Schulte, K. | Search bioRxiv

bioRxiv2024

Concurrent optogenetic motor mapping of multiple limbs in awake mice reveals cortical organization of coordinated movements

BackgroundMotor mapping allows for determining the macroscopic organization of motor circuits and corresponding motor movement representations on the cortex. Techniques such as intracortical microstimulation (ICMS) are robust, but can be time consuming and invasive, making them non-ideal for cortex-wide mapping or longitudinal studies. In contrast, optogenetic motor mapping offers a rapid and minimally invasive technique, enabling mapping with high spatiotemporal resolution. However, motor mapping has seen limited use in tracking 3-dimensonal, multi-limb movements in awake animals. This gap has left open questions regarding the underlying organizational principles of motor control of coordinated, ethologically relevant movements involving multiple limbs. ObjectiveOur first objective was to develop Multi-limb Optogenetic Motor Mapping (MOMM) to concurrently map motor movement representations of multiple limbs with high fidelity in awake mice. Having established MOMM, our next objective was determine whether maps of coordinated and ethologically relevant motor output were topographically organized on the cortex. MethodsWe combine optogenetic stimulation with a deep learning driven pose-estimation toolbox, DeepLabCut (DLC), and 3-dimentional triangulation to concurrently map motor movements of multiple limbs in awake mice. ResultsMOMM consistently revealed cortical topographies for all mapped features within and across mice. Many motor maps overlapped and were topographically similar. Several motor movement representations extended beyond cytoarchitecturally defined somatomotor cortex. Finer articulations of the forepaw resided within gross motor movement representations of the forelimb. Moreover, many cortical sites exhibited concurrent limb coactivation when photostimulated, prompting the identification of several cortical regions harboring coordinated and ethologically relevant movements. ConclusionsThe cortex appears to be topographically organized by motor programs, which are responsible for coordinated, multi-limbed, and behavioral-like movements.

neuroscience↗

bioRxiv2017

Toward Rapid Sequenced-Based Detection and Characterization of Causative Agents of Bacteremia

Rapid pathogen diagnosis and characterization performed by metagenomic DNA sequencing may permit physicians to better target therapies in order to improve patient outcomes. To this end, a novel sample-to-answer workflow was assembled to enable rapid clinical detection of causative pathogens of bacteremia in whole blood utilizing metagenomic sequence data captured by the MinION. Rapid lysis, nucleic acid purification, host depletion, and genomic DNA library preparation permitted the detection of multiple bacterial and fungal agents spiked into whole blood, with sequencing commencing within 40 minutes of sample receipt. A hybrid detection strategy utilizing targeted PCR detection of specific pathogens of concern was adopted to improve overall sensitivity. As a proof of concept, primers for relatively long amplicons (~ 1800 bp) were selected to enable the specific detection of Yersinia pestis. The resulting amplicon library was spiked onto the same sequencing flow cell used to perform genomic sequencing, permitting simultaneous pathogen detection via both targeted and untargeted sequencing workflows. Sensitivities on the order of 1x10^6 cells/mL and 1x10^5 cells/mL were achieved for untargeted and targeted detection, respectively, of Y. pestis genomes spiked into whole blood. Bacterial and fungal species present in the ZymoBIOMICS Microbial Community Standard were also detected when spiked at similar levels. Variable quality of sequence reads was observed between the transposase-based and ligation-based library preparation methods, demonstrating that the more time consuming ligation-based approach may be more appropriate for the workflow described herein. Overall, this approach provides a foundation from which future point of care platforms could be developed to permit characterization of bacteremia within hours of admittance into a clinical environment.\n\nAuthor SummaryCases of bacteremia in the U.S. present a significant clinical challenge, especially due to rising rates of antimicrobial resistant strains. Rapid diagnosis of the etiologic pathogen and underlying drug resistance genetic signatures between the first and second antibiotic dose should improve patient outcomes and may permit physicians to better target antibiotic therapies without turning to broad spectrum antibiotics, which may further propagate resistant strains. The methods described herein have been developed to enhance the real time nature of the MinION sequencer. DNA sequencing and real time analysis begin within 40 minutes of sample receipt (as opposed to hours or days for common clinical nucleic acid extraction or blood culture techniques). The incorporation of sensitivity enhancements, such as methylation-based pulldown of human DNA or PCR targeted for pathogens of interest, ensures that this assay can detect bacterial blood infections at clinically relevant levels. The pathogen-agnostic aspect of the assay could one day allow clinicians to identify any unknown bacterial, fungal, or viral DNA in a sample. Ultimately, this study serves as an important step toward establishing a pipeline to rapidly detect and characterize pathogens present in whole blood.

genomics↗