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2 Janelia Publications

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    12/01/19 | High-yield, automated intracellular electrophysiology in retinal pigment epithelia.
    Lewallen CF, Wan Q, Maminishkis A, Stoy W, Kolb I, Hotaling N, Bharti K, Forest CR
    Journal of Neuroscience Methods. 2019 Dec 01;328:108442. doi: 10.1016/j.jneumeth.2019.108442

    BACKGROUND: Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput.

    NEW METHOD: A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques.

    RESULTS: The automated algorithm improves yield per attempt from 17% (manually, n = 23) to 22% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 μm and electroporating (buzzing) just prior to this depth (5 μm) maximized yield.

    COMPARISON WITH EXISTING METHOD: Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods.

    CONCLUSIONS: This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.

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    04/26/19 | A neural circuit encoding the experience of copulation in female Drosophila.
    Shao L, Chung P, Wong A, Siwanowicz I, Kent CF, Long X, Heberlein U
    Neuron. 2019 Apr 26;102(5):1025. doi: 10.1016/j.neuron.2019.04.009

    Female behavior changes profoundly after mating. In Drosophila, the mechanisms underlying the long-term changes led by seminal products have been extensively studied. However, the effect of the sensory component of copulation on the female's internal state and behavior remains elusive. We pursued this question by dissociating the effect of coital sensory inputs from those of male ejaculate. We found that the sensory inputs of copulation cause a reduction of post-coital receptivity in females, referred to as the "copulation effect." We identified three layers of a neural circuit underlying this phenomenon. Abdominal neurons expressing the mechanosensory channel Piezo convey the signal of copulation to female-specific ascending neurons, LSANs, in the ventral nerve cord. LSANs relay this information to neurons expressing myoinhibitory peptides in the brain. We hereby provide a neural mechanism by which the experience of copulation facilitates females encoding their mating status, thus adjusting behavior to optimize reproduction.

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