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2529 Janelia Publications
Showing 931-940 of 2529 resultsEfforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely used technique for refined cell-type specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip(+)), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip(+) acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip(+) technology, we have developed a versatile set of LexAop-KZip(+) fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. The Killer Zipper significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.
Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation.
Destabilized nanobodies can be used to deliver fluorescent proteins and enzymes to specific targets inside cells.
Calcium imaging permits optical measurement of neural activity. Since intracellular calcium concentration is an indirect measurement of neural activity, computational tools are necessary to infer the true underlying spiking activity from fluorescence measurements. Bayesian model inversion can be used to solve this problem, but typically requires either computationally expensive MCMC sampling, or faster but approximate maximum-a-posteriori optimization. Here, we introduce a flexible algorithmic framework for fast, efficient and accurate extraction of neural spikes from imaging data. Using the framework of variational autoencoders, we propose to amortize inference by training a deep neural network to perform model inversion efficiently. The recognition network is trained to produce samples from the posterior distribution over spike trains. Once trained, performing inference amounts to a fast single forward pass through the network, without the need for iterative optimization or sampling. We show that amortization can be applied flexibly to a wide range of nonlinear generative models and significantly improves upon the state of the art in computation time, while achieving competitive accuracy. Our framework is also able to represent posterior distributions over spike-trains. We demonstrate the generality of our method by proposing the first probabilistic approach for separating backpropagating action potentials from putative synaptic inputs in calcium imaging of dendritic spines.
Optical flow is a key method used for quantitative motion estimation of biological structures in light microscopy. It has also been used as a key module in segmentation and tracking systems and is considered a mature technology in the field of computer vision. However, most of the research focused on 2D natural images, which are small in size and rich in edges and texture information. In contrast, 3D time-lapse recordings of biological specimens comprise up to several terabytes of image data and often exhibit complex object dynamics as well as blurring due to the point-spread-function of the microscope. Thus, new approaches to optical flow are required to improve performance for such data. We solve optical flow in large 3D time-lapse microscopy datasets by defining a Markov random field (MRF) over super-voxels in the foreground and applying motion smoothness constraints between super-voxels instead of voxel-wise. This model is tailored to the specific characteristics of light microscopy datasets: super-voxels help registration in textureless areas, the MRF over super-voxels efficiently propagates motion information between neighboring cells and the background subtraction and super-voxels reduce the dimensionality of the problem by an order of magnitude. We validate our approach on large 3D time-lapse datasets of Drosophila and zebrafish development by analyzing cell motion patterns. We show that our approach is, on average, 10 x faster than commonly used optical flow implementations in the Insight Tool-Kit (ITK) and reduces the average flow end point error by 50% in regions with complex dynamic processes, such as cell divisions.
Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
We review the principles of development and deployment of genetically encoded calcium indicators (GECIs) for the detection of neural activity. Our focus is on the popular GCaMP family of green GECIs, culminating in the recent release of the jGCaMP8 sensors, with dramatically improved kinetics relative to previous generations. We summarize the properties of GECIs in multiple color channels (blue, cyan, green, yellow, red, far-red) and highlight areas for further improvement. With their low-millisecond rise-times, the jGCaMP8 indicators allow new classes of experiments following neural activity in timeframes approaching the underlying computations. Abstract legend: GCaMP calcium sensors are widely used to report neuronal activity via fluorescence readout. This article is protected by copyright. All rights reserved.
MOTIVATION: Homology search for RNAs can use secondary structure information to increase power by modeling base pairs, as in covariance models, but the resulting computational costs are high. Typical acceleration strategies rely on at least one filtering stage using sequence-only search. RESULTS: Here we present the multi-segment CYK (MSCYK) filter, which implements a heuristic of ungapped structural alignment for RNA homology search. Compared to gapped alignment, this approximation has lower computation time requirements (O(N⁴) reduced to O(N³), and space requirements (O(N³) reduced to O(N²). A vector-parallel implementation of this method gives up to 100-fold speed-up; vector-parallel implementations of standard gapped alignment at two levels of precision give 3- and 6-fold speed-ups. These approaches are combined to create a filtering pipeline that scores RNA secondary structure at all stages, with results that are synergistic with existing methods.
Many important physiological processes operate at time and space scales far beyond those accessible to atom-realistic simulations, and yet discrete stochastic rather than continuum methods may best represent finite numbers of molecules interacting in complex cellular spaces. We describe and validate new tools and algorithms developed for a new version of the MCell simulation program (MCell3), which supports generalized Monte Carlo modeling of diffusion and chemical reaction in solution, on surfaces representing membranes, and combinations thereof. A new syntax for describing the spatial directionality of surface reactions is introduced, along with optimizations and algorithms that can substantially reduce computational costs (e.g., event scheduling, variable time and space steps). Examples for simple reactions in simple spaces are validated by comparison to analytic solutions. Thus we show how spatially realistic Monte Carlo simulations of biological systems can be far more cost-effective than often is assumed, and provide a level of accuracy and insight beyond that of continuum methods.
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.