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2685 Janelia Publications
Showing 1791-1800 of 2685 resultsThe Neurodata Without Borders (NWB) initiative promotes data standardization in neuroscience to increase research reproducibility and opportunities. In the first NWB pilot project, neurophysiologists and software developers produced a common data format for recordings and metadata of cellular electrophysiology and optical imaging experiments. The format specification, application programming interfaces, and sample datasets have been released.
Dopamine signals reward in animal brains. A single presentation of a sugar reward to Drosophila activates distinct subsets of dopamine neurons that independently induce short- and long-term olfactory memories (STM and LTM, respectively). In this study, we show that a recurrent reward circuit underlies the formation and consolidation of LTM. This feedback circuit is composed of a single class of reward-signaling dopamine neurons (PAM-α1) projecting to a restricted region of the mushroom body (MB), and a specific MB output cell type, MBON-α1, whose dendrites arborize that same MB compartment. Both MBON-α1 and PAM-α1 neurons are required during the acquisition and consolidation of appetitive LTM. MBON-α1 additionally mediates the retrieval of LTM, which is dependent on the dopamine receptor signaling in the MB α/β neurons. Our results suggest that a reward signal transforms a nascent memory trace into a stable LTM using a feedback circuit at the cost of memory specificity.
UNLABELLED: Sensorimotor delays decouple behaviors from the events that drive them. The brain compensates for these delays with predictive mechanisms, but the efficacy and timescale over which these mechanisms operate remain poorly understood. Here, we assess how prediction is used to compensate for prey movement that occurs during visuomotor processing. We obtained high-speed video records of freely moving, tongue-projecting salamanders catching walking prey, emulating natural foraging conditions. We found that tongue projections were preceded by a rapid head turn lasting ∼130 ms. This motor lag, combined with the ∼100 ms phototransduction delay at photopic light levels, gave a ∼230 ms visuomotor response delay during which prey typically moved approximately one body length. Tongue projections, however, did not significantly lag prey position but were highly accurate instead. Angular errors in tongue projection accuracy were consistent with a linear extrapolation model that predicted prey position at the time of tongue contact using the average prey motion during a ∼175 ms period one visual latency before the head movement. The model explained successful strikes where the tongue hit the fly, and unsuccessful strikes where the fly turned and the tongue hit a phantom location consistent with the fly's earlier trajectory. The model parameters, obtained from the data, agree with the temporal integration and latency of retinal responses proposed to contribute to motion extrapolation. These results show that the salamander predicts future prey position and that prediction significantly improves prey capture success over a broad range of prey speeds and light levels. SIGNIFICANCE STATEMENT: Neural processing delays cause actions to lag behind the events that elicit them. To cope with these delays, the brain predicts what will happen in the future. While neural circuits in the retina and beyond have been suggested to participate in such predictions, few behaviors have been explored sufficiently to constrain circuit function. Here we show that salamanders aim their tongues by using extrapolation to estimate future prey position, thereby compensating for internal delays from both visual and motor processing. Predictions made just before a prey turn resulted in the tongue being projected to a position consistent with the prey's pre-turn trajectory. These results define the computations and operating regimen for neural circuits that predict target motion.
Automated reconstruction of neural connectivity graphs from electron microscopy image stacks is an essential step towards large-scale neural circuit mapping. While significant progress has recently been made in automated segmentation of neurons and detection of synapses, the problem of synaptic partner assignment for polyadic (one-to-many) synapses, prevalent in the Drosophila brain, remains unsolved. In this contribution, we propose a method which automatically assigns pre- and postsynaptic roles to neurites adjacent to a synaptic site. The method constructs a probabilistic graphical model over potential synaptic partner pairs which includes factors to account for a high rate of one-to-many connections, as well as the possibility of the same neuron to be pre-synaptic in one synapse and post-synaptic in another. The algorithm has been validated on a publicly available stack of ssTEM images of Drosophila neural tissue and has been shown to reconstruct most of the synaptic relations correctly.
The Drosophila mushroom body (MB) is a key associative memory center that has also been implicated in the control of sleep. However, the identity of MB neurons underlying homeostatic sleep regulation, as well as the types of sleep signals generated by specific classes of MB neurons, has remained poorly understood. We recently identified two MB output neuron (MBON) classes whose axons convey sleep control signals from the MB to converge in the same downstream target region: a cholinergic sleep-promoting MBON class and a glutamatergic wake-promoting MBON class. Here, we deploy a combination of neurogenetic, behavioral, and physiological approaches to identify and mechanistically dissect sleep-controlling circuits of the MB. Our studies reveal the existence of two segregated excitatory synaptic microcircuits that propagate homeostatic sleep information from different populations of intrinsic MB "Kenyon cells" (KCs) to specific sleep-regulating MBONs: sleep-promoting KCs increase sleep by preferentially activating the cholinergic MBONs, while wake-promoting KCs decrease sleep by preferentially activating the glutamatergic MBONs. Importantly, activity of the sleep-promoting MB microcircuit is increased by sleep deprivation and is necessary for homeostatic rebound sleep (i.e., the increased sleep that occurs after, and in compensation for, sleep lost during deprivation). These studies reveal for the first time specific functional connections between subsets of KCs and particular MBONs and establish the identity of synaptic microcircuits underlying transmission of homeostatic sleep signals in the MB.
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
The Drosophila mushroom body (MB) is an associative learning network that is important for the control of sleep. We have recently identified particular intrinsic MB Kenyon cell (KC) classes that regulate sleep through synaptic activation of particular MB output neurons (MBONs) whose axons convey sleep control signals out of the MB to downstream target regions. Specifically, we found that sleep-promoting KCs increase sleep by preferentially activating cholinergic sleep-promoting MBONs, while wake-promoting KCs decrease sleep by preferentially activating glutamatergic wake-promoting MBONs. Here we use a combination of genetic and physiological approaches to identify wake-promoting dopaminergic neurons (DANs) that innervate the MB, and show that they activate wake-promoting MBONs. These studies reveal a dopaminergic sleep control mechanism that likely operates by modulation of KC-MBON microcircuits.
We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques.
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.
To stimulate progress in automating the reconstruction of neural circuits, we organized the first international challenge on 2D segmentation of electron microscopic (EM) images of the brain. Participants submitted boundary maps predicted for a test set of images, and were scored based on their agreement with a consensus of human expert annotations. The winning team had no prior experience with EM images, and employed a convolutional network. This “deep learning” approach has since become accepted as a standard for segmentation of EM images. The challenge has continued to accept submissions, and the best so far has resulted from cooperation between two teams. The challenge has probably saturated, as algorithms cannot progress beyond limits set by ambiguities inherent in 2D scoring and the size of the test dataset. Retrospective evaluation of the challenge scoring system reveals that it was not sufficiently robust to variations in the widths of neurite borders. We propose a solution to this problem, which should be useful for a future 3D segmentation challenge.