Proper identification of spinal cord levels is crucial for clinical-pathological and imaging studies in humans, but can be a challenge given technical limitations. We have previously demonstrated in non-primate models that the contours of the spinal ventral horn are determined by the position of motoneuron pools. These positions are preserved within and among individuals and can be used to identify lumbosacral spinal levels. Here we tested the hypothesis that this approach can be extended to identify monkey and human spinal levels. In 7 rhesus monkeys, we retrogradely labeled motoneuron pools that represent rostral, middle and caudal landmarks of the lumbosacral enlargement. We then aligned the lumbosacral enlargements among animals using absolute length, segmental level or a relative scale based upon rostral and caudal landmarks. Inter-animal matching of labeled motoneurons across the lumbosacral enlargement was most precise when using internal landmarks. We then reconstructed 3 human lumbosacral spinal cords, and aligned these based upon homologous internal landmarks. Changes in shape of the ventral horn were consistent among human subjects using this relative scale, despite marked differences in absolute length or age. These data suggest that the relative position of spinal motoneuron pools is conserved across species, including primates. Therefore, in clinical-pathological or imaging studies in humans, one can assign spinal cord levels to even single sections by matching ventral horn shape to standardized series.

Walking is a slow gait which is particularly adaptable to meet internal or external needs and is prone to maladaptive alterations that lead to gait disorders. Alterations can affect speed, but also style (the way one walks). While slowed speed may signify the presence of a problem, style represents the hallmark essential for clinical classification of gait disorders. However, it has been challenging to objectively capture key stylistic features while uncovering neural substrates driving these features. Here we revealed brainstem hotspots that drive strikingly different walking styles by employing an unbiased mapping assay that combines quantitative walking signatures with focal, cell type specific activation. We found that activation of inhibitory neurons that mapped to the ventromedial caudal pons induced slow motion-like style. Activation of excitatory neurons that mapped to the ventromedial upper medulla induced shuffle-like style. Contrasting shifts in walking signatures distinguished these styles. Activation of inhibitory and excitatory neurons outside these territories or of serotonergic neurons modulated walking speed, but without walking signature shifts. Consistent with their contrasting modulatory actions, hotspots for slow-motion and shuffle-like gaits preferentially innervated different substrates. These findings lay the basis for new avenues to study mechanisms underlying (mal)adaptive walking styles and gait disorders.

A method for capturing gait signatures in neurological conditions that allows comparison of human gait with animal models would be of great value in translational research. However, the velocity dependence of gait parameters and differences between quadruped and biped gait have made this comparison challenging. Here we present an approach that accounts for changes in velocity during walking and allows for translation across species. In mice, we represented spatial and temporal gait parameters as a function of velocity and established regression models that reproducibly capture the signatures of these relationships during walking. In experimental parkinsonism models, regression curves representing these relationships shifted from baseline, implicating changes in gait signatures, but with marked differences between models. Gait parameters in healthy human subjects followed similar strict velocity dependent relationships which were altered in Parkinson's patients in ways that resemble some but not all mouse models. This novel approach is suitable to quantify qualitative walking abnormalities related to CNS circuit dysfunction across species, identify appropriate animal models, and it provides important translational opportunities.

Clinical signs in Parkinson's disease (PD), including parkinsonian gait, are often asymmetric, but mechanisms underlying gait asymmetries in PD remain poorly understood. A translational toolkit, a set of standardized measures to capture gait asymmetries in relevant mouse models and patients, would greatly facilitate research efforts. We validated approaches to quantify asymmetries in placement and timing of limbs in mouse models of parkinsonism and human PD subjects at speeds that are relevant for human walking. In mice, we applied regression analysis to compare left and right gait metrics within a condition. To compare alternation ratios of left and right limbs before and after induction of parkinsonism, we used circular statistics. Both approaches revealed asymmetries in hind- and forelimb step length in a unilateral PD model, but not in bilateral or control models. In human subjects, a similar regression approach showed a step length asymmetry in the PD but not control group. Sub-analysis of cohorts with predominant postural instability-gait impairment and with predominant tremor revealed asymmetries for step length in both cohorts and for swing time only in the former cohort. This translational approach captures asymmetries of gait in mice and patients. Application revealed striking differences between models, and that spatial and temporal asymmetries may occur independently. This approach will be useful to investigate circuit mechanisms underlying the heterogeneity between models.

Reduced walking speed is a hallmark of functional decline in aging across species. An age-related change in walking style may represent an additional key marker signifying deterioration of the nervous system. Due to the speed dependence of gait metrics combined with slowing of gait during aging, it has been challenging to determine whether changes in gait metrics represent a change in style. In this longitudinal study we employed gait signatures to separate changes in walking style and speed in mice. We compared gait signatures at mature adult age with middle aged, old and geriatric time points and included female and male sub-cohorts to examine sex differences in nature or timing signature shifts. To determine whether gait signature shifts occurred independently from a decline in other mobility domains we measured balance and locomotor activity. We found that walking speed declined early, whereas gait signatures shifted very late during the aging process. Shifts represented longer swing time and stride length than expected for speed, as in slow motion, and were preceded by a decline in other mobility domains. The pattern of shifts was similar between female and male cohorts, but with sex differences in timing. We conclude that changes in walking style, speed and other mobility domains represent separate age-related phenomena. These findings call for careful, sex specific selection of type and timing of outcome measures in mechanistic or interventional studies. The pattern of age-related gait signature shifts is distinct from patterns seen in neurodegenerative conditions and may be a translatable marker for the end of the lifespan.

In patients with obstructive sleep apnea, airway obstruction during sleep produces hypercapnia, which in turn activates respiratory muscles that pump air into the lungs (e.g., the diaphragm) and that dilate and stabilize the upper airway (e.g., the genioglossus). We hypothesized that these responses are facilitated by glutamatergic neurons in the parabrachial complex (PB) that respond to hypercapnia and project to premotor and motor neurons that innervate the diaphragm and genioglossus muscles. To test this hypothesis, we combined c-Fos immunohistochemistry with in situ hybridization for vGluT2 or GAD67 or with retrograde tracing from the ventrolateral medullary region that contains phrenic premotor neurons, the phrenic motor nucleus in the C3-C5 spinal ventral horn, or the hypoglossal motor nucleus. We found that hypercapnia (10% CO2 for 2 hours) activated c-Fos expression in neurons in the external lateral, lateral crescent (PBcr), and Kölliker-Fuse (KF) PB subnuclei and that most of these neurons were glutamatergic and virtually none γ-aminobutyric acidergic. Numerous CO2 -responsive neurons in the KF and PBcr were labeled after retrograde tracer injection into the ventrolateral medulla or hypoglossal motor nuclei, and in the KF after injections into the spinal cord, making them candidates for mediating respiratory-facilitatory and upper-airway-stabilizing effects of hypercapnia.

Lower urinary tract symptoms (LUTS) are exceptionally common and debilitating, and they are likely caused or exacerbated by dysfunction of neural circuits controlling bladder function. An incomplete understanding of neural control of bladder function limits our ability to clinically address LUTS. Barrington's nucleus (Bar) provides descending control of bladder and sphincter function, and its glutamatergic neurons expressing corticotropin releasing hormone (BarCrh/Vglut2) are implicated in bladder control. However, it remains unclear whether this subset of Bar neurons is necessary for voiding, and the broader circuitry providing input to this control center remains largely unknown. Here, we examine the contribution to micturition behavior of BarCrh/Vglut2 neurons relative to the overall BarVglut2 population. First, we identify robust, excitatory synaptic input to Bar. Glutamatergic axons from the periaqueductal gray (PAG) and lateral hypothalamic area (LHA) intensely innervate and are functionally connected to Bar, and optogenetic stimulation of these axon terminals reliably provokes voiding. Similarly, optogenetic stimulation of BarVglut2 neurons triggers voiding, whereas stimulating the BarCrh/Vglut2 subpopulation causes bladder contraction, typically without voiding. Next, we genetically ablate either BarVglut2 or BarCrh/Vglut2 neurons and found that only BarVglut2 ablation replicates the profound urinary retention produced by conventional lesions in this region. Fiber photometry recordings reveal that BarVglut2 neuron activity precedes increased bladder pressure, while activity of BarCrh/Vglut2 is phase delayed. Finally, deleting Crh from Bar neurons has no effect on voiding and related bladder physiology. Our results help identify the circuitry that modulates Bar neuron activity and identify subtypes that may serve different roles in micturition.