Biology subjects

Havton, L. A.

Publications and source records attributed to Havton, L. A..

8 recordsLinked to original sources

bioRxiv2024

Comparative specialization of intrinsic cardiac neurons in humans, mice, and pigs

Intrinsic cardiac neurons (ICNs) play a crucial role in the proper functioning of the heart; yet a paucity of data pertaining to human ICNs exists. We took a multidisciplinary approach to complete a detailed cellular comparison of the structure and function of ICNs from mice, pigs, and humans. Immunohistochemistry of whole and sectioned ganglia, transmission electron microscopy, intracellular microelectrode recording and dye filling for quantitative morphometry were used to define the neurophysiology, histochemistry, and ultrastructure of these cells across species. The densely packed, smaller ICNs of mouse lacked dendrites, formed axosomatic connections, and had high synaptic efficacy constituting an obligatory synapse. At Pig ICNs, a convergence of subthreshold cholinergic inputs onto extensive dendritic arbors supported greater summation and integration of synaptic input. Human ICNs were tonically firing, with synaptic stimulation evoking large suprathreshold excitatory postsynaptic potentials like mouse, and subthreshold potentials like pig. Ultrastructural examination of synaptic terminals revealed conserved architecture, yet small clear vesicles (SCVs) were larger in pigs and humans. The presence and localization of ganglionic neuropeptides was distinct, with abundant VIP observed in human but not pig or mouse ganglia, and little SP or CGRP in pig ganglia. Action potential waveforms were similar, but human ICNs had larger after-hyperpolarizations. Intrinsic excitability differed; 93% of human cells were tonic, all pig neurons were phasic, and both phasic and tonic phenotypes were observed in mouse. In combination, this publicly accessible, multimodal atlas of ICNs from mice, pigs, and humans identifies similarities and differences in the evolution of ICNs.

neuroscience↗

bioRxiv2024

Sarm1 is not necessary for activation of neuron-intrinsic growth programs yet required for the Schwann cell repair response and peripheral nerve regeneration.

Upon peripheral nervous system (PNS) injury, severed axons undergo rapid SARM1-dependent Wallerian degeneration (WD). In mammals, the role of SARM1 in PNS regeneration, however, is unknown. Here we demonstrate that Sarm1 is not required for axotomy induced activation of neuron-intrinsic growth programs and axonal growth into a nerve crush site. However, in the distal nerve, Sarm1 is necessary for the timely induction of the Schwann cell (SC) repair response, nerve inflammation, myelin clearance, and regeneration of sensory and motor axons. In Sarm1-/- mice, regenerated fibers exhibit reduced axon caliber, defective nerve conduction, and recovery of motor function is delayed. The growth hostile environment of Sarm1-/- distal nerve tissue was demonstrated by grafting of Sarm1-/- nerve into WT recipients. SC lineage tracing in injured WT and Sarm1-/- mice revealed morphological differences. In the Sarm1-/- distal nerve, the appearance of p75NTR+, c-Jun+ SCs is significantly delayed. Ex vivo, p75NTR and c-Jun upregulation in Sarm1-/- nerves can be rescued by pharmacological inhibition of ErbB kinase. Together, our studies show that Sarm1 is not necessary for the activation of neuron intrinsic growth programs but in the distal nerve is required for the orchestration of cellular programs that underlie rapid axon extension.

neuroscience↗

bioRxiv2022

Ultra-flexible and Stretchable Intrafascicular Peripheral Nerve Recording Device with Axon-dimension, Cuff-less Microneedle Electrode Array

Peripheral nerve mapping tools with higher spatial resolution are needed to advance systems neuroscience, and potentially provide a closed-loop biomarker in neuromodulation applications. Two critical challenges of microscale neural interfaces are (i) how to apply them to small peripheral nerves, and (ii) how to minimize chronic reactivity. We developed a flexible microneedle nerve array (MINA), which is the first high-density penetrating electrode array made with axon-sized silicon microneedles embedded in low-modulus thin silicone. We present the design, fabrication, acute recording, and chronic reactivity to an implanted MINA. Distinctive units were identified in the rat peroneal nerve. We also demonstrate a long-term, cuff-free, and suture-free fixation manner using rose bengal as a light-activated adhesive for two timepoints. The tissue response at 1-week included a sham (N=5) and MINA-implanted (N=5) group, and the response at 6-week also included a sham (N=3) and MINA-implanted (N=4) group. These conditions were quantified in the left vagus nerve of rats using histomorphometry. Micro-CT was added to visualize and quantify tissue encapsulation around the implant. MINA demonstrated a reduction in encapsulation thickness over previously quantified interfascicular methods. Future challenges include techniques for precise insertion of the microneedle electrodes and demonstrating long-term recording.

neuroscience↗

bioRxiv2021

Screening for axon regeneration promoting compounds with human iPSC-derived motor neurons

CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human "injury in a dish" assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.

neuroscience↗

bioRxiv2020

Innervation and Neuronal Control of the Mammalian Sinoatrial Node: A Comprehensive Atlas

Cardiac function is under exquisite intrinsic cardiac neural control. Neuroablative techniques to modulate control of cardiac function are currently being studied in patients, albeit with variable and sometimes deleterious results. Recognizing the major gaps in our understanding of cardiac neural control, we sought to evaluate neural regulation of impulse initiation in the sinoatrial node as an initial discovery step. Here, we report an in-depth, multi-scale structural and functional characterization of the innervation of the sinoatrial node (SAN) by the right atrial ganglionated plexus (RAGP) in porcine and human hearts. Combining intersectional strategies including tissue clearing, immunohistochemical and ultrastructural techniques, we have delineated a comprehensive neuroanatomic atlas of the RAGP-SAN complex. The RAGP shows significant phenotypic diversity of neurons while maintaining predominant cholinergic innervation. Cellular and tissue-level electrophysiologic mapping and ablation studies demonstrate interconnected ganglia with synaptic convergence within the RAGP to modulate SAN automaticity, atrioventricular (AV) conduction and left ventricular (LV) contractility. For the first time, we demonstrate that intrinsic cardiac neurons influence the pacemaking site in the heart. This provides an experimental demonstration of a discrete neuronal population controlling a specific geographic region of the heart (SAN) that can serve as a framework for further exploration of other parts of the intrinsic cardiac nervous system (ICNS) in mammalian hearts and for developing targeted therapies.

neuroscience↗

bioRxiv2020

Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

The failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

neuroscience↗

bioRxiv2019

Endothelial CXCL5 negatively regulates myelination and repair after white matter stroke

Cerebral small vessel disease and resulting white matter pathologies are worsened by cardiovascular risk factors including obesity. The molecular changes in cerebral endothelial cells caused by chronic cerebrovascular risk factors remain unknown. We developed a novel approach for molecular profiling of chronically injured cerebral endothelial cells using cell-specific translating ribosome affinity purification (RiboTag) with RNA-seq in Tie2-Cre:RiboTag mice. We used this approach to identify the transcriptome of white matter endothelial cells after the onset of diet-induced obesity (DIO). DIO induces an IL-17B signaling pathway that acts on the cerebral endothelia through IL-17Rb to increase levels of both circulating CXCL5 and local endothelial expression of CXCL5 in both the DIO mouse model and in humans with imaging or pathologic evidence of cerebral small vessel disease. In the white matter, endothelial CXCL5 acts as a chemoattractant and promotes the association of oligodendrocyte progenitor cells (OPCs) with cerebral endothelia increasing vessel-associated OPC cell number and triggers OPC gene expression programs regulating migration and chemokine receptor activation. Targeted blockade of IL-17B with peripheral antibody administration reduced the population of vessel-associated OPCs by reducing endothelial CXCL5 expression. CXCL5-mediated sequestration of OPCs to white matter vasculature impairs OPC differentiation after a focal white matter ischemic lesion. DIO promotes a unique white matter endothelial-to-oligodendrocyte progenitor cell signaling pathway that compromises brain repair after stroke.

neuroscience↗

bioRxiv2018

Obesity blocks oligodendrocyte precursor cell differentiation and impedes repair after white matter stroke

Obesity is a growing public health problem that increases rates of white matter atrophy and increases the likelihood of ischemic lesions within white matter. However, the cellular and molecular mechanisms that regulate these changes are unknown. We hypothesized that obesity may alter oligodendrocytes and myelin priming white matter for worsening injury and repair responses after ischemia. C57Bl/6 mice fed a high fat diet (60% kcal from fat) show increased numbers of oligodendrocyte precursor cells (OPCs), decreased myelin thickness with elevated g-ratios, and shorter paranodal axonal segments, indicating accelerated myelin turnover. Fate mapping of OPCs in PDGFR-CreERT;Rpl22tm1.1Psam mice demonstrated that OPC differentiation rates are enhanced by obesity. Gene expression analyses using a novel oligodendrocyte staging assay demonstrated OPC differentiation is blocked by obesity in between the pre-myelinating and myelinating stage. Using a model of subcortical white matter stroke, the number of stroke-responsive OPCs in obese mice was increased after stroke. At early time points after ischemic white matter stroke, spatial mapping of stroke-responsive OPCs indicates that obesity leads to increased OPCs at the edge of ischemic white matter lesions. At later time points, obesity results in increased OPCs within the ischemic lesion while reducing the number of GST-{pi}-positive mature oligodendrocytes in the lesion core. These data indicate that obesity disrupts normal white matter biology by blocking oligodendrocyte differentiation, leads to an exaggerated response of OPCs to white matter ischemia, and limits remyelination after stroke.\n\nSignificance StatementObesity is a growing public health crisis that specifically increases the development of white matter lesions and silent brain infarcts. This relationship implies a direct cellular effect on white matter yet direct evidence is limited. We modeled diet-induced obesity in mice and studied the effect on oligodendrocyte biology and the response to focal white matter stroke. Adult-onset obesity results in thinner myelin, compromised axonal microdomain structure, and blocks the differentiation of OPCs, leaving the white matter with increased numbers of OPCs. After focal white matter stroke in obese mice, the early OPC response to stroke is exaggerated while late reparative OPC differentiation is blocked. These results suggest that obesity specifically blocks OPC differentiation with consequence on brain repair after stroke.

neuroscience↗