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14 Janelia Publications

Showing 1-10 of 14 results
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    10/05/23 | Conjoint specification of action by neocortex and striatum.
    Junchol Park , Peter Polidoro , Catia Fortunato , Jon Arnold , Brett Mensh , Juan A. Gallego , Joshua T. Dudman
    bioRxiv. 2023 Oct 05:. doi: 10.1101/2023.10.04.560957

    The interplay between two major forebrain structures - cortex and subcortical striatum - is critical for flexible, goal-directed action. Traditionally, it has been proposed that striatum is critical for selecting what type of action is initiated while the primary motor cortex is involved in the online control of movement execution. Recent data indicates that striatum may also be critical for specifying movement execution. These alternatives have been difficult to reconcile because when comparing very distinct actions, as in the vast majority of work to date, they make essentially indistinguishable predictions. Here, we develop quantitative models to reveal a somewhat paradoxical insight: only comparing neural activity during similar actions makes strongly distinguishing predictions. We thus developed a novel reach-to-pull task in which mice reliably selected between two similar, but distinct reach targets and pull forces. Simultaneous cortical and subcortical recordings were uniquely consistent with a model in which cortex and striatum jointly specify flexible parameters of action during movement execution.

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    05/17/23 | Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator
    Abdelfattah AS, Zheng J, Singh A, Huang Y, Reep D, Tsegaye G, Tsang A, Arthur BJ, Rehorova M, Olson CV, Shuai Y, Zhang L, Fu T, Milkie DE, Moya MV, Weber TD, Lemire AL, Baker CA, Falco N, Zheng Q, Grimm JB, Yip MC, Walpita D, Chase M, Campagnola L, Murphy GJ, Wong AM, Forest CR, Mertz J, Economo MN, Turner GC, Koyama M, Lin B, Betzig E, Novak O, Lavis LD, Svoboda K, Korff W, Chen T, Schreiter ER, Hasseman JP, Kolb I
    Neuron. 2023 May 17;111(10):1547-1563. doi: 10.1016/j.neuron.2023.03.009

    The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

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    11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
    Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
    bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

    Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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    11/02/22 | Cap-dependent translation initiation monitored in living cells.
    Gandin V, English BP, Freeman M, Leroux L, Preibisch S, Walpita D, Jaramillo M, Singer RH
    Nature Communications. 2022 Nov 02;13(1):6558. doi: 10.1038/s41467-022-34052-8

    mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5'cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.

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    09/03/22 | Motion of single molecular tethers reveals dynamic subdomains at ER-mitochondria contact sites
    Christopher J. Obara , Jonathon Nixon-Abell , Andrew S. Moore , Federica Riccio , David P. Hoffman , Gleb Shtengel , C. Shan Xu , Kathy Schaefer , H. Amalia Pasolli , Jean-Baptiste Masson , Harald F. Hess , Christopher P. Calderon , Craig Blackstone , Jennifer Lippincott-Schwartz
    bioRxiv. 2022 Sep 03:. doi: 10.1101/2022.09.03.505525

    To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites3. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle4,5. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation6,7, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

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    07/04/22 | Visualizing Synaptic Dopamine Efflux with a 2D Nanofilm.
    Chandima Bulumulla , Andrew T. Krasley , Deepika Walpita , Abraham G. Beyene
    eLife. 2022 Jul 04:. doi: 10.7554/eLife.78773

    Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional nanofilm that facilitates subcellular visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈3.2 µm (full width at half maximum) and are observed with a mean spatial frequency of 1 hotspot per ≈7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots colocalized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.

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    05/29/22 | Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.
    V. C. Wong , P.R. Houlihan , H. Liu , D. Walpita , M.C. DeSantis , Z. Liu , E. K. O’Shea
    bioRxiv. 2022 May 29:. doi: 10.1101/2022.05.29.493906

    AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing long-term potentiation (LTP) to increase synaptic transmission, but how AMPAR-containing vesicles are selectively trafficked to these synapses during LTP is not known. Here we developed a strategy to label AMPAR GluA1 subunits expressed from the endogenous loci of rat hippocampal neurons such that the motion of GluA1-containing vesicles in time-lapse sequences can be characterized using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of neuronal activity.

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    03/11/22 | Motor cortical output for skilled forelimb movement is selectively distributed across projection neuron classes.
    Park J, Phillips JW, Guo J, Martin KA, Hantman AW, Dudman JT
    Science Advances. 2022 Mar 11;8(10):eabj5167. doi: 10.1126/sciadv.abj5167

    The interaction of descending neocortical outputs and subcortical premotor circuits is critical for shaping skilled movements. Two broad classes of motor cortical output projection neurons provide input to many subcortical motor areas: pyramidal tract (PT) neurons, which project throughout the neuraxis, and intratelencephalic (IT) neurons, which project within the cortex and subcortical striatum. It is unclear whether these classes are functionally in series or whether each class carries distinct components of descending motor control signals. Here, we combine large-scale neural recordings across all layers of motor cortex with cell type-specific perturbations to study cortically dependent mouse motor behaviors: kinematically variable manipulation of a joystick and a kinematically precise reach-to-grasp. We find that striatum-projecting IT neuron activity preferentially represents amplitude, whereas pons-projecting PT neurons preferentially represent the variable direction of forelimb movements. Thus, separable components of descending motor cortical commands are distributed across motor cortical projection cell classes.

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    09/30/21 | The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease
    Aurelie de Rus Jacquet , Jenna L. Tancredi , Andrew L. Lemire , Michael C. DeSantis , Wei-Ping Li , Erin K. O’Shea
    eLife. 2021 Sep 30:. doi: https://doi.org/10.7554/eLife.73062

    Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson’s disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remains largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes, and we identify the abnormal accumulation of key PD-related proteins within multi vesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

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    04/01/21 | The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.
    Deo C, Abdelfattah AS, Bhargava HK, Berro AJ, Falco N, Farrants H, Moeyaert B, Chupanova M, Lavis LD, Schreiter ER
    Nature Chemical Biology. 2021 Apr 01:. doi: 10.1038/s41589-021-00775-w

    Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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