Silencing long ascending propriospinal neurons after spinal cord injury improves hindlimb stepping in the adult rat

  1. Courtney T Shepard
  2. Amanda M Pocratsky
  3. Brandon L Brown
  4. Morgan A Van Rijswijck
  5. Rachel M Zalla
  6. Darlene A Burke
  7. Johnny R Morehouse
  8. Amberley S Riegler
  9. Scott R Whittemore
  10. David SK Magnuson

  • Curated by eLife

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    Evaluation Summary:

    This paper will be of interest to neuroscientists in the fields of spinal motor control and spinal cord injury. The finding that silencing of spared lumbar to cervical long ascending propriospinal neurons after spinal cord injury enhances locomotor coordination is unexpected and well-supported by the data. This has potential implications for strategies aimed at promoting recovery of function after SCI.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Long ascending propriospinal neurons (LAPNs) are a subpopulation of spinal cord interneurons that directly connect the lumbar and cervical enlargements. Previously we showed, in uninjured animals, that conditionally silencing LAPNs disrupted left-right coordination of the hindlimbs and forelimbs in a context-dependent manner, demonstrating that LAPNs secure alternation of the fore- and hindlimb pairs during overground stepping. Given the ventrolateral location of LAPN axons in the spinal cord white matter, many likely remain intact following incomplete, contusive, thoracic spinal cord injury (SCI), suggesting a potential role in the recovery of stepping. Thus, we hypothesized that silencing LAPNs after SCI would disrupt recovered locomotion. Instead, we found that silencing spared LAPNs post-SCI improved locomotor function, including paw placement order and timing, and a decrease in the number of dorsal steps. Silencing also restored left-right hindlimb coordination and normalized spatiotemporal features of gait such as stance and swing time. However, hindlimb-forelimb coordination was not restored. These data indicate that the temporal information carried between the spinal enlargements by the spared LAPNs post-SCI is detrimental to recovered hindlimb locomotor function. These findings are an illustration of a post-SCI neuroanatomical-functional paradox and have implications for the development of neuronal- and axonal-protective therapeutic strategies and the clinical study/implementation of neuromodulation strategies.

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  1. Version published to 10.7554/elife.70058 on eLife
  2. eLife

    Evaluation Summary:

    This paper will be of interest to neuroscientists in the fields of spinal motor control and spinal cord injury. The finding that silencing of spared lumbar to cervical long ascending propriospinal neurons after spinal cord injury enhances locomotor coordination is unexpected and well-supported by the data. This has potential implications for strategies aimed at promoting recovery of function after SCI.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Experiments reported in this manuscript aimed to determine if long ascending propriospinal neurons (LAPN) located in the lumbar prominence facilitated locomotor recovery after a moderate severity spinal cord contusion in rats using measurements of gait and swimming ability. Prior studies from this group implicated LAPN in fine tuning coordination of forelimbs and hindlimb during locomotion. The authors used viral constructs to infect cell bodies of LAPN in lumbar spinal cord and their synapses in cervical spinal cord to express botulinum toxin when doxycycline was administered in drinking water. This technique appeared to infect both inhibitory and excitatory LAPN. Strengths of the manuscript include a very thorough gait analysis, evidence through histologic analysis that the desired viral infections occurred, and an experiment in which it was shown that effects of doxycycline on gait resolved when this medication was removed then reappeared when it was added back. Weaknesses include lack of controls that demonstrate lack of any effect of doxycycline on gait parameters in animals injected with control viruses to control for any unexpected effect on gait of the medication or infection of neurons with the virus.

    The authors were able to evaluate effects of synaptic silencing of LAPN after SCI. The data showed, unexpectedly, that silencing improved ipsilateral hindlimb gait parameters, left-right hindlimb coordination and forelimb hindlimb coordination.

    How the spinal cord is rewired after SCI remains poorly understood. The data presented in this manuscript will advance understanding of the role of LAPN in this process. An important question posed by the authors results is what the respective role(s) of the excitatory and inhibitory LAPN are in the biology they uncovered. Also of interest is whether this biology results from changes in local inputs to LAPN through the sensory nervous systems which could be interpreted as maladaptive given the authors results.

  4. eLife

    Reviewer #2 (Public Review):

    Shepard et al. investigated the role of long ascending propriospinal neurons (LAPNs) in recovery of locomotor function after a moderate spinal contusion injury. Their previous observations had determined that LAPNs have a context dependent function in coordinating fore- and hindlimbs patterns during locomotion. Surprisingly, silencing LAPNs after contusion SCI improved several parameter of locomotion.

    Strengths

    The novelty and unexpected results are certainly the mains strengths of this report. Given all previous evidence, mostly originating from their own work, they logically hypothesized that disrupting this usually spared pathway after contusion injuries would have a detrimental effect on functional recovery. Interestingly, interlimb coordination was improved after silencing of LAPNs with limit or no change to forelimb-hindlimb coordination patterns.
    They provide an extensive study of overground kinematics and swimming test. The detailed kinematics parameters provided the basis for their novel results. The addition of the swimming test provided conflicting results compared to the overground walking tests. This aspect was quite interesting and informative as swimming in rodents is a bipedal motor skill devoid of weight bearing. Hence, control of overground locomotion and swimming may be differentially dependent on LAPN activity as clearly illustrated here.

    Weaknesses

    Although the results are novel, the complete reliance on kinematic analysis limited the reach and conclusions of this manuscript. Their previous published work using these methods also used similar techniques for kinematics analysis. Two fundamental points of weakness here in relation to analysis of hindlimb movements after SCI is the lack of markers defining the knee but importantly the distal phalange of the toes. They defined their hindlimb segments as hip/knee and knee/ankle, when the knee joint was clearly not labelled. There are obvious issues with skin markers especially on the knee joint of rodents; however, given that virtually all of their evidence is based on kinematics, inclusion of such marker would have strengthen their analysis. Knee joint issues apart, the exclusion of a marker on the distal phalange of the toe is an equally critical variable in SCI locomotion. They described they placed markers on the “iliac crest, hip, ankle and toe”, but more precise location would have been helpful, e.g., head of the femur, lateral malleolus, etc. Especially after moderate contusion, toe dragging in different phases of stepping is a hallmark of deficits in this condition. This was partially illustrated by analysis of dorsal versus plantar stepping. However, toe dragging does not always result in dorsal stepping. From the figures and previous publications from the authors, the toe marker seems to have been placed between the metatarsal and the proximal phalange, which does not describe the location of the toes in relation to ground contact. The 2-dimensional analysis also limits any analysis of potential rotations.

    There was a complete lack of mechanisms, which is really disappointing. No anatomy or physiology studies were presented to substantiate or investigate possible mechanisms. Figure 2 is the only one showing anatomy, but it is limited to confirming lesion/spared tissue and location of viral vectors. Several of these issues were later mentioned in the Discussion, but this makes this manuscript purely descriptive.

  5. eLife

    Reviewer #3 (Public Review):

    The manuscript by Shepard et al determines the effects of silencing long ascending propriospinal neurons after spinal cord injury. The authors use an elegant reversable silencing strategy that involves a dual virus system to specifically label long ascending propriospinal neurons (LAPNs). This follows a recent study by the group which demonstrated that silencing these neurons in intact rats, resulted in disrupted left-right coordination between the hindlimbs (and between the forelimbs), in addition to alterations in coordination of diagonal forelimb-hindlimb pairs, all seen only in certain contexts (Procratsky et al 2020). Here, the same silencing strategy is used but to determine the functional role these neurons play during locomotor recovery post-incomplete SCI. The reversibility of the silencing was fully leveraged in order to examine the functional consequences of LAPN loss both pre- and post-SCI (multiple times) in the same subject. It was expected that the silencing of the LAPNs spared after incomplete SCI would further disrupt locomotion but the opposite occurred, hindlimb coordination improved. Both the improvement in left-right alternation of the hindlimbs and the lack of major detrimental effect on forelimb-hindlimb coordination are surprising. Having spared fiber tracts leading to worse functional outcomes has interesting implications for strategies to restore function after SCI.

    Overall, this is a well-written manuscript with data that are clearly presented and support the conclusions. However, some aspects of the analysis should be clarified. Possible functional explanations for the findings are discussed and it may be possible to narrow down some of these possibilities with additional histological data.

  6. Version published to 10.1101/2021.05.18.444653v1 on bioRxiv