Silencing long-descending inter-enlargement propriospinal neurons improves hindlimb stepping after contusive spinal cord injuries.

Spinal locomotor circuitry is comprised of rhythm generating centers, one for each limb, that are interconnected by local and long-distance propriospinal neurons thought to carry temporal information necessary for interlimb coordination and gait control. We showed previously that conditional silencing of the long ascending propriospinal neurons (LAPNs) that project from the lumbar to the cervical rhythmogenic centers (L1/L2 to C6), disrupts right-left alternation of both the forelimbs and hindlimbs without significantly disrupting other fundamental aspects of interlimb and speed-dependent coordination (Pocratsky et al., 2020). Subsequently, we showed that silencing the LAPNs after a moderate thoracic contusive spinal cord injury (SCI) resulted in better recovered locomotor function (Shepard et al., 2021). In this research advance, we focus on the descending equivalent to the LAPNs, the long descending propriospinal neurons (LDPNs) that have cell bodies at C6 and terminals at L2. We found that conditional silencing of the LDPNs in the intact adult rat resulted in a disrupted alternation of each limb pair (forelimbs and hindlimbs) and after a thoracic contusion SCI significantly improved locomotor function. These observations lead us to speculate that the LAPNs and LDPNs have similar roles in the exchange of temporal information between the cervical and lumbar rhythm generating centers, but that the partial disruption of the pathway after SCI limits the independent function of the lumbar circuitry. Silencing the LAPNs or LDPNs effectively permits or frees-up the lumbar circuitry to function independently.

Unintentional Effects from Housing Enhancement Resulting in Functional Improvement in Spinal Cord-Injured Mice.

It is well established that both positive and negative housing conditions of laboratory animals can affect behavioral, biochemical, and physiological responses. Housing enhancements have been shown to have beneficial effects on locomotor outcomes in rodents with spinal cord injury (SCI). Subsequent to an unplanned housing enhancement of the addition of a balcony to home cages by animal care personnel at a research facility, a retrospective analysis of multiple SCI studies was performed to determine whether outcomes differed before (four studies, N = 28) and after (four studies, N = 23) the addition of the balcony. Locomotor and morphological differences were compared after a mild-moderate T9 spinal cord contusion injury in wild-type mice. Post-injury assessments of locomotor function for 6 weeks included Basso Mouse Scale (BMS) and treadmill kinematic assessments (week 6). Balcony-housed mice showed greater improvements not only in basic locomotor functions (weight-supported stepping, balance) compared to those in standard housing, but also surpassed mice in standard housing without the balcony in higher-order locomotor recovery outcomes, including BMS late-stage recovery measures (paw, tail, and trunk indices). Additionally, balcony-housed mice had overall higher BMS scores, consistently attained more BMS subscores, and had better treadmill track width and stride length compared to those with no balcony. The housing enhancement of a balcony led to unforeseen consequences and unexpected higher recovery outcomes compared to mice in standard housing. This retrospective study highlights the importance of housing conditions in the key outcomes of locomotor recovery after incomplete contusive SCIs in mice.

Direct Ryanodine Receptor-2 Knockout in Primary Afferent Fibers Modestly Affects Neurological Recovery after Contusive Spinal Cord Injury.

Neuronal ryanodine receptors (RyR) release calcium from internal stores and play a key role in synaptic plasticity, learning, and memory. Dysregulation of RyR function contributes to neurodegeneration and negatively impacts neurological recovery after spinal cord injury (SCI). However, the individual role of RyR isoforms and the underlying mechanisms remain poorly understood. To determine whether RyR2 plays a direct role in axonal fate and functional recovery after SCI, we bred Advillin-Cre: tdTomato (Ai9) reporter mice with "floxed" RyR2 mice to directly knock out (KO) RyR2 function in dorsal root ganglion neurons and their spinal projections. Adult 6- to 8-week-old RyR2KO and littermate controls were subjected to a contusive SCI and their dorsal column axons were imaged in vivo using two-photon excitation microscopy. We found that direct RyR2KO in dorsal column primary afferents did not significantly alter secondary axonal degeneration after SCI. We next assessed behavioral recovery after SCI and found that direct RyR2KO in primary afferents worsened open-field locomotor scores (Basso Mouse Scale subscore) compared to littermate controls. However, both TreadScan™ gait analysis and overground kinematic gait analysis tests revealed subtle, but no fundamental, differences in gait patterns between the two groups after SCI. Subsequent removal of spared afferent fibers using a dorsal column crush revealed similar outcomes in both groups. Analysis of primary afferents at the lumbar (L3-L5) level similarly revealed no noticeable differences between groups. Together, our results support a modest contribution of dorsal column primary afferent RyR2 in neurological recovery after SCI.

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

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.

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day.

The circadian gene expression rhythmicity drives diurnal oscillations of physiological processes that may determine the injury response. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such influences on recovery after spinal cord injury (SCI) are unknown. We report that mice receiving moderate, T9 contusive SCI at ZT0 (zeitgeber time 0, time of lights on) and ZT12 (time of lights off) showed similar hindlimb function recovery in the Basso mouse scale (BMS) over a 6 week post-injury period. In an independent study, no significant differences in BMS were observed after SCI at ZT18 vs. ZT6. However, the ladder walking test revealed modestly improved performance for ZT18 vs. ZT6 mice at week 6 after injury. Consistent with those minor effects on functional recovery, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Likewise, blood-spinal cord barrier disruption and neuroinflammation appeared similar when analyzed at 1 week post injury at ZT6 or ZT18. Therefore, locomotor recovery after thoracic contusive SCI is not substantively modulated by the time of day at which the neurotrauma occurred.

Long ascending propriospinal neurons provide flexible, context-specific control of interlimb coordination.

Within the cervical and lumbar spinal enlargements, central pattern generator (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that inter-connect the lumbar and cervical CPGs disrupts left-right limb coupling of each limb pair in the adult rat during overground locomotion on a high-friction surface. These perturbations occurred independent of the locomotor rhythm, intralimb coordination, and speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. These data reveal surprising flexibility and context-dependence in the control of interlimb coordination during locomotion.

Reversible silencing of lumbar spinal interneurons unmasks a task-specific network for securing hindlimb alternation.

Neural circuitry in the lumbar spinal cord governs two principal features of locomotion, rhythm and pattern, which reflect intra- and interlimb movement. These features are functionally organized into a hierarchy that precisely controls stepping in a stereotypic, speed-dependent fashion. Here, we show that a specific component of the locomotor pattern can be independently manipulated. Silencing spinal L2 interneurons that project to L5 selectively disrupts hindlimb alternation allowing a continuum of walking to hopping to emerge from the otherwise intact network. This perturbation, which is independent of speed and occurs spontaneously with each step, does not disrupt multi-joint movements or forelimb alternation, nor does it translate to a non-weight-bearing locomotor activity. Both the underlying rhythm and the usual relationship between speed and spatiotemporal characteristics of stepping persist. These data illustrate that hindlimb alternation can be manipulated independently from other core features of stepping, revealing a striking freedom in an otherwise precisely controlled system.

Gait analysis in normal and spinal contused mice using the TreadScan system.

Advances in spinal cord injury (SCI) research are dependent on quality animal models, which in turn rely on sensitive outcome measures able to detect functional differences in animals following injury. To date, most measurements of dysfunction following SCI rely either on the subjective rating of observers or the slow throughput of manual gait assessment. The present study compares the gait of normal and contusion-injured mice using the TreadScan system. TreadScan utilizes a transparent treadmill belt and a high-speed camera to capture the footprints of animals and automatically analyze gait characteristics. Adult female C57Bl/6 mice were introduced to the treadmill prior to receiving either a standardized mild, moderate, or sham contusion spinal cord injury. TreadScan gait analyses were performed weekly for 10 weeks and compared with scores on the Basso Mouse Scale (BMS). Results indicate that this software successfully differentiates sham animals from injured animals on a number of gait characteristics, including hindlimb swing time, stride length, toe spread, and track width. Differences were found between mild and moderate contusion injuries, indicating a high degree of sensitivity within the system. Rear track width, a measure of the animal's hindlimb base of support, correlated strongly both with spared white matter percentage and with terminal BMS. TreadScan allows for an objective and rapid behavioral assessment of locomotor function following mild-moderate contusive SCI, where the majority of mice still exhibit hindlimb weight support and plantar paw placement during stepping.