Online control of reach accuracy in mice.

Reaching movements, as a basic yet complex motor behavior, are a foundational model system in neuroscience. In particular, there has been a significant recent expansion of investigation into the neural circuit mechanisms of reach behavior in mice. Nevertheless, quantification of mouse reach kinematics remains lacking, limiting comparison to the primate literature. In this study, we quantitatively demonstrate the homology of mouse reach kinematics to primate reach and also discover novel late-phase correlational structure that implies online control. Overall, our results highlight the decelerative phase of reach as important in driving successful outcome. Specifically, we develop and implement a novel statistical machine-learning algorithm to identify kinematic features associated with successful reaches and find that late-phase kinematics are most predictive of outcome, signifying online reach control as opposed to preplanning. Moreover, we identify and characterize late-phase kinematic adjustments that are yoked to midflight position and velocity of the limb, allowing for dynamic correction of initial variability, with head-fixed reaches being less dependent on position in comparison to freely behaving reaches. Furthermore, consecutive reaches exhibit positional error correction but not hot-handedness, implying opponent regulation of motor variability. Overall, our results establish foundational mouse reach kinematics in the context of neuroscientific investigation, characterizing mouse reach production as an active process that relies on dynamic online control mechanisms.NEW & NOTEWORTHY Mice use reaching movements to grasp and manipulate objects in their environment, similar to primates. To better establish mouse reach as a model for motor control, we implement several analytical frameworks, from basic kinematic relationships to statistical machine learning, to quantify mouse reach, finding many canonical features of primate reaches are conserved in mice, as well as evidence for midflight course corrections, expanding the utility of mouse reach paradigms for motor control studies.

Ephrin-B2 overexpression predicts for poor prognosis and response to therapy in solid tumors.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease-free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma (n = 186), and bladder urothelial carcinoma (n = 410). In HNSCC patients, high-EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient-derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc.

Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.

BACKGROUND: Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro. METHODS: The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting. RESULTS: We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others. CONCLUSIONS: Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.

Using a mixed-reality headset to elicit and track clinically relevant movement in the clinic.

Background: 21st century neurology will require scalable and quantitative tools that can improve neurologic evaluations over telehealth and expand access to care. Commercially available mixed-reality headsets allow for simultaneous presentation of stimuli via holograms projected into the real world and objective and quantitative measurement of hand movement, eye movement, and phonation. Methods: We created 6 tasks designed to mimic standard neurologic assessments and administered them to a single participant via the Microsoft HoloLens 2 mixed-reality headset. The tasks assessed postural hand tremor, finger tapping, pronation and supination of hands, hand and eye tracking of a center-out task, hand and eye tracking of a random motion task, and vocal assessment. Findings: We show the utility of the HoloLens for commonly used neurological exams. First, we demonstrate that headset-derived holograms can project hand movements and objects in 3D space, providing a method to accurately and reproducibly present test stimuli to reduce test-test variability. Second, we found that participant hand movements closely matched holographic stimuli using a variety of metrics calculated on recorded movement data. Third, we showed that the HoloLens can record and playback exam tasks for visual inspection, sharing with other medical providers, and future analysis. Fourth, we showed that vocal recordings and analysis could be used to profile vocal characteristics over time. Together, this demonstrates the versatility of mixed reality headsets and possible applications for neurological assessment. Interpretation: Administering components of the neurologic exam via a self-contained and commercially available mixed-reality headset has numerous benefits including detailed kinematic quantification, reproducible stimuli presentation from test to test, and can be self-administered expanding access to neurological care and saving hospital time and money.

Cerebellar associative learning underlies skilled reach adaptation.

The cerebellum is hypothesized to refine movement through online adjustments. We examined how such predictive control may be generated using a mouse reach paradigm, testing whether the cerebellum uses within-reach information as a predictor to adjust reach kinematics. We first identified a population-level response in Purkinje cells that scales inversely with reach velocity, pointing to the cerebellar cortex as a potential site linking kinematic predictors and anticipatory control. Next, we showed that mice can learn to compensate for a predictable reach perturbation caused by repeated, closed-loop optogenetic stimulation of pontocerebellar mossy fiber inputs. Both neural and behavioral readouts showed adaptation to position-locked mossy fiber perturbations and exhibited aftereffects when stimulation was removed. Surprisingly, position-randomized stimulation schedules drove partial adaptation but no opposing aftereffects. A model that recapitulated these findings suggests that the cerebellum may decipher cause-and-effect relationships through time-dependent generalization mechanisms.

A dual Purkinje cell rate and synchrony code sculpts reach kinematics.

Cerebellar Purkinje cells (PCs) encode movement kinematics in their population firing rates. Firing rate suppression is hypothesized to disinhibit neurons in the cerebellar nuclei, promoting adaptive movement adjustments. Debates persist, however, about whether a second disinhibitory mechanism, PC simple spike synchrony, is a relevant population code. We addressed this question by relating PC rate and synchrony patterns recorded with high density probes, to mouse reach kinematics. We discovered behavioral correlates of PC synchrony that align with a known causal relationship between activity in cerebellar output. Reach deceleration was positively correlated with both Purkinje firing rate decreases and synchrony, consistent with both mechanisms disinhibiting target neurons, which are known to adjust reach velocity. Direct tests of the contribution of each coding scheme to nuclear firing using dynamic clamp, combining physiological rate and synchrony patterns ex vivo, confirmed that physiological levels of PC simple spike synchrony are highly facilitatory for nuclear firing. These findings suggest that PC firing rate and synchrony collaborate to exert fine control of movement.

Presynaptic PRRT2 Deficiency Causes Cerebellar Dysfunction and Paroxysmal Kinesigenic Dyskinesia.

PRRT2 loss-of-function mutations have been associated with familial paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions and choreoathetosis, and benign familial infantile seizures. Dystonia is the foremost involuntary movement disorder manifest by patients with PKD. Using a lacZ reporter and quantitative reverse-transcriptase PCR, we mapped the temporal and spatial distribution of Prrt2 in mouse brain and showed the highest levels of expression in cerebellar cortex. Further investigation into PRRT2 localization within the cerebellar cortex revealed that Prrt2 transcripts reside in granule cells but not Purkinje cells or interneurons within cerebellar cortex, and PRRT2 is presynaptically localized in the molecular layer. Analysis of synapses in the cerebellar molecular layer via electron microscopy showed that Prrt2-/- mice have increased numbers of docked vesicles but decreased vesicle numbers overall. In addition to impaired performance on several motor tasks, approximately 5% of Prrt2-/- mice exhibited overt PKD with clear face validity manifest as dystonia. In Prrt2 mutants, we found reduced parallel fiber facilitation at parallel fiber-Purkinje cell synapses, reduced Purkinje cell excitability, and normal cerebellar nuclear excitability, establishing a potential mechanism by which altered cerebellar activity promotes disinhibition of the cerebellar nuclei, driving motor abnormalities in PKD. Overall, our findings replicate, refine, and expand upon previous work with PRRT2 mouse models, contribute to understanding of paroxysmal disorders of the nervous system, and provide mechanistic insight into the role of cerebellar cortical dysfunction in dystonia.

Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma.

Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.