal roots that innervate lumbosacral segments. b , Chronophotography showing the transitioning from sitting to walking in a representative participant. c , 18 FDG-PET projected onto a personalized model of the spinal cord elaborated from high-resolution MRI (participant ID DM002), showing the metabolic activity of the spinal cord—expressed as standardized uptake value (SUV bw )—in response to walking before and after EES REHAB . d , Bar plots reporting the relative change in normalized FDG-PET metabolic activity during walking before and after EES REHAB , the lower limb motor scores, and the distance covered during the 6-min walk test ( n = 9; metabolic activity mixed-effects model: t = −3.2, P = 0.002; lower limb motor scores, paired samples two-tailed t -test: t = 3.7, P = 0.0063; distance covered, paired samples two-tailed t -test: t = 3.5; P = 0.0076). e , Left, body weight support system enabling overground walking in mice, with implantable electrodes to deliver EES. Right, spinal cord visualization of projections from neurons in the motor cortex and glutamatergic (vGluT2 ON ) neurons in the reticular formation, traced with AAV5-CAG-COMET-GFP and AAV5-CAG-DIO-COMET-tdTomato, respectively. Scale bars, 1 mm. f , Chronophotography of representative mice with SCI only (SCI, EES OFF ) or SCI with EES REHAB (EES REHAB , EES OFF ). g , Lumbar spinal cord expression of cFos following walking with EES ON following SCI or SCI with EES REHAB . Scale bars, 500 μm. h , Walking performance of uninjured mice ( n = 3), mice with SCI ( n = 10), and mice with SCI and EES REHAB tested with EES OFF ( n = 10) or EES ON ( n = 10) (one-way ANOVA; Tukey’s honest significant difference for SCI versus EES REHAB →EES OFF : P = 3.3 × 10 –11 ). i , The number of neurons expressing cFos(cFos ON ) (mice with SCI with EES ON , n = 4; mice with EES REHAB and EES ON , n = 4; independent samples two-tailed t -test: t = –5.7; P = 0.001). h , i , Bars show mean ± s.e.m. with individual points overlaid. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following AAV plasmids were used and detailed sequence information is available as detailed or upon request: AAVDj-hSyn-flex-mGFP-2A-synaptophysin-mRuby (Stanford Vector Core Facility, reference AAV DJ GVVC-AAV-100), AAV5-CAG-DIO-COMET-tdTomato and AAV5CAG-COMET-GFP (a gift from M. Tuszynski), AAV5-Syn-flex-ChrimsonR-tdT (Addgene 62723), AAV5-CAG-flex-Jaws-KGC-GFP-ER2 (Addgene 84445), AAV5-hSyn-DIO-hm4D (Gi)-mCherry (Addgene 44362), AAV5-hSyn-DIO-hm3D (Gq)-mCherry (Addgene 44361), AAV5-CAG-flex-tdTomato (a gift from S. Arber), AAV5CAG-flex-human diphtheria toxin receptor (DTR) (a gift from S. Arber), AAV5-DIO-TC66T-2A-eGFP-2A-oG (GT3) (Salk Institute) and AAV5-hSyn-DIO-TVAP2A-EGFP-2A-oG (a gift from T. Karayannis).

Techniques: Activity Assay, Two Tailed Test, Expressing

a , Three-dimensional visualization and quantification of the contusion SCI. Photographs show multiple 3D views of a contused spinal cord that has been cleared using uDISCO . The extent of spinal cord damage was quantified from coronal sections immunolabeled against glial fibrillary acidic protein (GFAP). The relative amount of spared tissues was quantified at the lesion epicentre and at regular intervals from the lesion epicentre in the rostral and caudal directions, as reported in the line plots. Sparing was consistent across groups (mean = 10.6%, n = 5 mice per group; independent samples two-tailed t-test on lesion epicenter, t = 0.5, P = 0.61). The impact of the contusion SCI on descending pathways compared to an uninjured spinal cord was visualized in whole brain-spinal cord preparations cleared with CLARITY. Projections from neurons located in the leg region of the motor cortex (AAV5-CAG-COMET-GFP) and from GluT2 ON neurons located in the reticular formation (AAV5-CAGDIO-COMET-tdTomato) were labeled with virus infusions, as indicated in the photograph. Insets show the complete interruption of corticospinal fibers and partial preservation of reticulospinal fibers. b , Adaptations of the technological features of EES REHAB in mice, including a new robotic body weight support interface with 1g accuracy and a pair of electrodes attached to L2 and S1 spinal segments to deliver EES. Kinematic recordings of whole-body movements were mapped onto a 3D model of the mouse including bones and skin contours to generate realistic visualization of leg movements . The line shows the trajectory of the lower limb endpoint (toe) while circles indicate the maximum step height. Representative leg movements are shown concomitantly to the electromyographic (EMG) activity of the tibialis anterior, recorded from chronically implanted bipolar electrodes into the muscle. At one week, EES was insufficient to reactivate the spinal cord to a level that enabled walking in mice. Consequently, a small bolus of agonists to 5H1A/7 (8-OH-DPAT, 0.05-0.2 mg/kg) and 5HT2A receptors (quipazine 0.2-0.3 mg/kg) was administered to augment the response of the spinal cord . The combination of EES and 5-HT agonists enabled all the mice to walk overground as early as one week after the contusion SCI. 5-HT agonists were used to enable sustained walking during EES REHAB , but the amount of drug was progressively decreased over the course of the recovery, and eventually suppressed. Final testing was performed without 5-HT agonists. c , We noticed that compared to rats and humans, EES was not as effective to enable walking in mice with SCI. Inspection of muscle responses to EES revealed that the stimulation recruited motor nerves (efferents) at relatively low amplitudes. This off-target stimulation of the ventral roots is due to the relatively small size of the mouse spinal cord. We previously showed that high-frequency bursts are effective to maximize the activation of large-diameter afferent fibers targeted by EES . To adapt this stimulation protocol to mice, we elaborated a computational model of the mouse spinal cord . The model includes a spiking neural network model of muscle spindle feedback circuits for a pair of antagonistic muscles. Response in motor neurons are shown for two amplitudes of EES that correspond to the recruitment of 20% and 60% of the entire afferent population. Simulations suggested that, compared to conventional protocols (40 Hz), high-frequency burst stimulation (carrier frequency 600 Hz, modulating frequency 30 Hz) leads to a summation of synaptic events into motor neurons, which augment the probability to activate muscles via the recruitment of afferents compared to efferents. To confirm these findings experimentally, we compared conventional stimulation protocols to high-frequency burst stimulation during stepping on a treadmill in mice with contusion SCI. Reconstructed leg movements including foot trajectories are shown for each experimental condition at two amplitudes of EES. High frequency burst stimulation increased the therapeutic window through which stimulation could be delivered. Concretely, step height scaled linearly up to 1.5× motor threshold using burst stimulation protocols, and stimulation could be delivered effectively up to 2.5× motor threshold. In contrast, conventional stimulation at 40 Hz facilitated stepping around motor threshold, but additional increase in amplitude that would be necessary to promote robust stepping led to tonic activation of leg muscle activation and thus cessation of stepping. d , To model the volitional control of stepping observed in humans during EES ON , we manipulated cortical activity with optogenetics. We expressed channelrhodopsin in the neurons of the leg motor cortex using targeted injections of AAV5-hSyn-ChR2 ( n = 4 mice). Mice with contusion SCI were recorded during stepping on a treadmill with EES ON (no 5-HT agonists). Photostimulation of the motor cortex induced a significant increase of the step height that scaled up with laser intensity, as shown in the bar plots ( n = 4 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 0.0003, P = 0.025, respectively). e , Leg movements and muscle activity during overground walking without any intervention or support recorded at four weeks after a contusion SCI in a mouse that did not undergo EES REHAB and in a mouse that underwent EES REHAB . Locomotor performance was quantified using principal component analysis applied to 80 gait parameters calculated from kinematic recordings (Supplementary Table ). In this denoised space, each dot represents a gait cycle ( n = 20 per mouse, n = 9 mice per group). Larger dots represent the mean of each experimental group. The first principal component (PC1) distinguished gaits from mice with SCI that did not undergo EES REHAB from mice that underwent EES REHAB . Locomotor performances were thus quantified as the scores on PC1 (reported in Fig. ). Analysis of factor loadings on PC1 revealed that the percentage of paw dragging, the extent of whole-limb oscillation (virtual limb connecting the hip to the toe) and step height were the parameters that showed the highest correlation with PC1. Bars report the mean values of these gait parameters ( n = 9 mice per group; statistics indicate Tukey HSD tests following oneway ANOVA, P = 2.1 × 10 –9 , P = 7.4 × 10 –6 , P = 0.0055, respectively). Data from mice that underwent EES REHAB are shown during EES OFF and EES ON . f , Photographs show whole mouse spinal cords before and after processing with iDISCO+ , , during which the spinal cords underwent immunolabeling of cFos followed by clearing. The spinal cords were imaged with the mesoSPIM lightsheet microscope . Representative microscopy images show a raw coronal optical slice of the cFos labelling in the spinal cord and after application of automated 3D nuclear spot detection. Images were then reconstructed to visualize the entire lumbar spinal cord. g , 3D cFos quantifications were confirmed using immunohistochemistry and labelling for cFos on coronal sections of the lumbar spinal cord, as illustrated in the representative photographs of spinal cord sections from mice with SCI and mice that followed EES REHAB . The bar plot reports the mean number of cFos labeled cells per section in the spinal grey matter across the whole section, in Lamina I–III (dorsal), in Lamina IV–VI (intermediate), in Lamina VII–IX (ventral) and in Lamina X (central canal) ( n = 4 mice per group; independent samples two-tailed t-test, t = –2.7, P = 0.042; t = 0.60, P = 0.57; t = 3.7, P = 0.010; t = 1.2, P = 0.27; t = 1.0, P = 0.35, respectively). 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.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Three-dimensional visualization and quantification of the contusion SCI. Photographs show multiple 3D views of a contused spinal cord that has been cleared using uDISCO . The extent of spinal cord damage was quantified from coronal sections immunolabeled against glial fibrillary acidic protein (GFAP). The relative amount of spared tissues was quantified at the lesion epicentre and at regular intervals from the lesion epicentre in the rostral and caudal directions, as reported in the line plots. Sparing was consistent across groups (mean = 10.6%, n = 5 mice per group; independent samples two-tailed t-test on lesion epicenter, t = 0.5, P = 0.61). The impact of the contusion SCI on descending pathways compared to an uninjured spinal cord was visualized in whole brain-spinal cord preparations cleared with CLARITY. Projections from neurons located in the leg region of the motor cortex (AAV5-CAG-COMET-GFP) and from GluT2 ON neurons located in the reticular formation (AAV5-CAGDIO-COMET-tdTomato) were labeled with virus infusions, as indicated in the photograph. Insets show the complete interruption of corticospinal fibers and partial preservation of reticulospinal fibers. b , Adaptations of the technological features of EES REHAB in mice, including a new robotic body weight support interface with 1g accuracy and a pair of electrodes attached to L2 and S1 spinal segments to deliver EES. Kinematic recordings of whole-body movements were mapped onto a 3D model of the mouse including bones and skin contours to generate realistic visualization of leg movements . The line shows the trajectory of the lower limb endpoint (toe) while circles indicate the maximum step height. Representative leg movements are shown concomitantly to the electromyographic (EMG) activity of the tibialis anterior, recorded from chronically implanted bipolar electrodes into the muscle. At one week, EES was insufficient to reactivate the spinal cord to a level that enabled walking in mice. Consequently, a small bolus of agonists to 5H1A/7 (8-OH-DPAT, 0.05-0.2 mg/kg) and 5HT2A receptors (quipazine 0.2-0.3 mg/kg) was administered to augment the response of the spinal cord . The combination of EES and 5-HT agonists enabled all the mice to walk overground as early as one week after the contusion SCI. 5-HT agonists were used to enable sustained walking during EES REHAB , but the amount of drug was progressively decreased over the course of the recovery, and eventually suppressed. Final testing was performed without 5-HT agonists. c , We noticed that compared to rats and humans, EES was not as effective to enable walking in mice with SCI. Inspection of muscle responses to EES revealed that the stimulation recruited motor nerves (efferents) at relatively low amplitudes. This off-target stimulation of the ventral roots is due to the relatively small size of the mouse spinal cord. We previously showed that high-frequency bursts are effective to maximize the activation of large-diameter afferent fibers targeted by EES . To adapt this stimulation protocol to mice, we elaborated a computational model of the mouse spinal cord . The model includes a spiking neural network model of muscle spindle feedback circuits for a pair of antagonistic muscles. Response in motor neurons are shown for two amplitudes of EES that correspond to the recruitment of 20% and 60% of the entire afferent population. Simulations suggested that, compared to conventional protocols (40 Hz), high-frequency burst stimulation (carrier frequency 600 Hz, modulating frequency 30 Hz) leads to a summation of synaptic events into motor neurons, which augment the probability to activate muscles via the recruitment of afferents compared to efferents. To confirm these findings experimentally, we compared conventional stimulation protocols to high-frequency burst stimulation during stepping on a treadmill in mice with contusion SCI. Reconstructed leg movements including foot trajectories are shown for each experimental condition at two amplitudes of EES. High frequency burst stimulation increased the therapeutic window through which stimulation could be delivered. Concretely, step height scaled linearly up to 1.5× motor threshold using burst stimulation protocols, and stimulation could be delivered effectively up to 2.5× motor threshold. In contrast, conventional stimulation at 40 Hz facilitated stepping around motor threshold, but additional increase in amplitude that would be necessary to promote robust stepping led to tonic activation of leg muscle activation and thus cessation of stepping. d , To model the volitional control of stepping observed in humans during EES ON , we manipulated cortical activity with optogenetics. We expressed channelrhodopsin in the neurons of the leg motor cortex using targeted injections of AAV5-hSyn-ChR2 ( n = 4 mice). Mice with contusion SCI were recorded during stepping on a treadmill with EES ON (no 5-HT agonists). Photostimulation of the motor cortex induced a significant increase of the step height that scaled up with laser intensity, as shown in the bar plots ( n = 4 mice per group; statistics indicate Tukey HSD tests following one-way ANOVA, P = 0.0003, P = 0.025, respectively). e , Leg movements and muscle activity during overground walking without any intervention or support recorded at four weeks after a contusion SCI in a mouse that did not undergo EES REHAB and in a mouse that underwent EES REHAB . Locomotor performance was quantified using principal component analysis applied to 80 gait parameters calculated from kinematic recordings (Supplementary Table ). In this denoised space, each dot represents a gait cycle ( n = 20 per mouse, n = 9 mice per group). Larger dots represent the mean of each experimental group. The first principal component (PC1) distinguished gaits from mice with SCI that did not undergo EES REHAB from mice that underwent EES REHAB . Locomotor performances were thus quantified as the scores on PC1 (reported in Fig. ). Analysis of factor loadings on PC1 revealed that the percentage of paw dragging, the extent of whole-limb oscillation (virtual limb connecting the hip to the toe) and step height were the parameters that showed the highest correlation with PC1. Bars report the mean values of these gait parameters ( n = 9 mice per group; statistics indicate Tukey HSD tests following oneway ANOVA, P = 2.1 × 10 –9 , P = 7.4 × 10 –6 , P = 0.0055, respectively). Data from mice that underwent EES REHAB are shown during EES OFF and EES ON . f , Photographs show whole mouse spinal cords before and after processing with iDISCO+ , , during which the spinal cords underwent immunolabeling of cFos followed by clearing. The spinal cords were imaged with the mesoSPIM lightsheet microscope . Representative microscopy images show a raw coronal optical slice of the cFos labelling in the spinal cord and after application of automated 3D nuclear spot detection. Images were then reconstructed to visualize the entire lumbar spinal cord. g , 3D cFos quantifications were confirmed using immunohistochemistry and labelling for cFos on coronal sections of the lumbar spinal cord, as illustrated in the representative photographs of spinal cord sections from mice with SCI and mice that followed EES REHAB . The bar plot reports the mean number of cFos labeled cells per section in the spinal grey matter across the whole section, in Lamina I–III (dorsal), in Lamina IV–VI (intermediate), in Lamina VII–IX (ventral) and in Lamina X (central canal) ( n = 4 mice per group; independent samples two-tailed t-test, t = –2.7, P = 0.042; t = 0.60, P = 0.57; t = 3.7, P = 0.010; t = 1.2, P = 0.27; t = 1.0, P = 0.35, respectively). 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.

Techniques: Immunolabeling, Two Tailed Test, Labeling, Preserving, Activity Assay, Activation Assay, Optogenetics, Microscopy, Immunohistochemistry

a , UMAP visualization of 20,990 neurons, coloured by Augur cell type prioritization, identifying perturbation-responsive subpopulations in a representative experimental comparison. b , Identification of perturbation-responsive subpopulations across experimental comparisons with Augur. Top, clustering tree of neuronal subpopulations. Middle, heat map showing scaled areas under the curve (AUCs) for individual comparisons: (1) SCI versus SCI →EES::walking ; (2) SCI versus EES REHAB ; (3) EES REHAB versus EES REHAB →cortex ; (4) EES REHAB versus EES REHAB →EES ; (5) EES REHAB →EES::walking versus SCI →EES::walking ; (6) EES REHAB versus EES REHAB →EES::walking . Bottom, distribution of AUCs across all comparisons. c , Spatial visualization of 22,127 barcodes in the common coordinate space coloured by Magellan spatial prioritization for the same representative experimental comparison as in a . d , Synaptic inputs and outputs from SC Vsx2::Hoxa10 neurons. Inputs: SC Vsx2::Hoxa10 neurons and their projections. Projections from vGi neurons (AAV5-CAG-COMET-GFP) and large-diameter afferent neurons (PV cre::Advillin:tdTomato ) onto SC Vsx2::Hoxa10 neurons. Outputs: synaptic appositions from SC Vsx2::Hoxa10 neurons (AAV-Dj-hSyn-flex-mGFP-2A-synaptophysin-mRuby) onto glutamatergic, GABAergic and motor neurons. C, caudal; R, rostral; PV, parvalbumin; syn, synaptophysin. e , Single-unit recordings of optotagged SC Vsx2::Hoxa10 neurons. Responses of an optotagged single unit to optogenetic stimulations. Right, the number of units responding to each type of stimulation. f , Number of vGluT1 synapses from large-diameter afferents apposing SC Vsx2::Hoxa10 neurons ( n = 6 out of 10 mice per group; independent samples two-tailed t -test: t = 4.9, P = 0.002). g , Number of reticulospinal neurons projecting to SC Vsx2::Hoxa10 neurons ( n = 4 mice per group; independent samples two-tailed t -test: t = 4.8, P = 0.0029). h , Percentage of motor neurons, glutamatergic and GABAergic neurons receiving at least one synapse from SC Vsx2::Hoxa10 neurons ( n = 6; one-way analysis of variance (ANOVA); choline acetyltransferase (ChAT): F = 0.45, P = 0.39; glutamatergic: F = 0.52, P = 0.76; GABAergic: statistics indicate Tukey’s honest significant difference tests: uninjured versus SCI: P = 5.2 × 10 −6; SCI versus EES REHAB : P = 1.8 × 10 −5 ). i , Fluorescent intensity of cFos in Vsx2 cre neurons following walking with EES ON ( n = 5 out of 3 mice per group; independent samples two-tailed t -test: t = 5.7, P = 0.0013). f – i , Bars show mean ± s.e.m. with individual points overlaid. AU, arbitrary units.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , UMAP visualization of 20,990 neurons, coloured by Augur cell type prioritization, identifying perturbation-responsive subpopulations in a representative experimental comparison. b , Identification of perturbation-responsive subpopulations across experimental comparisons with Augur. Top, clustering tree of neuronal subpopulations. Middle, heat map showing scaled areas under the curve (AUCs) for individual comparisons: (1) SCI versus SCI →EES::walking ; (2) SCI versus EES REHAB ; (3) EES REHAB versus EES REHAB →cortex ; (4) EES REHAB versus EES REHAB →EES ; (5) EES REHAB →EES::walking versus SCI →EES::walking ; (6) EES REHAB versus EES REHAB →EES::walking . Bottom, distribution of AUCs across all comparisons. c , Spatial visualization of 22,127 barcodes in the common coordinate space coloured by Magellan spatial prioritization for the same representative experimental comparison as in a . d , Synaptic inputs and outputs from SC Vsx2::Hoxa10 neurons. Inputs: SC Vsx2::Hoxa10 neurons and their projections. Projections from vGi neurons (AAV5-CAG-COMET-GFP) and large-diameter afferent neurons (PV cre::Advillin:tdTomato ) onto SC Vsx2::Hoxa10 neurons. Outputs: synaptic appositions from SC Vsx2::Hoxa10 neurons (AAV-Dj-hSyn-flex-mGFP-2A-synaptophysin-mRuby) onto glutamatergic, GABAergic and motor neurons. C, caudal; R, rostral; PV, parvalbumin; syn, synaptophysin. e , Single-unit recordings of optotagged SC Vsx2::Hoxa10 neurons. Responses of an optotagged single unit to optogenetic stimulations. Right, the number of units responding to each type of stimulation. f , Number of vGluT1 synapses from large-diameter afferents apposing SC Vsx2::Hoxa10 neurons ( n = 6 out of 10 mice per group; independent samples two-tailed t -test: t = 4.9, P = 0.002). g , Number of reticulospinal neurons projecting to SC Vsx2::Hoxa10 neurons ( n = 4 mice per group; independent samples two-tailed t -test: t = 4.8, P = 0.0029). h , Percentage of motor neurons, glutamatergic and GABAergic neurons receiving at least one synapse from SC Vsx2::Hoxa10 neurons ( n = 6; one-way analysis of variance (ANOVA); choline acetyltransferase (ChAT): F = 0.45, P = 0.39; glutamatergic: F = 0.52, P = 0.76; GABAergic: statistics indicate Tukey’s honest significant difference tests: uninjured versus SCI: P = 5.2 × 10 −6; SCI versus EES REHAB : P = 1.8 × 10 −5 ). i , Fluorescent intensity of cFos in Vsx2 cre neurons following walking with EES ON ( n = 5 out of 3 mice per group; independent samples two-tailed t -test: t = 5.7, P = 0.0013). f – i , Bars show mean ± s.e.m. with individual points overlaid. AU, arbitrary units.

Techniques: Two Tailed Test

a , Schematic detailing the experimental set-up to study synaptic inputs to the SC Vsx2::Hoxa10 neurons. Monosynaptically restricted EnvA pseudotyped and G-protein deleted rabies expressing mCherry revealed reticulospinal neurons in the vGi and PV ON neurons in the DRG give direct inputs to SC Vsx2::Hoxa10 neurons. b , Schematic detailing the experimental set-up to study synaptic inputs to the SC Vsx2::Hoxa10 neurons. PV ON afferent fibres are labeled in the transgenic PV Cre::Advillin:tdTomato mice. Injections targeted to the vGi labeled descending reticulospinal projections. Vsx2 ON neurons were labeled with RNAScope ISH. c , Schematic detailing the experimental set-up to study outputs from SC Vsx2::Hoxa10 neurons. SC Vsx2::Hoxa1 0 neurons were traced with AAVDj-hSyn-Flex-mGFPfp-2A-Synaptophysin-mRuby that labels the axons and pre-synaptic terminals. Synaptic appositions from SC Vsx2::Hoxa10 neurons on to glutamatergic, GABAergic and motor neurons, labeled using RNAScope ISH were quantified. Bar graphs represent the mean with individual data points overlaid. Error bars reflect the standard error of the mean.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Schematic detailing the experimental set-up to study synaptic inputs to the SC Vsx2::Hoxa10 neurons. Monosynaptically restricted EnvA pseudotyped and G-protein deleted rabies expressing mCherry revealed reticulospinal neurons in the vGi and PV ON neurons in the DRG give direct inputs to SC Vsx2::Hoxa10 neurons. b , Schematic detailing the experimental set-up to study synaptic inputs to the SC Vsx2::Hoxa10 neurons. PV ON afferent fibres are labeled in the transgenic PV Cre::Advillin:tdTomato mice. Injections targeted to the vGi labeled descending reticulospinal projections. Vsx2 ON neurons were labeled with RNAScope ISH. c , Schematic detailing the experimental set-up to study outputs from SC Vsx2::Hoxa10 neurons. SC Vsx2::Hoxa1 0 neurons were traced with AAVDj-hSyn-Flex-mGFPfp-2A-Synaptophysin-mRuby that labels the axons and pre-synaptic terminals. Synaptic appositions from SC Vsx2::Hoxa10 neurons on to glutamatergic, GABAergic and motor neurons, labeled using RNAScope ISH were quantified. Bar graphs represent the mean with individual data points overlaid. Error bars reflect the standard error of the mean.

Techniques: Expressing, Labeling, Transgenic Assay

a , Optimization of the timing to identify supraspinal neurons connected to SC Vsx2::Hoxa10 neurons using rabies viruses. Tracing was conducted using infusions of Cre-dependent G-deleted EnvA rabies or Cre-dependent pseudorabies (PRV) into the lumbar spinal cord of Vsx2 Cre mice. For pseudorabies experiments, tissue was harvested at 2, 2.5, 3, 3.5 and 4 days after the infusion. Sagittal brain sections were used for 3D reconstructions of brains in Neurolucida, shown here for key timepoints. Quantifications report the number of neurons identified in each region of the brain and brainstem after rabies infusion, and at different timepoints after pseudorabies infusions. At 3.5 days days after pseudorabies injections, neurons were labeled in new brain regions compared to regions labeled with monosynaptically-restricted rabies, suggesting that secondorder neurons became transfected by the pseudorabies. We thus used a window of 3 days to study the connectome of SC Vsx2::Hoxa10 neurons after SCI. b , Transverse spinal cord section from a Vsx2 Cre mouse showing Cre-dependent expression of ChrimsonR in SC Vsx2::Hoxa10 and the tract resulting from the insertion of one electrode shank. Inset shows the cell bodies of SC Vsx2::Hoxa10 neurons in the vicinity of the tract. The schematic displays the 4-shank, 64-channel silicon probe used for single-unit recordings. The histogram reports the timing of spikes with respect to the onset of photostimulation for the SC Vsx2::Hoxa10 optotagged single unit shown in Fig. . The waveforms display spontaneous (gray) vs. optogenetically-evoked (red) spikes from an SC Vsx2::Hoxa10 optotagged single unit. Data are shown as mean traces with standard error of the mean ribbons. The traces on the right were obtained from the same recording site during EES (top) and stimulation of the reticular formation (bottom). In these trials, two single-units responded to EES, whereas only one of these two units also responded to stimulation of the reticular formation (C56). The plot on the right reports the latencies of spikes from SC Vsx2::Hoxa10 optotagged single units following EES across all trials (150 trials, top) and stimulation of the reticular stimulation (100 trials, bottom). Neurons that consistently responded to stimulations with short latencies (putatively monosynaptic) are highlighted in red. c , To evaluate the response of the spinal cord to EES, we measured muscle responses in the tibialis anterior when delivering a single pulse of EES. We tested uninjured mice, mice with chronic SCI, and mice that had undergone EES REHAB . Mice with chronic SCI exhibited abnormal long-latency responses (range: 10 to 20 ms), which are highlighted within the grey area and quantified in the bar plots as integral of root mean square (RMS) ( n = 6 mice per group; statistics indicate Tukey HSD tests of the key comparison following one-way ANOVA, P = 0.0001). d , The evaluations reported in c for mice were also conducted in the cohort of human participants. Three of the participants (DM002, GO004, HT008) showed abnormal long-latency responses (range: 50 to 100 ms) to single-pulse of EES before EES REHAB . EES REHAB nearly completely abolished these responses. The bar plot reports the normalised amplitude of these responses before and after EES REHAB ( n = 3, trials > 5/session; mixed-effects model, t = –6.40, P = 2.9 × 10 –8 ). e, f , The role of SC Vsx2::Hoxa10 neurons in the reorganization of muscle responses to EES was evaluated using Cre-dependent expression of Gi, e, or Gq DREADDs in SC Vsx2::Hoxa10 neurons, f. The timelines summarize the timing of the various experimental procedures. The photographs illustrate the expression of DREADD receptors in SC Vsx2::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) SC Vsx2::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 SC Vsx2::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 Vsx2 Cre mice to evaluate the short-term or immediate impact of silencing SC Vsx2::Hoxa10 neurons, and targeted injection of AAV-flex-DTR to evaluate the long-term impact of the complete ablation of SC Vsx2::Hoxa10 neurons. Locomotor performance was evaluated during overground walking using the procedures detailed in Extended Data Fig. (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 SC Vsx2::Hoxa10 neurons per tissue section (SC Vsx2::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.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Optimization of the timing to identify supraspinal neurons connected to SC Vsx2::Hoxa10 neurons using rabies viruses. Tracing was conducted using infusions of Cre-dependent G-deleted EnvA rabies or Cre-dependent pseudorabies (PRV) into the lumbar spinal cord of Vsx2 Cre mice. For pseudorabies experiments, tissue was harvested at 2, 2.5, 3, 3.5 and 4 days after the infusion. Sagittal brain sections were used for 3D reconstructions of brains in Neurolucida, shown here for key timepoints. Quantifications report the number of neurons identified in each region of the brain and brainstem after rabies infusion, and at different timepoints after pseudorabies infusions. At 3.5 days days after pseudorabies injections, neurons were labeled in new brain regions compared to regions labeled with monosynaptically-restricted rabies, suggesting that secondorder neurons became transfected by the pseudorabies. We thus used a window of 3 days to study the connectome of SC Vsx2::Hoxa10 neurons after SCI. b , Transverse spinal cord section from a Vsx2 Cre mouse showing Cre-dependent expression of ChrimsonR in SC Vsx2::Hoxa10 and the tract resulting from the insertion of one electrode shank. Inset shows the cell bodies of SC Vsx2::Hoxa10 neurons in the vicinity of the tract. The schematic displays the 4-shank, 64-channel silicon probe used for single-unit recordings. The histogram reports the timing of spikes with respect to the onset of photostimulation for the SC Vsx2::Hoxa10 optotagged single unit shown in Fig. . The waveforms display spontaneous (gray) vs. optogenetically-evoked (red) spikes from an SC Vsx2::Hoxa10 optotagged single unit. Data are shown as mean traces with standard error of the mean ribbons. The traces on the right were obtained from the same recording site during EES (top) and stimulation of the reticular formation (bottom). In these trials, two single-units responded to EES, whereas only one of these two units also responded to stimulation of the reticular formation (C56). The plot on the right reports the latencies of spikes from SC Vsx2::Hoxa10 optotagged single units following EES across all trials (150 trials, top) and stimulation of the reticular stimulation (100 trials, bottom). Neurons that consistently responded to stimulations with short latencies (putatively monosynaptic) are highlighted in red. c , To evaluate the response of the spinal cord to EES, we measured muscle responses in the tibialis anterior when delivering a single pulse of EES. We tested uninjured mice, mice with chronic SCI, and mice that had undergone EES REHAB . Mice with chronic SCI exhibited abnormal long-latency responses (range: 10 to 20 ms), which are highlighted within the grey area and quantified in the bar plots as integral of root mean square (RMS) ( n = 6 mice per group; statistics indicate Tukey HSD tests of the key comparison following one-way ANOVA, P = 0.0001). d , The evaluations reported in c for mice were also conducted in the cohort of human participants. Three of the participants (DM002, GO004, HT008) showed abnormal long-latency responses (range: 50 to 100 ms) to single-pulse of EES before EES REHAB . EES REHAB nearly completely abolished these responses. The bar plot reports the normalised amplitude of these responses before and after EES REHAB ( n = 3, trials > 5/session; mixed-effects model, t = –6.40, P = 2.9 × 10 –8 ). e, f , The role of SC Vsx2::Hoxa10 neurons in the reorganization of muscle responses to EES was evaluated using Cre-dependent expression of Gi, e, or Gq DREADDs in SC Vsx2::Hoxa10 neurons, f. The timelines summarize the timing of the various experimental procedures. The photographs illustrate the expression of DREADD receptors in SC Vsx2::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) SC Vsx2::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 SC Vsx2::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 Vsx2 Cre mice to evaluate the short-term or immediate impact of silencing SC Vsx2::Hoxa10 neurons, and targeted injection of AAV-flex-DTR to evaluate the long-term impact of the complete ablation of SC Vsx2::Hoxa10 neurons. Locomotor performance was evaluated during overground walking using the procedures detailed in Extended Data Fig. (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 SC Vsx2::Hoxa10 neurons per tissue section (SC Vsx2::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.

Techniques: Labeling, Transfection, Expressing, Two Tailed Test, Injection

a , Exploded diagram of a new wireless optoelectronic system that integrates on the same implant red-shifted microLEDs to deliver deeply-penetrating photostimulation and electrodes to deliver EES. The zoom shows the microLEDs and electrodes for EES. b , Optogenetic silencing of SC Vsx2::Hoxa10 neurons in mice that underwent four weeks of EES REHAB . Experiments and data are shown using the same conventions as in Extended Data Figs. and ( 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 SC Vsx2::Hoxa10 neurons with Gi in mice that underwent four weeks of EES REHAB ( 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 SC Vsx2::Hoxa10 neurons with Gq in mice tested at four weeks post-SCI (no EES REHAB ) with EES OFF and EES ON ( 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 SC Vsx2::Hoxa10 neurons (Gi, CNO in drinking water) in mice that underwent four weeks of EES REHAB , chronic activation of SC Vsx2::Hoxa10 neurons (Gq, CNO in drinking water) in mice after SCI, and chronic activation of SC Vsx2::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 SC Vsx2::Hoxa10 neurons during EES REHAB altered the connectome and projectome of SC Vsx2::Hoxa10 neurons compared to mice that underwent EES REHAB . 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 Vsx2 Cre mice. The density of vGluT1 synapses apposing SC Vsx2::Hoxa10 neurons was quantified to evaluate the synaptic projection from large-diameter afferent fibers onto SC Vsx2::Hoxa10 neurons. The projectome of SC Vsx2::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 SC Vsx2::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 SC Vsx2::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 SC Vsx2::Hoxa10 neurons on the natural recovery of mice with thoracic lateral hemisection SCI was evaluated as in Extended Data Fig. , 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 SC Vsx2::Hoxa10 neurons in the lumbar spinal cord of Vsx2 Cre 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 SC Vsx2::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 SC Vsx2::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.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Exploded diagram of a new wireless optoelectronic system that integrates on the same implant red-shifted microLEDs to deliver deeply-penetrating photostimulation and electrodes to deliver EES. The zoom shows the microLEDs and electrodes for EES. b , Optogenetic silencing of SC Vsx2::Hoxa10 neurons in mice that underwent four weeks of EES REHAB . Experiments and data are shown using the same conventions as in Extended Data Figs. and ( 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 SC Vsx2::Hoxa10 neurons with Gi in mice that underwent four weeks of EES REHAB ( 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 SC Vsx2::Hoxa10 neurons with Gq in mice tested at four weeks post-SCI (no EES REHAB ) with EES OFF and EES ON ( 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 SC Vsx2::Hoxa10 neurons (Gi, CNO in drinking water) in mice that underwent four weeks of EES REHAB , chronic activation of SC Vsx2::Hoxa10 neurons (Gq, CNO in drinking water) in mice after SCI, and chronic activation of SC Vsx2::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 SC Vsx2::Hoxa10 neurons during EES REHAB altered the connectome and projectome of SC Vsx2::Hoxa10 neurons compared to mice that underwent EES REHAB . 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 Vsx2 Cre mice. The density of vGluT1 synapses apposing SC Vsx2::Hoxa10 neurons was quantified to evaluate the synaptic projection from large-diameter afferent fibers onto SC Vsx2::Hoxa10 neurons. The projectome of SC Vsx2::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 SC Vsx2::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 SC Vsx2::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 SC Vsx2::Hoxa10 neurons on the natural recovery of mice with thoracic lateral hemisection SCI was evaluated as in Extended Data Fig. , 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 SC Vsx2::Hoxa10 neurons in the lumbar spinal cord of Vsx2 Cre 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 SC Vsx2::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 SC Vsx2::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.

Techniques: Activation Assay, Labeling, Two Tailed Test, Microscopy, Standard Deviation

a , Implantable optoelectronic device to deliver EES and photostimulation. After EES REHAB , chronophotography of walking during EES ON in a representative mouse. Red-shifted light is delivered for a few seconds to silence SC Vsx2::Hoxa10 neurons (AAV5-Syn-flex-ChrimsonR-tdTomato; n = 4; Tukey’s honest significant difference tests following repeated measures one-way analysis of variance (ANOVA): P = 0.0023). b , Chronophotography of walking after EES REHAB in a representative mouse with EES ON before and after chemogenetic silencing of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm4D-(Gi)-mCherry; n = 4; paired samples two-tailed t -test; t = −21.3, P = 0.0002). c , Chronophotography of walking in representative mice with no EES REHAB , with EES OFF before and after chemogenetic activation of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm3D-(Gq)-mCherry; n = 4; paired samples two-tailed t -test; t = 5.3, P = 0.0013). d , After EES REHAB , chronophotography of walking in representative mice with EES ON . SC Vsx2::Hoxa10 neurons were silenced during the entire period of EES REHAB ( n = 5; independent samples two-tailed t -test: t = −3.5, P = 0.008). e , Chronophotography of representative mice walking without EES after recovery from a lateral hemisection SCI. Kinematic limb reconstruction is overlaid. Right, same condition, but SC Vsx2::Hoxa10 neurons located in lumbar segments were ablated before the SCI (AAV5-CAG-flex-DTR; n = 5 per group; independent samples two-tailed t -test: t = 5.9, P = 0.0004). a – e , Bars show mean ± s.e.m. with individual points overlaid.

Journal: Nature

Article Title: The neurons that restore walking after paralysis

doi: 10.1038/s41586-022-05385-7

Figure Lengend Snippet: a , Implantable optoelectronic device to deliver EES and photostimulation. After EES REHAB , chronophotography of walking during EES ON in a representative mouse. Red-shifted light is delivered for a few seconds to silence SC Vsx2::Hoxa10 neurons (AAV5-Syn-flex-ChrimsonR-tdTomato; n = 4; Tukey’s honest significant difference tests following repeated measures one-way analysis of variance (ANOVA): P = 0.0023). b , Chronophotography of walking after EES REHAB in a representative mouse with EES ON before and after chemogenetic silencing of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm4D-(Gi)-mCherry; n = 4; paired samples two-tailed t -test; t = −21.3, P = 0.0002). c , Chronophotography of walking in representative mice with no EES REHAB , with EES OFF before and after chemogenetic activation of SC Vsx2::Hoxa10 neurons (AAV5-hSyn-DIO-hm3D-(Gq)-mCherry; n = 4; paired samples two-tailed t -test; t = 5.3, P = 0.0013). d , After EES REHAB , chronophotography of walking in representative mice with EES ON . SC Vsx2::Hoxa10 neurons were silenced during the entire period of EES REHAB ( n = 5; independent samples two-tailed t -test: t = −3.5, P = 0.008). e , Chronophotography of representative mice walking without EES after recovery from a lateral hemisection SCI. Kinematic limb reconstruction is overlaid. Right, same condition, but SC Vsx2::Hoxa10 neurons located in lumbar segments were ablated before the SCI (AAV5-CAG-flex-DTR; n = 5 per group; independent samples two-tailed t -test: t = 5.9, P = 0.0004). a – e , Bars show mean ± s.e.m. with individual points overlaid.

Techniques: Two Tailed Test, Activation Assay

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