Biology subjects

Bushong, E. A.

Publications and source records attributed to Bushong, E. A..

13 recordsLinked to original sources

bioRxiv2026

Resilience to neuronal hyperactivity and restoration of the neuroimmune interactome by blocking fibrin-induced microglia activation in Alzheimers disease

Cerebrovascular pathology and neuronal network dysfunction are early features of Alzheimers disease (AD) associated with neuroinflammation and cognitive decline, but the vascular and immune triggers of neuronal hyperactivity remain largely unknown. Here, we show that the blood coagulation protein fibrin disrupts microglia-neuron interactions, promoting neuronal hyperactivity in an AD mouse model. Genetic elimination of the fibrin inflammatory domain reduced neuronal hyperactivity, restored dynamic microglial interactions with active neurons and protected from high-risk decision making in 5XFAD mice. Leveraging the transcriptional signatures of microglia and inhibitory and excitatory neurons, a ligand-receptor atlas revealed fibrin-dependent disruption of innate immune and glutamatergic signaling between microglia and neurons in AD mice. Patients with AD also showed a correlation of cerebrospinal fluid (CSF) fibrinogen levels with biomarkers of inflammation, vascular and synaptic dysfunction. Thus, resilience to neuronal hyperactivity and restoration of the neuroimmune interactome by targeting fibrin may have therapeutic implications for Alzheimers disease and related conditions. There is a companion manuscript submitted to bioRxiv (Lauderdale et al., 2026) HighlightsO_LIVascular-microglia axis drives neuronal hyperactivity C_LIO_LIFibrin inflammatory activity disrupts the microglia-neuron interactome C_LIO_LIMicroglia activation by fibrin impairs decision-making in AD mice C_LIO_LISynaptic dysfunction and immune biomarkers correlate with CSF fibrinogen in AD patients C_LI

neuroscience↗

bioRxiv2024

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via RiboTag, morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

neuroscience↗

bioRxiv2023

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. SignificanceIn this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.

neuroscience↗

bioRxiv2021

Systematic morphological and morphometric analysis of identified olfactory receptor neurons in Drosophila melanogaster

The biophysical properties of sensory neurons are influenced by their morphometric and morphological features, whose precise measurements require high-quality volume electron microscopy (EM). However, systematic surveys of these nanoscale characteristics for identified neurons are scarce. Here, we characterize the morphology of Drosophila olfactory receptor neurons (ORNs) across the majority of genetically identified sensory hairs. By analyzing serial block-face electron microscopy (SBEM) images of cryofixed antennal tissues, we compile an extensive morphometric dataset based on 122 reconstructed 3D models of 33 identified ORN types. In addition, we observe multiple novel features--including extracellular vacuoles within sensillum lumen, intricate dendritic branching, mitochondria enrichment in select ORNs, novel sensillum types, and empty sensilla containing no neurons--which raise new questions pertinent to cell biology and sensory neurobiology. Our systematic survey is critical for future investigations into how the size and shape of sensory neurons influence their responses, sensitivity and circuit function.

neuroscience↗

bioRxiv2021

Morphological principles of neuronal mitochondria

In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like ATP and heat often represent mitochondria as idealized geometries and therefore can miscalculate the metabolic fluxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial TEM tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.6 nanometer isotropic voxels. The resulting in silico representations were subsequently quantified by differential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs that are present across this population of mitochondria; observations which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons.

neuroscience↗

bioRxiv2020

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently-labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. Our approach allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglia cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

developmental biology↗

bioRxiv2020

Aβ-induced synaptic injury is mediated by presynaptic expression of amyloid precursor protein (APP) in hippocampal neurons

The patterns of A{beta}-induced synaptic injury were examined after targeting of the amyloid precursor protein (APP) preferentially to either CA1 or CA3 neurons using Cre-lox technology combined with tetracycline-regulated expression. Both CA1- and CA3-APP-expressing transgenic mouse lines exhibited reduction in long-term potentiation (LTP) only when APP was expressed in neurons presynaptic to the recording site, whereas LTP remained comparable to wild-type mice when APP was expressed in postsynaptic neurons. As quantified by both light and electron microscopy, this orientation-specific impairment in synaptic plasticity was mirrored by synaptic loss in regions receiving axonal inputs from neurons expressing APP. Furthermore, A(plaque deposition also occurred only in the postsynaptic axonal fields of APP-expressing neurons. These deficits were reversed not only with doxycycline to inhibit APP expression but also with {gamma}-secretase and Fyn kinase inhibitors, supporting the interpretation that the observed synaptic injury was mediated by A{beta}. Taken together, these results demonstrate that APP/A{beta}-induced synaptic toxicity is preferentially initiated by signaling of presynaptically expressed APP to the postsynaptic compartment.

neuroscience↗

bioRxiv2020

Combined membrane potential imaging and connectome of behavioral circuits in an annelid worm

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced all of DE-3s processes. Further, we traced back the processes of all of DE-3s presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all of the behaviors were widely distributed over DE-3s branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

neuroscience↗

bioRxiv2019

Mitochondrial morphology provides a mechanism for energy buffering at synapses

Mitochondria as the main energy suppliers of eukaryotic cells are highly dynamic organelles that fuse, divide and are transported along the cytoskeleton to ensure cellular energy homeostasis. While these processes are well established, substantial evidence indicates that the internal structure is also highly variable in dependence on metabolic conditions. However, a quantitative mechanistic understanding of how mitochondrial morphology affects energetic states is still elusive. To address this question, we here present an agent-based dynamic model using three-dimensional morphologies from electron microscopy tomography which considers the molecular dynamics of the main ATP production components. We apply our modeling approach to mitochondria at the synapse which is the largest energy consumer within the brain. Interestingly, comparing the spatiotemporal simulations with a corresponding space-independent approach, we find minor space dependence when the system relaxes toward equilibrium but a qualitative difference in fluctuating environments. These results suggest that internal mitochondrial morphology is not only optimized for ATP production but also provides a mechanism for energy buffering and may represent a mechanism for cellular robustness.

biophysics↗

bioRxiv2018

Asymmetric ephaptic inhibition between compartmentalized olfactory receptor neurons

In the Drosophila antenna, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this unusual form of lateral inhibition remain unclear. Here we use recordings from pairs of sensilla impaled by the same tungsten electrode to prove that direct electrical (\"ephaptic\") interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, we find that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.

neuroscience↗

bioRxiv2018

Verifying, Challenging, and Discovering New Synapses among Fully EM-Reconstructed Neurons in the Leech Ganglion

Neural circuits underpin the production of animal behavior, largely based upon the precise pattern of synaptic connectivity among the neurons involved. For large numbers of neurons, determining such \"connectomes\" by direct physiological means is difficult, as physiological accessibility is ultimately required to verify and characterize the function of synapses. We collected a volume of images spanning an entire ganglion of the juvenile leech nervous system via serial blockface electron microscopy (SBEM). We validated this approach by reconstructing a well-characterized circuit of motor neurons involved in the swimming behavior of the leech by locating the synapses among them. We confirm that there are multiple synaptic contacts between connected pairs of neurons in the leech, and that these synapses are widely distributed across the region of neuropil in which the neurons arbors overlap. We verified the anatomical existence of connections that had been described physiologically among longitudinal muscle motor neurons. We also found that some physiological connections were not present anatomically. We then drew upon the SBEM dataset to design additional physiological experiments. We reconstructed an uncharacterized neuron and one of its presynaptic partners identified from the SBEM dataset. We subsequently interrogated this cell pair via intracellular electrophysiology in an adult ganglion and found that the anatomically-discovered synapse was also functional physiologically. Our findings demonstrate the value of combining a connectomics approach with electrophysiology in the leech nervous system.\n\nSignificance StatementThe function of any nervous system depends on the arrangement of its component neurons into circuits. Determining this precise pattern requires an account of which neurons are linked by synapses, and where. Here we use serial electron microscopy to confirm, challenge, and discover synapses in the neuropil of one ganglion from a juvenile leech. Relying on the homology of the ganglion from animal to animal, we demonstrate that we can identify synapses we knew existed from previous physiological work, and that we can confirm a new anatomically-discovered synapse by subsequently recording from the same neurons in a different animal. Here we show how analyses of anatomical detail and physiologically determined interactions complementarily yield insight into how neural circuits produce behavior.

neuroscience↗

bioRxiv2018

CDeep3M - Plug-and-Play cloud based deep learning for image segmentation of light, electron and X-ray microscopy

As biological imaging datasets increase in size, deep neural networks are considered vital tools for efficient image segmentation. While a number of different network architectures have been developed for segmenting even the most challenging biological images, community access is still limited by the difficulty of setting up complex computational environments and processing pipelines, and the availability of compute resources. Here, we address these bottlenecks, providing a ready-to-use image segmentation solution for any lab, with a pre-configured, publicly available, cloud-based deep convolutional neural network on Amazon Web Services (AWS). We provide simple instructions for training and applying CDeep3M for segmentation of large and complex 2D and 3D microscopy datasets of diverse biomedical imaging modalities.

cell biology↗

bioRxiv2018

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation and high temporal resolution. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.

biophysics↗