Biology subjects

Lu, I.

Publications and source records attributed to Lu, I..

4 recordsLinked to original sources

bioRxiv2026

BLIMPS: a technique for tandem biosensor imaging across multiple populations of presynaptic terminals, using lattice light sheet microscopy.

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca2+ channels and Ca2+ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca2+ dynamics at the soma is often performed, the imaging of Ca2+ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca2+ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca2+ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca2+ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

neuroscience↗

bioRxiv2025

Presynaptic GABAB autoreceptors suppress neurotransmitter release during repetitive stimulation via the Gβγ-SNARE pathway.

GABAergic signaling provides the brains primary inhibitory mechanism with defects linked to epilepsy, anxiety, depression, insomnia, schizophrenia and neurodegeneration. A key regulatory mechanism is autoinhibition of GABA release during repetitive activity via presynaptic Gi/o-coupled GABAB receptors, supporting synaptic tuning and memory formation, and limiting neurotransmitter spillover. Exogenous GABAB receptor agonists reduce presynaptic Ca2+ entry by inhibiting calcium channels. However, using transgenic mice expressing a mutant SNAP25 with diminished ability to bind G{beta}{gamma} (SNAP25{Delta}3), we show that suppression by GABAB autoreceptors requires intact G{beta}{gamma}-SNARE interactions. Imaging of presynaptic Ca2+ transients in GABAergic axons showed no GABA-mediated autoreceptor suppression of Ca2+ entry during stimulus trains. In contrast, application of the exogenous GABAB receptor agonist baclofen profoundly inhibited Ca2+ entry, which could be partially reversed by exogenously elevating cAMP, indicating a complementary role of inhibition of adenylyl cyclase. Baclofen reduced spontaneous IPSC frequency and amplitude and both effects were diminished in SNAP25{Delta}3 mice, consistent with inhibition at Ca2+ channels and SNARE complexes. Physiological GABA-mediated and exogenous GABAB receptor activation thus produce distinct outcomes on GABAergic neurotransmission, indicating that synthetic drug application to neurons does not faithfully recapitulate endogenous signaling pathways. We conclude that endogenous rapid GABAB autoreceptor signaling inhibits neurotransmitter release primarily by G{beta}{gamma}-mediated inhibitions of SNARE mechanisms, whereas prolonged agonist application additionally suppresses Ca2+ influx via cAMP signaling.

neuroscience↗

bioRxiv2024

Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell derived sensory neurons

Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human pluripotent stem cells (hPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a novel population of hPSC-derived sensory neurons to previously published datasets and find this novel population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology with respect to voltage-gated sodium (Nav) and potassium channels (Kv), and ligand-gated ionotropic GABA and P2X receptors. Overall, we find this population of hPSC-derived sensory neurons to be of relatively high fidelity, and suitable for interrogating numerous potential pain targets on a fully humanized platform.

cell biology↗

bioRxiv2021

Single-Domain SARS-CoV-2 S1 and RBD Antibodies Isolated from Immunized Llama Effectively Bind Targets of the Wuhan, UK, and South African Strains in vitro

The spreading of SARS-CoV-2 variants has become a major challenge of the current fight against the pandemic. Of particular concerns are the strains that have arisen from the United Kingdom (UK) and South Africa. The UK variant spreads rapidly and is projected to overtake the original strain in the US as early as in March 2021, while the South African variant appears to evade some effects of the current vaccines. Potential false-negative diagnosis using currently available antigen kits that may not recognize these variants could cause another wave of community infection. Therefore, it is imperative that antibodies used in the detection kits are validated for binding against these variants. Here we report that the nanoantibodies (nAbs in our terminology, also referred to as VHH fragments, single domain antibodies, nanobodies) that we have developed for rapid antigen detection test bind the receptor binding domain (RBD) of the S1 protein from the original COVID-SARS-2 virus as well as those from the UK and South African variants. This finding validates our antibodies used in our assay for the detection of these major variant strains.

immunology↗