An integrated workflow for 2D and 3D posture analysis during vestibular system testing in mice.

Introduction: Posture extraction from videos is fundamental to many real-world applications, including health screenings. In this study, we extend the utility and specificity of a well-established protocol, the balance beam, for examining balance and active motor coordination in adult mice of both sexes. Objectives: The primary objective of this study is to design a workflow for analyzing the postures of mice walking on a balance beam. Methods: We developed new tools and scripts based on the FluoRender architecture, which can interact with DeepLabCut (DLC) through Python code. Notably, twenty input videos were divided into four feature point groups (head, body, tail, and feet), based on camera positions relative to the balance beam (left and right), and viewing angles (90° and 45° from the beam). We determined key feature points on the mouse to track posture in a still video frame. We extracted a standard walk cycle (SWC) by focusing on foot movements, which were computed by a weighted average of the extracted walk cycles. The correlation of each walk cycle to the SWC was used as the weight. Results: We learned that positions of the camera angles significantly improved the performance of 2D pose estimation (90°) and 3D (45°). Comparing the SWCs from age-matched mice, we found a consistent pattern of supporting feet on the beam. Two feet were consistently on the beam followed by three feet and another three feet in a 2-3-3 pattern. However, this pattern can be mirrored among individual subjects. A subtle phase shift of foot movement was also observed from the SWCs. Furthermore, we compared the SWCs with speed values to reveal anomalies in mouse walk postures. Some anomalies can be explained as the start or finish of the traversal, while others may be correlated to the distractions of the test environment, which will need further investigation. Conclusion: Our posture analysis workflow improves the classical behavioral testing and analysis, allowing the detection of subtle, but significant differences in vestibular function and motor coordination.

Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels.

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage-sensing mechanisms that trigger channel gating. Here, we define the parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of eight to nine amino acids in the S4 becomes exposed upon voltage sensing domain activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

Topoisomerase IIß Selectively Regulates Motor Neuron Identity and Peripheral Connectivity through Hox/Pbx-Dependent Transcriptional Programs.

Vital motor functions, such as respiration and locomotion, rely on the ability of spinal motor neurons (MNs) to acquire stereotypical positions in the ventral spinal cord and to project with high precision to their peripheral targets. These key properties of MNs emerge during development through transcriptional programs that dictate their subtype identity and connectivity; however, the molecular mechanisms that establish the transcriptional landscape necessary for MN specification are not fully understood. Here, we show that the enzyme topoisomerase IIß (Top2ß) controls MN migration and connectivity. Surprisingly, Top2ß is not required for MN generation or survival but has a selective role in columnar specification. In the absence of Top2ß, phrenic MN identity is eroded, while other motor columns are partially preserved but fail to cluster to their proper position. In Top2ß-/- mice, peripheral connectivity is impaired as MNs exhibit a profound deficit in terminal branching. These defects likely result from the insufficient activation of Hox/Pbx-dependent transcriptional programs as Hox and Pbx genes are downregulated in the absence of Top2ß. Top2ß mutants recapitulate many aspects of Pbx mutant mice, such as MN disorganization and defects in medial motor column (MMC) specification. Our findings indicate that Top2ß, a gene implicated in neurodevelopmental diseases such as autism spectrum disorders, plays a critical, cell-specific role in the assembly of motor circuits.

A Latent Propriospinal Network Can Restore Diaphragm Function after High Cervical Spinal Cord Injury.

Spinal cord injury (SCI) above cervical level 4 disrupts descending axons from the medulla that innervate phrenic motor neurons, causing permanent paralysis of the diaphragm. Using an ex vivo preparation in neonatal mice, we have identified an excitatory spinal network that can direct phrenic motor bursting in the absence of medullary input. After complete cervical SCI, blockade of fast inhibitory synaptic transmission caused spontaneous, bilaterally coordinated phrenic bursting. Here, spinal cord glutamatergic neurons were both sufficient and necessary for the induction of phrenic bursts. Direct stimulation of phrenic motor neurons was insufficient to evoke burst activity. Transection and pharmacological manipulations showed that this spinal network acts independently of medullary circuits that normally generate inspiration, suggesting a distinct non-respiratory function. We further show that this "latent" network can be harnessed to restore diaphragm function after high cervical SCI in adult mice and rats.

The Feasibility of Spectral-Domain Optical Coherence Tomography Grading of Anterior Chamber Inflammation in a Rabbit Model of Anterior Uveitis.

PURPOSE: To determine the feasibility and accuracy of spectral-domain optical coherence tomography (SD-OCT) based grading of anterior chamber cell, using aqueous sampling as a standard, in a rabbit model of anterior uveitis. METHODS: Adult Dutch-belted rabbits were preimmunized with M. tuberculosis (Tb) H37RA antigen, 1 week prior to induction of anterior uveitis with an intracameral injection of Tb antigen. The anterior chamber was imaged with SD-OCT, followed by a slit lamp examination. Two independent, trained graders recorded their estimate of anterior chamber cell count using the Standardization of Uveitis Nomenclature (SUN) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer. Using the aqueous cell density as a standard, correlation with SD-OCT counts were compared to those with SUN scores. RESULTS: Overall, SD-OCT correlated well with the hemocytometer counts (Spearman coefficient = 0.53, P < 0.001) compared with SUN grading and hemocytometer counts (Spearman coefficient = 0.02, P = 0.88). The correlation improved to 0.65 (P < 0.001) when we excluded eyes with corneal thickness ≥ 470 µm. Eyes with corneal thickness ≥ 470 µm exhibited the greatest degree of ocular inflammation and corneal opacity. CONCLUSIONS: In our rabbit model, SD-OCT grading of anterior chamber cell correlated significantly better with aqueous cell counts, compared to traditional slit lamp grading. Spectral-domain optical coherence tomography grading of anterior chamber cell may be a good alternative to SUN grading. Although SUN grading remains the clinical gold standard, alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response.