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.

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.

Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons.

The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).