Biology subjects

Ng, L.

Publications and source records attributed to Ng, L..

27 records · Page 2Linked to original sources

bioRxiv2020

High fidelity electrophysiological, morphological, and transcriptomic cell characterization using a refined Patch-seq protocol

The Patch-seq approach is a powerful variation of the standard patch clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at a scale and quality that can be integrated with high-throughput dissociated cell transcriptomic data, we have optimized the technique by identifying and refining key factors that contribute to the efficient collection of high-quality data. To rapidly generate high-quality electrophysiology data, we developed patch clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized a substantial improvement in transcriptomic data quality when the nucleus was extracted following the recording. For morphology success, the importance of maximizing the neurons membrane integrity during the extraction of the nucleus was much more critical to success than varying the duration of the electrophysiology recording. We compiled the lab protocol with the analysis and acquisition software at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate Patch-seq data across diverse mammalian species and that is compatible with recent large-scale publicly available Allen Institute Patch-seq datasets.

neuroscience↗

bioRxiv2020

Notch-Directed Germ Cell Proliferation Is Mediated by Proteoglycan-Dependent Transcription

Notch receptors are essential membrane-bound regulators of cell proliferation and differentiation in metazoa. In the nematode Caenorhabditis elegans, correct expression of GLP-1 (germline proliferation-1), a germline-expressed Notch receptor, is important for germ cell maintenance. However, mechanisms that regulate GLP-1 expression are undefined. Here, we demonstrate that an AP-2 transcription factor (APTF-2) regulates GLP-1 expression through calcium-dependent binding to a conserved motif in the glp-1 promoter. Our data reveals that SDN-1 (syndecan-1), a transmembrane proteoglycan, regulates a TRP calcium channel in the soma to modulate the interaction between APTF-2 and glp-1 promoter - thus providing a potential communication nexus between the germline and its somatic environment to control germ cell fate decisions.

developmental biology↗

bioRxiv2020

TIP60 acetylates H2AZ and regulates doxorubicin-induced DNA damage sensitivity through RAD51 transcription

TIP60, a lysine acetyltransferase and H2AZ, a histone H2A variant are involved in transcription and DNA repair. Recent studies suggest that H2AZ acetylation is dependent on TIP60. Here, we show that TIP60 acetylates both isoforms of H2AZ in vitro and in cells. Utilizing ChIP-seq and RNA-seq to identify the genes regulated by TIP60-dependent acetylation of H2AZ, we find that TIP60-dependent acetylation of H2AZ correlates with the expression of genes involved in DNA damage repair, amongst several other pathways. In line with this, TIP60-depleted cells exhibit increased sensitivity to the DNA damage-inducing drug doxorubicin. Restoring the expression level of RAD51, one of the genes involved in the DNA damage repair pathway, partially rescues the doxorubicin sensitivity due to TIP60 depletion. Overall, our study uncovers a role for TIP60 in regulating doxorubicin-induced DNA damage sensitivity in a manner dependent on RAD51 transcription.

molecular biology↗

bioRxiv2020

Regional, layer, and cell-class specific connectivity of the mouse default mode network

The evolutionarily conserved default mode network (DMN) is characterized by temporally correlated activity between brain regions during resting states. The DMN has emerged as a selectively vulnerable network in multiple disorders, so understanding its anatomical composition will provide fundamental insight into how its function is impacted by disease. Reproducible rodent analogs of the human DMN offer an opportunity to investigate the underlying brain regions and structural connectivity (SC) with high spatial and cell type resolution. Here, we performed systematic analyses using mouse resting state functional magnetic resonance imaging to identify the DMN and whole brain axonal tracing data, co-registered to the 3D Allen Mouse Common Coordinate Framework reference atlas. We identified the specific, predominantly cortical, brain regions comprising the mouse DMN and report preferential SC between these regions. Next, at the cell class level, we report that cortical layer (L) 2/3 neurons in DMN regions project almost exclusively to other DMN regions, whereas L5 neurons project to targets both in and out of the DMN. We then test the hypothesis that in- and out-DMN projection patterns originate from distinct L5 neuron sub-classes using an intersectional viral tracing strategy to label all the axons from neurons defined by a single target. In the ventral retrosplenial cortex, a core DMN region, we found two L5 projection types related to the DMN and mapped them to unique transcriptomically-defined cell types. Together, our results provide a multi-scale description of the anatomical correlates of the mouse DMN.

neuroscience↗

bioRxiv2020

Multi-modal characterization and simulation of human epileptic circuitry

Temporal lobe epilepsy is the fourth most common neurological disorder with about 40% of patients not responding to pharmacological treatment. Increased cellular loss in the hippocampus is linked to disease severity and pathological phenotypes such as heightened seizure propensity. While the hippocampus is the target of therapeutic interventions such as temporal lobe resection, the impact of the disease at the cellular level remains unclear in humans. Here we show that properties of hippocampal granule cells change with disease progression as measured in living, resected hippocampal tissue excised from epilepsy patients. We show that granule cells increase excitability and shorten response latency while also enlarging in cellular volume, surface area and spine density. Single-cell RNA sequencing combined with simulations ascribe the observed electrophysiological changes to gradual modification in three key ion channel conductances: BK, Cav2.2 and Kir2.1. In a bio-realistic computational network model, we show that the changes related to disease progression bring the circuit into a more excitable state. In turn, we observe that by reversing these changes in the three key conductances produces a less excitable, "early disease-like" state. These results provide mechanistic understanding of epilepsy in humans and will inform future therapies such as viral gene delivery to reverse the course of the disorder.

neuroscience↗

bioRxiv2019

Trimester-specific Zika virus infection affects placental responses in women

Zika virus (ZIKV) infection during pregnancy is associated with neurologic birth defects, but the effects on placental development are unclear. Full-term placentas from three women, each infected with ZIKV during specific pregnancy trimesters, were harvested for anatomic, immunologic and transcriptomic analysis. In this study, each woman exhibited a unique immune response, but they collectively diverged from healthy controls with raised IL-1RA, IP-10, EGF and RANTES expression, and neutrophil numbers during the acute infection phase. Although ZIKV NS3 antigens co-localized to placental Hofbauer cells, the placentas showed no anatomical defects. Transcriptomic analysis of samples from the placentas revealed that infection during trimester 1 caused a disparate cellular response centered on differential eIF2 signaling, mitochondrial dysfunction and oxidative phosphorylation. These findings should translate to improve clinical prenatal screening procedures for virus-infected pregnant patients.

immunology↗

bioRxiv2019

Complete single neuron reconstruction reveals morphological diversity in molecularly defined claustral and cortical neuron types

Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

neuroscience↗

bioRxiv2019

NWB:N 2.0: An Accessible Data Standard for Neurophysiology

Neurodata Without Borders: Neurophysiology (NWB:N) is a data standard for neurophysiology, providing neuroscientists with a common standard to share, archive, use, and build common analysis tools for neurophysiology data. With NWB:N version 2.0 (NWB:N 2.0) we made significant advances towards creating a usable standard, software ecosystem, and vibrant community for standardizing neurophysiology data. In this manuscript we focus in particular on the NWB:N data standard schema and present advances towards creating an accessible data standard for neurophysiology.

neuroscience↗

bioRxiv2018

The organization of intracortical connections by layer and cell class in the mouse brain

The mammalian cortex is a laminar structure composed of many cell types densely interconnected in complex ways. Recent systematic efforts to map the mouse mesoscale connectome provide comprehensive projection data on interareal connections, but not at the level of specific cell classes or layers within cortical areas. We present here a significant expansion of the Allen Mouse Brain Connectivity Atlas, with [~]1,000 new axonal projection mapping experiments across nearly all isocortical areas in 49 Cre driver lines. Using 13 lines selective for cortical layer-specific projection neuron classes, we identify the differential contribution of each layer/class to the overall intracortical connectivity patterns. We find layer 5 (L5) projection neurons account for essentially all intracortical outputs. L2/3, L4, and L6 neurons contact a subset of the L5 cortical targets. We also describe the most common axon lamination patterns in cortical targets. Most patterns are consistent with previous anatomical rules used to determine hierarchical position between cortical areas (feedforward, feedback), with notable exceptions. While diverse target lamination patterns arise from every source layer/class, L2/3 and L4 neurons are primarily associated with feedforward type projection patterns and L6 with feedback. L5 has both feedforward and feedback projection patterns. Finally, network analyses revealed a modular organization of the intracortical connectome. By labeling interareal and intermodule connections as feedforward or feedback, we present an integrated view of the intracortical connectome as a hierarchical network.

neuroscience↗