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1661 Janelia Publications

Showing 1-10 of 1661 results
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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Bock Lab
11/06/19 | A neural circuit arbitrates between persistence and withdrawal in hungry drosophila.
Sayin S, De Backer J, Siju KP, Wosniack ME, Lewis LP, Frisch L, Gansen B, Schlegel P, Edmondson-Stait A, Sharifi N, Fisher CB, Calle-Schuler SA, Lauritzen JS, Bock DD, Costa M, Jefferis GS, Gjorgjieva J, Grunwald Kadow IC
Neuron. 2019 Nov 6;104(3):544-58. doi: 10.1016/j.neuron.2019.07.028

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive.

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11/06/19 | Interactions between Dpr11 and DIP-γ control selection of amacrine neurons in color vision circuits.
Menon KP, Kulkarni V, Takemura S, Anaya M, Zinn K
eLife. 2019 Nov 06;8:. doi: 10.7554/eLife.48935

R7 UV photoreceptors (PRs) are divided into yellow (y) and pale (p) subtypes. yR7 PRs express the Dpr11 cell surface protein and are presynaptic to Dm8 amacrine neurons (yDm8) that express Dpr11's binding partner DIP-g, while pR7 PRs synapse onto DIP-g-negative pDm8. Dpr11 and DIP-g expression patterns define 'yellow' and 'pale' color vision circuits. We examined Dm8 neurons in these circuits by electron microscopic reconstruction and expansion microscopy. and mutations affect the morphologies of yDm8 distal ('home column') dendrites. yDm8 neurons are generated in excess during development and compete for presynaptic yR7 PRs, and interactions between Dpr11 and DIP-g are required for yDm8 survival. These interactions also allow yDm8 neurons to select yR7 PRs as their appropriate home column partners. yDm8 and pDm8 neurons do not normally compete for survival signals or R7 partners, but can be forced to do so by manipulation of R7 subtype fate.

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11/05/19 | 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 05:. doi: 10.1016/j.cell.2019.10.036
11/04/19 | Zebrafish neuroscience: Using artificial neural networks to help understand brains.
Ahrens MB
Current Biology. 2019 Nov 04;29(21):R1138-R1140. doi: 10.1016/j.cub.2019.09.039

Brains are notoriously hard to understand, and neuroscientists need all the tools they can get their hands on to have a realistic shot at it. Advances in machine learning are proving instrumental, illustrated by their recent use to shed light on navigational strategies implemented by zebrafish brains.

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10/30/19 | Functional clustering of dendritic activity during decision-making.
Kerlin A, Boaz M, Flickinger D, MacLennan BJ, Dean MB, Davis C, Spruston N, Svoboda K
Elife. 2019 Oct 30;8:. doi: 10.7554/eLife.46966

The active properties of dendrites can support local nonlinear operations, but previous imaging and electrophysiological measurements have produced conflicting views regarding the prevalence and selectivity of local nonlinearities in vivo. We imaged calcium signals in pyramidal cell dendrites in the motor cortex of mice performing a tactile decision task. A custom microscope allowed us to image the soma and up to 300 μm of contiguous dendrite at 15 Hz, while resolving individual spines. New analysis methods were used to estimate the frequency and spatial scales of activity in dendritic branches and spines. The majority of dendritic calcium transients were coincident with global events. However, task-associated calcium signals in dendrites and spines were compartmentalized by dendritic branching and clustered within branches over approximately 10 μm. Diverse behavior-related signals were intermingled and distributed throughout the dendritic arbor, potentially supporting a large learning capacity in individual neurons.

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10/23/19 | Recruitment of GABAergic interneurons in the barrel cortex during active tactile behavior.
Yu J, Hu H, Agmon A, Svoboda K
Neuron. 2019 Oct 23;104(2):412-27. doi: 10.1016/j.neuron.2019.07.027

Neural computation involves diverse types of GABAergic inhibitory interneurons that are integrated with excitatory (E) neurons into precisely structured circuits. To understand how each neuron type shapes sensory representations, we measured firing patterns of defined types of neurons in the barrel cortex while mice performed an active, whisker-dependent object localization task. Touch excited fast-spiking (FS) interneurons at short latency, followed by activation of E neurons and somatostatin-expressing (SST) interneurons. Touch only weakly modulated vasoactive intestinal polypeptide-expressing (VIP) interneurons. Voluntary whisker movement activated FS neurons in the ventral posteromedial nucleus (VPM) target layers, a subset of SST neurons and a majority of VIP neurons. Together, FS neurons track thalamic input, mediating feedforward inhibition. SST neurons monitor local excitation, providing feedback inhibition. VIP neurons are activated by non-sensory inputs, disinhibiting E and FS neurons. Our data reveal rules of recruitment for interneuron types during behavior, providing foundations for understanding computation in cortical microcircuits.

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10/23/19 | Unlimited genetic switches for cell-type-specific manipulation.
Garcia-Marques J, Yang C, Espinosa-Medina I, Mok K, Koyama M, Lee T
Neuron. 2019 Oct 23;104(2):227-38. doi:

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

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10/18/19 | A bidirectional network for appetite control in larval zebrafish.
Wee CL, Song EY, Johnson RE, Ailani D, Randlett O, Kim J, Nikitchenko M, Bahl A, Yang C, Ahrens MB, Kawakami K, Engert F, Kunes S
Elife. 2019 Oct 18;8:. doi: 10.7554/eLife.43775

Medial and lateral hypothalamic loci are known to suppress and enhance appetite, respectively, but the dynamics and functional significance of their interaction have yet to be explored. Here we report that, in larval zebrafish, primarily serotonergic neurons of the ventromedial caudal hypothalamus (cH) become increasingly active during food deprivation, whereas activity in the lateral hypothalamus (LH) is reduced. Exposure to food sensory and consummatory cues reverses the activity patterns of these two nuclei, consistent with their representation of opposing internal hunger states. Baseline activity is restored as food-deprived animals return to satiety via voracious feeding. The antagonistic relationship and functional importance of cH and LH activity patterns were confirmed by targeted stimulation and ablation of cH neurons. Collectively, the data allow us to propose a model in which these hypothalamic nuclei regulate different phases of hunger and satiety and coordinate energy balance via antagonistic control of distinct behavioral outputs.

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10/18/19 | Drosulfakinin signaling in fruitless circuitry antagonizes P1 neurons to regulate sexual arousal in Drosophila.
Wu S, Guo C, Zhao H, Sun M, Chen J, Han C, Peng Q, Qiao H, Peng P, Liu Y, Luo SD, Pan Y
Nature Communications. 2019 Oct 18;10(1):4770. doi: 10.1038/s41467-019-12758-6

Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. We found that Dsk neurons physiologically interact with male-specific P1 neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. We further found that the DSK-2 peptide functions through its receptor CCKLR-17D3 to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P1 neurons to balance arousal levels and modulate sexual behaviors.

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