Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

general_search_page-panel_pane_1 | views_panes

5 Janelia Publications

Showing 1-5 of 5 results
Your Criteria:
    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.

    View Publication Page
    10/26/22 | Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy
    Joshua L. Lillvis , Hideo Otsuna , Xiaoyu Ding , Igor Pisarev , Takashi Kawase , Jennifer Colonell , Konrad Rokicki , Cristian Goina , Ruixuan Gao , Amy Hu , Kaiyu Wang , John Bogovic , Daniel E. Milkie , Edward S. Boyden , Stephan Saalfeld , Paul W. Tillberg , Barry J. Dickson
    eLife. 2022 Oct 26:. doi: 10.7554/eLife.81248

    Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

    View Publication Page
    10/22/22 | Multimodal mapping of cell types and projections in the central nucleus of the amygdala
    Yuhan Wang , Sabine Krabbe , Mark Eddison , Fredrick E. Henry , Greg Fleishman , Andrew L. Lemire , Lihua Wang , Wyatt Korff , Paul W. Tillberg , Andreas Lüthi , Scott M. Sternson
    bioRxiv. 2022 Oct 22:. doi: 10.1101/2022.10.19.512845

    The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-Seq) to classify molecularly defined cell types in the CEA and identified marker-genes to map the location of these neuronal types using expansion assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ∼30,000 molecularly defined CEA neurons. Our study revealed spatio-molecular organization of the CEA, with medial and lateral CEA associated with distinct cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker-gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

    View Publication Page
    02/18/22 | Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
    Hugo G.J. Damstra , Boaz Mohar , Mark Eddison , Anna Akhmanova , Lukas C. Kapitein , Paul W. Tillberg
    eLife. 2022 Feb 18:. doi: https://doi.org/10.1101/2021.02.03.428837

    Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

    View Publication Page
    02/15/22 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission
    Abhi Aggarwal , Rui Liu , Yang Chen , Amelia J Ralowicz , Samuel J Bergerson , Filip Tomaska , Timothy L Hanson , Jeremy P Hasseman , Daniel Reep , Getahun Tsegaye , Pantong Yao , Xiang Ji , Marinus Kloos , Deepika Walpita , Ronak Patel , Paul W Tilberg , Boaz Mohar , GENIE , Loren L Looger , Jonathan S Marvin , Michael B Hoppa , Arthur Konnerth , David Kleinfeld , Eric R Schreiter , Kaspar Podgorski
    bioRxiv PrePrint. 2022 Feb 15:. doi: 10.1101/2022.02.13.480251

    The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

    View Publication Page