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202 Publications

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    11/23/22 | The 3D ultrastructure of the chordotonal organs in the antenna of a microwasp remains complex although simplified.
    Diakova AV, Makarova AA, Pang S, Xu CS, Hess H, Polilov AA
    Scientific Reports. 2022 Nov 23;12(1):20172. doi: 10.1038/s41598-022-24390-4

    Insect antennae are astonishingly versatile and have multiple sensory modalities. Audition, detection of airflow, and graviception are combined in the antennal chordotonal organs. The miniaturization of these complex multisensory organs has never been investigated. Here we present a comprehensive study of the structure and scaling of the antennal chordotonal organs of the extremely miniaturized parasitoid wasp Megaphragma viggianii based on 3D electron microscopy. Johnston's organ of M. viggianii consists of 19 amphinematic scolopidia (95 cells); the central organ consists of five scolopidia (20 cells). Plesiomorphic composition includes one accessory cell per scolopidium, but in M. viggianii this ratio is only 0.3. Scolopale rods in Johnston's organ have a unique structure. Allometric analyses demonstrate the effects of scaling on the antennal chordotonal organs in insects. Our results not only shed light on the universal principles of miniaturization of sense organs, but also provide context for future interpretation of the M. viggianii connectome.

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    Looger Lab
    11/20/22 | Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics.
    Beatty ZG, Muthusamy AK, Unger EK, Dougherty DA, Tian L, Looger LL, Shivange AV, Bera K, Lester HA, Nichols AL
    Bio-Protocol. 2022 Nov 20;12(22):. doi: 10.21769/BioProtoc.4551

    Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

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    11/18/22 | TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
    Espinosa-Medina I, Feliciano D, Belmonte-Mateos C, Linda Miyares R, Garcia-Marques J, Foster B, Lindo S, Pujades C, Koyama M, Lee T
    Neuron. 2022 Nov 18:. doi: 10.1016/j.neuron.2022.10.035

    During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

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    11/27/22 | ACC neural ensemble dynamics are structured by strategy prevalence
    Mikhail Proskurin , Maxim Manakov , Alla Y. Karpova
    bioRxiv. 2022 Nov 17:. doi: 10.1101/2022.11.17.516909

    Medial frontal cortical areas are thought to play a critical role in the brain's ability to flexibly deploy strategies that are effective in complex settings. Still, the specific circuit computations that underpin this foundational aspect of intelligence remain unclear. Here, by examining neural ensemble activity in rats that sample different strategies in a self-guided search for latent task structure, we demonstrate a robust tracking of individual strategy prevalence in the anterior cingulate cortex (ACC), especially in an area homologous to primate area 32D. Prevalence encoding in the ACC is wide-scale, independent of reward delivery, and persists through a substantial ensemble reorganization that tags ACC representations with contextual content. Our findings argue that ACC ensemble dynamics is structured by a summary statistic of recent behavioral choices, raising the possibility that ACC plays a role in estimating - through statistical learning - which actions promote the occurrence of events in the environment.

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    11/17/22 | RS-FISH: Precise, interactive, fast, and scalable FISH spot detection
    Ella Bahry , Laura Breimann , Marwan Zouinkhi , Leo Epstein , Klim Kolyvanov , Xi Long , Kyle I S Harrington , Timothée Lionnet , Stephan Preibisch
    Nature Methods. 2022 Nov 17:. doi: 10.1038/s41592-022-01669-y

    Fluorescent in-situ hybridization (FISH)-based methods are powerful tools to study molecular processes with subcellular resolution, relying on accurate identification and localization of diffraction-limited spots in microscopy images. We developed the Radial Symmetry-FISH (RS-FISH) software that accurately, robustly, and quickly detects single-molecule spots in two and three dimensions, making it applicable to several key assays, including single-molecule FISH (smFISH), spatial transcriptomics, and spatial genomics. RS-FISH allows interactive parameter tuning and scales to large sets of images as well as tera-byte sized image volumes such as entire brain scans using straight-forward distributed processing on workstations, clusters, and in the cloud.

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    11/15/22 | Channel-independent function of UNC-9/Innexin in spatial arrangement of GABAergic synapses in .
    Hendi A, Niu L, Snow AW, Ikegami R, Wang Z, Mizumoto K
    eLife. 2022 Nov 15;11:. doi: 10.7554/eLife.80555

    Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in , in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

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    11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
    Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
    bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

    Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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    07/21/10 | Droplet freezing, docking, and the exchange of immiscible phase and surfactant around frozen droplets.
    Sgro AE, Chiu DT
    Lab Chip. 07/2010;10(14):1873-7. doi: 10.1039/c001108h

    This paper describes a platform for cooling microfluidic chips so as to freeze aqueous droplets flowing in oil. Using a whole-chip cooling chamber, we can control the ambient temperature surrounding a microfluidic chip and induce cooling and freezing inside the channels. When combined with a droplet generation and droplet docking chip, this platform allows for the facile freezing of droplets immobilized in resistance-based docks. Depending on the design and shape of the docks, the frozen droplets can either be trapped stably in the docks or be released because deformed non-frozen aqueous droplets turn spherical when frozen, and thus can become dislodged from the docks. Additionally, using this chamber and chip combination we are able to exchange immiscible phases and surfactants surrounding the frozen droplets. The materials and methods are inexpensive and easily accessible to microfluidics researchers, making this a simple addition to an existing microfluidic platform.

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    11/10/22 | Robotic Multi-Probe-Single-Actuator Inchworm Neural Microdrive
    Smith R, Kolb I, Tanaka S, Lee A, Harris T, Barbic M
    eLife. 2022 Nov 10:. doi: 10.7554/eLife.71876

    Electrophysiology is one of the major experimental techniques used in neuroscience. The favorable spatial and temporal resolution as well as the increasingly larger site counts of brain recording electrodes contribute to the popularity and importance of electrophysiology in neuroscience. Such electrodes are typically mechanically placed in the brain to perform acute or chronic freely moving animal measurements. The micro positioners currently used for such tasks employ a single translator per independent probe being placed into the targeted brain region, leading to significant size and weight restrictions. To overcome this limitation, we have developed a miniature robotic multi-probe neural microdrive that utilizes novel phase-change-material-filled resistive heater micro-grippers. The microscopic dimensions, gentle gripping action, independent electronic actuation control, and high packing density of the grippers allow for micrometer-precision independent positioning of multiple arbitrarily shaped parallel neural electrodes with only a single piezo actuator in an inchworm motor configuration. This multi-probe-single-actuator design allows for significant size and weight reduction, as well as remote control and potential automation of the microdrive. We demonstrate accurate placement of multiple independent recording electrodes into the CA1 region of the rat hippocampus in vivo in acute and chronic settings. Thus, our robotic neural microdrive technology is applicable towards basic neuroscience and clinical studies, as well as other multi-probe or multi-sensor micro-positioning applications.

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    11/08/22 | Robust cell identity specifications through transitions in the collective state of growing developmental systems
    Stanoev A, Koseska A
    Current Opinion in Systems Biology. 2022 Nov 08;31:100437. doi: 10.1016/j.coisb.2022.100437

    Mammalian development is characterized with transitions from homogeneous populations of precursor to heterogeneous population of specified cells. We review here the main dynamical mechanisms through which such transitions are conceptualized, and discuss that the differentiation timing, robust cell-type proportions and recovery upon perturbation are emergent property of proliferating and communicating cell populations. We argue that studying developmental systems using transitions in collective system states is necessary to describe observed experimental features, and propose additionally the basis of a novel analytical method to deduce the relationship between single-cell dynamics and the collective, symmetry-broken states in cellular populations.

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