Long ascending propriospinal neurons provide flexible, context-specific control of interlimb coordination
- Department of Anatomical Sciences and Neurobiology, University of Louisville, United States
- Kentucky Spinal Cord Injury Research Center, University of Louisville, United States
- Department of Neurological Surgery, University of Louisville, United States
- Speed School of Engineering, University of Louisville, United States
- Cardiovascular Innovation Institute, Department of Physiology and Biophysics, University of Louisville, United States
Figures
Figure 1 with 1 supplement
Histological detection of putatively silenced long ascending propriospinal neurons (LAPNs).
(a–b) Experimental design (see Materials and methods for details). (c–f) Volume rendered, high magnification images showing enhanced eTeNT.EGFP putative fibers (green) surrounding NeuN-stained …
Figure 1—figure supplement 1
Long ascending propriospinal neurons (LAPNs) are a bilaterally distributed pathway throughout the rostral lumbar enlargement with modest local projections.
(a–i) Intrasegmental distribution profile of LAPNs. Scatter plot of (a) ipsilateral (n = 1,069) and (b) commissural-projecting LAPNs (n = 1,379) at spinal L1 with overlaid contour map (N = 11 …
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Figure 1—figure supplement 1—source data 1
contains the source data for CTB labeled cell body counts.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Silencing long ascending propriospinal neurons (LAPNs) disrupts intra-girdle movements during overground stepping.
(a–c) Representative swing-stance graphs of stepping behaviors observed at control time points. Left: orange = homolateral HL-FL movements (out-of-phase, 0.5), blue = diagonal HL-FL movements …
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Figure 2—source data 1
Contains the source data for step ratio measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig2-data1-v2.xlsx
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Figure 2—source data 2
Contains the source data for the magnitude of change of step ratio measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig2-data2-v2.xlsx
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Figure 2—source data 3
Contains the source data for the interlimb coordination measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Silencing long ascending propriospinal neurons (LAPNs) disrupts interlimb coordination during overground locomotion.
(a–d) Individual time point comparisons for silencing-induced changes in interlimb coordination. Time points: BL = Baseline, PD1 = Pre-Dox1, D1D3 = Dox1 On-Day 3, D1D5 = Dox1 On-Day 5, D1D8 = Dox1 On…
Figure 3
Intralimb coordination and postural control endures despite silencing-induced generalized interlimb discoordination.
(a) Three-segment (iliac crest-hip, hip-ankle, ankle-toe), two-angle model of intralimb coordination. Five phases of step cycle illustrated with corresponding hindlimb range-of-motion …
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Figure 3—source data 1
Contains the source data for trunk angle measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig3-data1-v2.xlsx
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Figure 3—source data 2
Contains the source data for intralimb range-of-motion.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig3-data2-v2.xlsx
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Figure 3—source data 3
Contains the source data for intralimb coordination measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig3-data3-v2.xlsx
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Figure 3—source data 4
Contains the source data for foot faults on the narrow beam.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig3-data4-v2.xlsx
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Figure 3—source data 5
Contains the source data for hindlimb base-of-support.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig3-data5-v2.xlsx
Figure 4 with 3 supplements
Silencing long ascending propriospinal neurons (LAPNs) disrupts interlimb coordination independent from the salient features of locomotion.
(a) Schematic illustrating the speed-dependent gaits with representative swing-stance graphs (purple = walk trot; blue = gallop; green = half-bound; yellow = full-bound). (b) Schematic illustrating …
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Figure 4—source data 1
Contains the source data for stereotypical gait measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig4-data1-v2.xlsx
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Figure 4—source data 2
Contains the source data for phase/frequency relationship.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig4-data2-v2.xlsx
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Figure 4—source data 3
Contains the source data for spatiotemporal relationship.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig4-data3-v2.xlsx
Figure 4—figure supplement 1
Stereotypic limb coupling patterns for speed-dependent, volitionally expressed locomotor gaits.
(a) Schematic illustrating custom-built long tank to analyze volitionally-expressed locomotor gaits (see methods for details). (b–e) Schematics and representative swing-stance graphs illustrating …
Figure 4—figure supplement 2
Salient features of locomotion remain unaffected during silencing-induced disruption to interlimb coordination.
(a–l) The fundamental relationship between speed and various speed-dependent spatiotemporal features of locomotion remained intact during silencing-induced changes to hindlimb-forelimb coordination …
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Figure 4—figure supplement 2—source data 1
Contains the source data for FL-HL spatiotemporal measures.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Rhythmic locomotor output within and between limb girdles remains coupled during long ascending propriospinal neuron (LAPN) silencing.
(a) Silencing LAPNs does not affect the underlying stride duration of the forelimbs (p>0.05; repeated measures ANOVA with speed as a co-variate followed by Sidák post hoc t-tests where appropriate) …
Figure 5 with 2 supplements
Silencing-induced disruption to interlimb coordination occurs in a task-specific, context-driven manner.
(a) Intra-girdle left-right coordination was affected to a greater extent during overground stepping as compared to treadmill during long ascending propriospinal neuron (LAPN) silencing (forelimbs, …
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Figure 5—source data 1
Contains the source data for phase during exploratory walking.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig5-data1-v2.xlsx
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Figure 5—source data 2
Contains the source data for phase during treadmill walking.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig5-data2-v2.xlsx
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Figure 5—source data 3
Contains the source data for phase on different surfaces.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig5-data3-v2.xlsx
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Figure 5—source data 4
Contains the source data for phase during swimming.
- https://cdn.elifesciences.org/articles/53565/elife-53565-fig5-data4-v2.xlsx
Figure 5—figure supplement 1
Interlimb coordination is disrupted in a context-driven manner.
Left-right forelimb (a,d,g,j,m), left-right hindlimb (b,e,h,k,n,p), and diagonal hindlimb-forelimb coordination (c,f,i,l,o) were assessed across the various behavioral contexts. Left-right forelimb …
Figure 5—figure supplement 2
Silencing-induced changes to overground stepping occurred at a speed range which is shared across all behavioral contexts.
Hindlimb stride time vs speed (a–j), stride frequency vs speed (k–t), and stride length vs speed (u–dd) relationships observed at Control and DoxOn long ascending propriospinal neuron (LAPN) …
Videos
Video 1
Conditionally silencing long ascending propriospinal neurons (LAPNs) disrupts interlimb coordination during overground stepping.
DoxOn videos shown from two independent experiments, three separate animals at Dox1On Day 8 of LAPN silencing. Videos shown from the same animal at 1x, 0.5x, and 0.25x speed.
Video 2
Silencing long ascending propriospinal neurons (LAPNs) disrupts interlimb coordination during overground stepping but not during treadmill-based locomotion.
Videos shown from the same animal at 1x, 0.5x, and 0.25x speed during overground and treadmill stepping at Dox1OnDays 4 and 5.
Video 3
Interlimb coordination is not affected during exploratory-like stepping behavior.
Videos shown from the same animal at the same DoxOn time point at 1x, 0.5x, and 0.25x speed.
Video 4
Silencing long ascending propriospinal neurons (LAPNs) selectively disrupts interlimb coordination when animals are locomoting on a coated, but not smooth stepping surface.
Videos shown from the same animal at the Control and DoxOn time points at 1x and 0.5x speed.
Video 5
Silencing long ascending propriospinal neurons (LAPNs) does not disrupt left-right hindlimb alternation during swimming.
Videos shown from the same animal at 1x, 0.5x, and 0.25x speed.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (female Sprague-Dawley rats) | Envigo | 200–220 g, approximately 10–12 weeks old | ||
| Antibody (GFP) | Rabbit IgG | Abcam ab290 | 1:5000 | |
| Antibody (NeuN) | Guinea pig IgG | Millipore ABN90P | 1:500 | |
| Antibody (NeuN) | Mouse IgM | Millipore MAB377 | 1:500 | |
| Antibody (neurofilament) | Mouse IgM | Sigma N5264 | 1:30,000 | |
| Antibody (synaptophysin) | Mouse IgM | Millipore MAB5258-50UG | 1:10,000 | |
| Antibody (vesicular glutamate transporter 2) | Guinea pig IgG | Millipore AB2251-I | 1:5000 | |
| Antibody (vesicular GABA transporter) | Goat igG | Frontier Institute VGAT-Go-Af620 | 1:500 | |
| Antibody (non-immune sera) | Rabbit IgG | Jackson ImmunoResearch #711-005-152 | 1:5000 | |
| Antibody (secondary AlexaFluor 488) | Rabbit IgG | Jackson ImmunoResearch # 711-545-152 | 1:200 | |
| Antibody (secondary AlexaFluor 594) | Guinea pig IgG | Jackson ImmunoResearch #706-585-148 | 1:200 | |
| Antibody (secondary AlexaFluor 594) | Mouse IgG | Jackson ImmunoResearch # 715-585-150 | 1:200 | |
| Antibody (secondary AlexaFluor 647) | Mouse IgG | Jackson ImmunoResearch # 715-605-151 | 1:200 | |
| Antibody (secondary AlexaFluor 647) | Guinea pig IgG | Jackson ImmunoResearch # 706-546-148 | 1:200 | |
| Antibody (secondary AlexaFluor 647) | Goat IgG | Jackson ImmunoResearch # 705-605-147 | 1:200 | |
| HiRet-TRE-EGFP.eTeNT | Generous gift from Tadashi Isa | 1.6 × 107 vp/ml | ||
| AAV2-CMV-rtTAV16 | Generous gift from Tadashi Isa | 4.8 × 1012 vp/ml | ||
| HiRet-Cre | Generous gift from Zhigang He | 1.6 × 1012 vp/ml | ||
| AAV2-CAG-FLEx-GFP | UNC Vector Core | 3.5 × 1012 vp/ml | ||
| Chemical compound (Sylgard) | Sylgard-coated surface | Sylgard 184 Silicone Elastomer Kit, Dow Corning | ||
| Chemical compound (cholera toxin B subunit conjugate) | CTB-488 | Invitrogen/Molecular Probes C-34775 | 1.5% solution in sterile saline | |
| Chemical compound (cholera toxin B subunit conjugate) | CTB-594 | Invitrogen/Molecular Probes C-34777 | 1.5% solution in sterile saline | |
| Chemical compound (cholera toxin B subunit conjugate) | CTB-647 | Invitrogen/Molecular Probes C-34778 | 1.5% solution in sterile saline |
Additional files
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Supplementary file 1
The fundamental relationship between speed and the various speed-dependent locomotor parameters remains intact despite the silencing-induced disruption to interlimb coordination.
Time point comparisons reveal that silencing long ascending propriospinal neurons (LAPNs) did not affect the underlying foundation relationship between speed and stance duration, stride duration, and stride length at the fore- and hindlimbs, respectively. Running the same comparisons on the curated dataset of Dox-induced affected stepping yielded similar results (‘Dox-induced affected step’). Pearson correlation coefficients (r) and p values shown post-Bonferroni correction for multiple comparisons.
- https://cdn.elifesciences.org/articles/53565/elife-53565-supp1-v2.docx
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Supplementary file 2
Silencing long ascending propriospinal neurons (LAPNs) functionally uncouples the intra-girdle left-right limb pairs during overground locomotion.
Interlimb coordination data were analyzed using Watson’s non-parametric two-sample U (Orlovskiĭ et al., 1999) test (Critical value of Watson’s U2 = 0.1869; Appendix D, TableD.44) (Zar, 1974)
- https://cdn.elifesciences.org/articles/53565/elife-53565-supp2-v2.docx
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Supplementary file 3
Silencing long ascending propriospinal neurons (LAPNs) disrupts interlimb coordination in select behavioral contexts.
Interlimb coordination data were analyzed using Watson’s non-parametric two-sample U (Orlovskiĭ et al., 1999) test (Critical value of Watson’s U2 = 0.1869; Appendix D, TableD.44) (Zar, 1974)
- https://cdn.elifesciences.org/articles/53565/elife-53565-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53565/elife-53565-transrepform-v2.docx
