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1702 Janelia Publications

Showing 31-40 of 1702 results
12/11/19 | The computation of directional selectivity in the OFF motion pathway.
Gruntman E, Romani S, Reiser MB
eLife. 2019 Dec 11;8:. doi: 10.7554/eLife.50706

In flies, the direction of moving ON and OFF features is computed separately. T4 (ON) and T5 (OFF) are the first neurons in their respective pathways to extract a directionally selective response from their non-selective inputs. Our recent study of T4 found that the integration of offset depolarizing and hyperpolarizing inputs is critical for the generation of directional selectivity. However, T5s lack small-field inhibitory inputs, suggesting they may use a different mechanism. Here we used whole-cell recordings of T5 neurons and found a similar receptive field structure: fast depolarization and persistent, spatially offset hyperpolarization. By assaying pairwise interactions of local stimulation across the receptive field, we found no amplifying responses, only suppressive responses to the non-preferred motion direction. We then evaluated passive, biophysical models and found that a model using direct inhibition, but not the removal of excitation, can accurately predict T5 responses to a range of moving stimuli.

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12/04/19 | Preparation and co-culture of iPSC-derived dopaminergic neurons and astrocytes.
de Rus Jacquet A
Current Protocols in Cell Biology. 2019 Dec 04;85(1):e98. doi: 10.1002/cpcb.98

Induced pluripotent stem cell (iPSC)-based models are powerful tools to study neurodegenerative diseases such as Parkinson's disease. The differentiation of patient-derived neurons and astrocytes allows investigation of the molecular mechanisms responsible for disease onset and development. In particular, these two cell types can be mono- or co-cultured to study the influence of cell-autonomous and non-cell-autonomous contributors to neurodegenerative diseases. We developed a streamlined procedure to produce high-quality/high-purity cultures of dopaminergic neurons and astrocytes that originate from the same population of midbrain floor-plate progenitors. This unit describes differentiation, quality control, culture parameters, and troubleshooting tips to ensure the highest quality and reproducibility of research results. © 2019 The Authors. Basic Protocol 1: Differentiation of iPSCs into midbrain-patterned neural progenitor cells Support Protocol: Quality control of neural progenitor cells Basic Protocol 2: Differentiation of neural progenitor cells into astrocytes Basic Protocol 3: Differentiation of neural progenitor cells into dopaminergic neurons Basic Protocol 4: Co-culture of iPSC-derived neurons and astrocytes.

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12/01/19 | A neuronal pathway that commands deceleration in Drosophila larval light-avoidance.
Gong C, Ouyang Z, Zhao W, Wang J, Li K, Zhou P, Zhao T, Zheng N, Gong Z
Neuroscience Bulletin. 2019 Dec 1;35(6):. doi: 10.1007/s12264-019-00349-w

When facing a sudden danger or aversive condition while engaged in on-going forward motion, animals transiently slow down and make a turn to escape. The neural mechanisms underlying stimulation-induced deceleration in avoidance behavior are largely unknown. Here, we report that in Drosophila larvae, light-induced deceleration was commanded by a continuous neural pathway that included prothoracicotropic hormone neurons, eclosion hormone neurons, and tyrosine decarboxylase 2 motor neurons (the PET pathway). Inhibiting neurons in the PET pathway led to defects in light-avoidance due to insufficient deceleration and head casting. On the other hand, activation of PET pathway neurons specifically caused immediate deceleration in larval locomotion. Our findings reveal a neural substrate for the emergent deceleration response and provide a new understanding of the relationship between behavioral modules in animal avoidance responses.

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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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12/01/19 | Neuropixels data-acquisition system: A scalable platform for parallel recording of 10 000+ electrophysiological signals.
Putzeys J, Musa S, Mora Lopez C, Raducanu BC, Carton A, De Ceulaer J, Karsh B, Siegle JH, Van Helleputte N, Harris TD, Dutta B
IEEE Transactions on Biomedical Circuits and Systems. 2019 Dec 01;13(6):1635-1644. doi: 10.1109/TBCAS.2019.2943077

Although CMOS fabrication has enabled a quick evolution in the design of high-density neural probes and neural-recording chips, the scaling and miniaturization of the complete data-acquisition systems has happened at a slower pace. This is mainly due to the complexity and the many requirements that change depending on the specific experimental settings. In essence, the fundamental challenge of a neural-recording system is getting the signals describing the largest possible set of neurons out of the brain and down to data storage for analysis. This requires a complete system optimization that considers the physical, electrical, thermal and signal-processing requirements, while accounting for available technology, manufacturing constraints and budget. Here we present a scalable and open-standards-based open-source data-acquisition system capable of recording from over 10,000 channels of raw neural data simultaneously. The components and their interfaces have been optimized to ensure robustness and minimum invasiveness in small-rodent electrophysiology.

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11/27/19 | Cryo-EM structure of the human FLCN-FNIP2-Rag-Ragulator complex.
Shen K, Rogala KB, Chou H, Huang RK, Yu Z, Sabatini DM
Cell. 2019 Nov 27;179(6):1319-29. doi: 10.1016/j.cell.2019.10.036
11/25/19 | In situ structure determination at nanometer resolution using TYGRESS.
Song K, Shang Z, Fu X, Lou X, Grigorieff N, Nicastro D
Nature Methods. 2019 Nov 25:. doi: 10.1038/s41592-019-0651-0

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

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11/25/19 | Two-photon imaging with silicon photomultipliers.
Modi MN, Daie K, Turner GC, Podgorski K
Optics Express. 2019 Nov 25;27(24):35830-35841. doi: 10.1364/OE.27.035830

We compared performance of recently developed silicon photomultipliers (SiPMs) to GaAsP photomultiplier tubes (PMTs) for two-photon imaging of neural activity. Despite higher dark counts, SiPMs match or exceed the signal-to-noise ratio of PMTs at photon rates encountered in typical calcium imaging experiments due to their low pulse height variability. At higher photon rates encountered during high-speed voltage imaging, SiPMs substantially outperform PMTs.

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11/22/19 | TwoLumps ascending neurons mediate touch-evoked reversal of walking direction in Drosophila.
Sen R, Wang K, Dickson BJ
Current Biology. 2019 Nov 22;29(24):4337-44. doi: 10.1016/j.cub.2019.11.004

External cues, including touch, enable walking animals to flexibly maneuver around obstacles and extricate themselves from dead-ends (for reviews, see [1-3]). In a screen for neurons that enable Drosophila melanogaster to retreat when it encounters a dead-end, we identified a pair of ascending neurons, the TwoLumps Ascending (TLA) neurons. Silencing TLA activity impairs backward locomotion, whereas optogenetic activation triggers backward walking. TLA-induced reversal is mediated in part by the Moonwalker Descending Neurons (MDNs) [4], which receive excitatory input from the TLAs. Silencing the TLAs decreases the extent to which freely walking flies back up upon encountering a physical barrier in the dark, and TLAs show calcium responses to optogenetic activation of neurons expressing the mechanosensory channel NOMPC. We infer that TLAs convey feedforward mechanosensory stimuli to transiently activate MDNs in response to anterior body touch.

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11/20/19 | Generation of stable heading representations in diverse visual scenes.
Kim SS, Hermundstad AM, Romani S, Abbott LF, Jayaraman V
Nature. 2019 Nov 20;576(7785):126-31. doi: 10.1038/s41586-019-1767-1

Many animals rely on an internal heading representation when navigating in varied environments. How this representation is linked to the sensory cues that define different surroundings is unclear. In the fly brain, heading is represented by 'compass' neurons that innervate a ring-shaped structure known as the ellipsoid body. Each compass neuron receives inputs from 'ring' neurons that are selective for particular visual features; this combination provides an ideal substrate for the extraction of directional information from a visual scene. Here we combine two-photon calcium imaging and optogenetics in tethered flying flies with circuit modelling, and show how the correlated activity of compass and visual neurons drives plasticity, which flexibly transforms two-dimensional visual cues into a stable heading representation. We also describe how this plasticity enables the fly to convert a partial heading representation, established from orienting within part of a novel setting, into a complete heading representation. Our results provide mechanistic insight into the memory-related computations that are essential for flexible navigation in varied surroundings.

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