Biology subjects

Barber, K.

Publications and source records attributed to Barber, K..

8 recordsLinked to original sources

bioRxiv2026

Cellular-resolution Three-photon Ultra-Deep Imaging (TrUDI) of neuronalpopulation activity at 2 mm depth in the mammalian brain

Extending the depth reach of multi-photon microscopy for imaging neuronal population activity in the scattering mammalian brain is an exponentially hard problem. Three-photon (3p) microscopy has enabled imaging of neuroactivity at depths up to ~1.2 mm in the mouse brain. However, the fundamental depth limit of 3p imaging, as set by the depth at which the out-of-focus background fluorescence exceeds the in-focus fluorescence signal, is well beyond 2 mm. Reaching this fundamental depth limit in 3p imaging has been hampered by insufficient in-focus fluorescence signal arising from suboptimal optical design and acquisition conditions. Here, we demonstrate a Three-photon Ultra-Deep Imaging (TrUDI) system that approaches this fundamental depth limit via systematic and principled optimization of the in-focus fluorescence signal. TrUDI optimally matches the parameters of the excitation laser, the optical system, and the acquisition to linear and non-linear limits for safe laser exposure. At the same time, TrUDI remains compatible with the spatial and temporal requirements for single-cell resolution calcium imaging, and its parameters are tuned to the photo-physics of 3p excitation and the scattering properties of the mouse brain tissue. Our approach results in a ~250-fold increase in detected fluorescence photons at the same laser power as in previous approaches. We use TrUDI to demonstrate in vivo cellular-resolution recordings of neuronal population activity within fields of view of ~230 x 230 um2 at 8.6 Hz at tissue depths of up to ~2 mm in mouse cortex and hippocampus.

neuroscience↗

bioRxiv2026

Neuropil aggrecan, not perineuronal nets, closes the critical period for visual plasticity

Perineuronal nets (PNNs) are widely reported to close developmental critical periods and restrict experience-dependent plasticity. Here we tested this model by selectively eliminating gene expression for aggrecan (Acan), an essential component of PNNs. In visual cortex, PNNs predominantly ensheath parvalbumin-positive (PV+) interneurons. Deletion of Acan in inhibitory neurons eliminated PNNs but did not prevent closure of the critical period. By comparison, deletion of Acan in all neurons, or only in excitatory forebrain neurons, sustained critical-period plasticity in adult mice. Visual plasticity in adults was associated with reduced cortical excitatory synaptic inputs onto layer 2/3 PV+ interneurons and increased expression of some immediate early genes in visual cortex but normal response strength and tuning properties of layer 2/3 excitatory neurons. These findings rectify long-standing models that misattribute closure of the critical period to PNNs and identify that aggrecan expressed by excitatory neurons resident in the surrounding neuropil limits visual plasticity.

neuroscience↗

bioRxiv2025

End-to-end automation of repeat-target cryo-EM structure determination in CryoSPARC

Single particle cryo-EM is a valuable and growing technique for life science and drug discovery. Currently, obtaining state-of-the-art results from cryo-EM data analysis requires a human in the loop to analyze intermediate results and make image processing decisions. This bottleneck limits the achievable throughput of structure determination, especially in high-throughput settings such as structure-based drug design. In this work, we develop an end-to-end automation strategy for repeat-target structure determination using new tools in CryoSPARC. We demonstrate completely hands-off processing of 21 challenging G protein-coupled receptor (GPCR) datasets. In 17 of 21 cases, automated processing meets or exceeds published resolution and map quality and, in several cases, provides significant improvement in receptor and ligand density that allows improved model building. Our results on both active and inactive state GPCRs show that our automation strategy generalizes easily to new target classes, and that complete automation of data processing is straightforward to achieve in CryoSPARC. We provide downloadable CryoSPARC Workflow files so that users can import, replicate, adapt and extend our automated workflow for their own targets, enabling cryo-EM to be applied at larger scales and to answer larger biological questions.

biophysics↗

bioRxiv2022

hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

developmental biology↗

bioRxiv2021

Volumetric Calcium Imaging of 1 Million Neurons Across Cortical Regions at Cellular Resolution Using Light Beads Microscopy

Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 108 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3x5x0.5 mm3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4x6x0.5 mm3 volumes at ~2Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain.

neuroscience↗

bioRxiv2020

Age-Associated Insolubility of Parkin in Human Midbrain is Linked to Redox Balance and Sequestration of Reactive Dopamine Metabolites

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

neuroscience↗

bioRxiv2020

Drosophila model of Anti-retroviral therapy Induced Peripheral Neuropathy and Nociceptive Hypersensitivity

The success of antiretroviral therapy (ART) has improved the survival of HIV-infected patients significantly. However, significant numbers of patients on ART whose HIV disease is well controlled show peripheral sensory neuropathy (PSN), suggesting that ART may cause PSN. Although the nucleoside reverse transcriptase inhibitors (NRTIs), one of the vital components of ART, are thought to contribute to PSN, the mechanisms underlying the PSN induced by NRTIs are unclear. In this study, we developed a Drosophila model of NRTI-induced PSN that recapitulates the salient features observed in patients undergoing ART: PSN and nociceptive hypersensitivity. Furthermore, our data demonstrate that pathways known to suppress PSN induced by chemotherapeutic drugs are ineffective in suppressing the PSN or nociception induced by NRTIs. Instead, we found that increased dynamics of a peripheral sensory neuron may underlie NRTI-induced PSN and nociception. Our model provides a solid platform in which to investigate further mechanisms of ART-induced PSN and nociceptive hypersensitivity.

neuroscience↗

bioRxiv2018

Synergistic Recruitment of UbcH7~Ub and Phosphorylated Ubl Domain Triggers Parkin Activation

The mechanism of activation and ubiquitin conjugation by the E3 ligase parkin is pivotal to understand the molecular pathology of early-onset Parkinsons disease. Parkin is normally autoinhibited but is activated by the kinase PINK1 that phosphorylates parkins N-terminal ubiquitin-like (pUbl) domain and ubiquitin. How these alter the structure of parkin to allow recruitment of an E2~Ub conjugate to enhance ubiquitination is an unresolved question. We present the structure of an incoming E2~Ub conjugate with the phospho-ubiquitin bound C-terminus of parkin (R0RBR). We show the UbcH7~Ub conjugate is recruited by R0RBR parkin in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR experiments indicate there is re-modelling near the RING0/RING2 interface remote from the E2-binding site. This, and parkin phosphorylation lead to rapid reactivity of the RING2(Rcat) catalytic cysteine in parkin, needed for ubiquitin transfer. Parkin phosphorylation also leads to relocation and weak interaction of the pUbl domain with the RING0 domain that is enhanced upon E2~Ub recruitment indicating these events act synergistically to drive parkin activity.

biochemistry↗